Thunnus thynnus, approximately 15 to 60 kg, including juveniles and adult fishes) collected from the Mediterranean Sea through the metabarcoding of two gene regions (cytochrome c oxidase subunit I (COI) and the ribosomal 18S-V1V2 region). The identified prey taxa and their relative read abundances (RRAs) estimated using COI results were in line with the findings of morphologically based inventories simultaneously performed on the same set of tuna samples. In both cases (and with the same rankings), the prey taxa included anchovy , sardine , sprat , mackerel  and cephalopods . Another striking result was the detection, based on 18S , of a high prevalence and diversity of gelatinous organisms (RRA = 27.1%), including cnidarians (6.7%), salps (11.7%), and ctenophores (8.7%), the latter increasing with the size of the predator. These results thus support the hypothesis of the role of gelatinous prey in the diet of Atlantic bluefin tuna, suggesting that this species is even more generalist and opportunistic than previously thought. This study further confirms that DNA metabarcoding can be a powerful tool for assessing the diet and trophodynamics of top marine predators.Studies of the diet, feeding habits and trophic activity of top marine predators are essential for understanding their trophodynamics. The main direct method used for such studies thus far has been morphological inventories of stomach contents. This approach presents limitations such as missing gelatinous prey, which are usually digested too quickly to be detectable. Here, we analysed the stomachs of 48 Atlantic bluefin tuna ( Large pelagic fishes are top predators that contribute to the stability and persistence of marine ecosystems through top-down control . InformaSeveral methods can be used to identify the diets of these fish: direct morphological inventories of stomach contents, indirect reconstruction through the biochemical analysis of tissue, and indirect assessment through telemetry surveys . Direct Thunnus thynnus) and Pacific bluefin tuna  demonstrated the power to identify fish prey down to the species level, especially for larvae  according to a presence-absence inventory as well as associated metrics  to describe the ABFT diet. Our second objective was to assess the importance of gelatinous species in the ABFT diet and potential differences between ABFT juveniles and adults due to differences in foraging behaviour. Finally, we also tested the usefulness of blocking primers for minimizing predator sequences and optimizing the sequencing depth for prey in molecular analyses of tuna stomach contents.https://fisheries.msc.org/). All the stomachs were collected at the landings port  from fish captured between late July and early December of 2011 to 2014. Individual fishes were measured  and weighed (±0.1 kg) while stomach contents were weighed, and completely and partially intact prey were identified to the lowest possible taxonomic level (J < 30 kg and M > 30 kg) and mouth length (8.8 cm–17 cm), as an indicator of overall length, and these metrics did not vary significantly compared to the sampling set that was used in the morphological study.ABFTs were caught in the Gulf of Lions  by local small-scale fishery using longlines or handlines, which recently become the second bluefin tuna fishery in the world to achieve the Marine Stewartship council (MSC) certification , and molecular water to the final volume. Additionally, all COI reactions included an additional 1 mM MgCl2 (1.2 ul/25 mM), which together with the 1.5 mM MgCl2 contained in the Mix-Phusion resulted in 2.5 mM MgCl2. The PCR cycling conditions based on Mus musculus) DNA to improve decontamination (see bioinformatic methods), and index sequencing control.For DNA extraction, the stomachs were only slightly thawed to facilitate the dissociation of stomach contents while maintaining the lowest possible count of host cells and, thus, minimizing contamination by host (tuna) DNA. For every stomach, all laboratory surfaces were cleaned with bleach, and the upper part of the stomach was carefully incised with a scalpel to open the stomach, after which its complete contents were emptied into a  bleach-disinfected blender, where they were mixed for 30 s to 2 min until completely homogenized. We performed this step on only unfrozen  stomach contents to limit the natural heating during blending to a marginal level, in order to limit DNA degradation during this process. Approximately 1 g of the homogenized mixture was then used for DNA extraction. In addition to mechanical maceration, lysis with proteinase K proved necessary, likely due to the high protein content of the mixture of nearly intact fish prey. For extraction, the best results were thus obtained using a lysis step followed by the NucleoSpin Tissue Kit . This kit was thus used for all DNA extractions, following the manufacturer’s instructions, except that DNA elution was performed twice in 50 µl of preheated (70 °C) molecular water. Two empty NucleoSpin columns were added to the extraction series and used as extraction controls. PCR was performed separately for two barcoding gene regions : COI  can cause the host DNA to monopolize part of the sequencing capacity, lowering the effective sequencing depth and hindering the assessment of prey diversity. However, the use of blocking primers specific to the host including a three-carbon spacer (C3) modification at the 3 ′ end to specifically amplify host DNA shown good results in avoiding this potential pitfall and optimizing the sequencing of prey . Therefo GenBank . An in sDatabase  to optimData were analysed following the bioinformatic pipeline described by decontam R package , known to occur when amplifying COI with universal primers  for 18S-V1V2 ribosomal sequences and Midori  for COI. of 0.5; ), with i of 0.5;  to accou of 0.5; . To remos , the prs  matches. COI was shown not to be diagnostic for all distinct tuna species  and the weighted percentage of occurrence (wPOO) of prey taxa, both with a minimum 1% occurrence threshold. Second, to test the accuracy of the semi-quantitative information that the number of reads could deliver, RRA  was estiThe data for all gene regions were analysed in R using the vegan package  with the family set to ‘binomial’ to predict the presence/absence of each prey taxon (dependent variable) depending on the independent variable “year of catch”  as well as the linked (because both were correlated with size) factors “predator body mass”  and “mouth length” . This ‘full’ model as well as the ‘null’ model was tested against forward and backward selection, and the model was chosen according to the best AIC  and testSRR134477398–SRR13447844, with BioSamples SAMN17349101–SAMN17349148, ID PRJNA692564, respectively.All raw sequences have been stored in the Sequence Read Archive (SRA) under accession numbers Metazoans were detected in 45 and 44 of the 48 ABFT stomachs analysed on the basis of COI and 18S sequences, respectively. Two of the three remaining ABFT stomachs were nearly empty, as noted during dissection-homogenization, while we have no obvious explanation for the failure of detection in the third stomach. The number of generated reads was 40 to 100,952  for 18S, with 142 to 27,237 identifications after stringent cleaning and 1329 to 53,998 taxonomic identifications , leading to the identification of 832 to 41,528 sequences , including barcodes for several algae and other nonmetazoans  from the following taxa Apicomplexa, Dinoflagellata, Fungi, Ochrophyta, Phragmoplastophyta, Protalveolata, and Viridiplantae. Thus, the original metazoan RRA was 58.2%, with nonmetazoans accounting for slightly less than half the RRA but excluded from further analyses as they were not the target of this study.The potential metazoan prey detected using the 18S and COI sequences are summarized in The identified metazoan group with the highest diversity of species/taxa see  was arthEngraulis encrasicolus (RRA = 43.4%), 38 Sardina pilchardus (RRA = 30%), 32 Sprattus sprattus (RRA = 8%) and 21 Scomber colias (RRA = 7%). Vertebrates were also the most abundant taxonomic group detected with 18SV1 (RRA = 61.6), but most sequence were assigned to Alosa alosa , which is unlikely based on the limitations of species identification combined with the relatively high abundance of sequences of this particular species in reference databases.The second most diverse taxonomic group (but detected the most frequently) was vertebrates see also , which mCreseis, and one Bivalvia (RAA > 0.01%).The genetic approach also allowed the detection of soft-bodied potential prey items, with 15 different cnidarian taxa detected from the COI and 18S data  or overall. For example, both morphological and COI analyses indicated that fish were the dominant prey, with four common and clearly identified fish species being recorded  calculated per year showed the same trends as the relative abundance (RA) based on the morphological data from rded see . Among t COI see .p = 0.0016 for COI testing weight at euclidian distribution, all others with p < 0.001), which is likely to affect the correlation analysis. The two factors were, however, correlated, as they are both proxies of ABFT size. General linear models (GLMs) showed that ABFT weight/size and year of catch were significantly related to the presence/absence of anchovy, arthropods, and Ctenophora values (coefficient estimate and std. error) given in In the analysis of compositional dissimilarity, none of the available fish descriptors  showed any significant relationship with the stomach contents  was developed and tested within this study. Nearly all samples analysed produced a high number of Thunnus sequences. However, in the 10 samples analysed with the blocking primer, no Thunnus DNA was found, showing the high efficiency of the primer (see To avoid the possible overdetection of predator DNA (ABFT sequences in this case), a blocking primer for the COI gene region of imer see .in silico analyses suggested a low amount of (if any) undesired blocking , as only 92 species  among the tested 110,726 species in the Midori database could be blocked when accepting a blocking effect with up to three mismatches. The exact list, including the number of mismatches, is provided in Thunnus species might potentially prevent the amplification of sequences from 8 other Scombridae if some mismatches were accepted. However, the detection of Scomber colias was positive with or without blocking primer in the present study, suggesting that the blocking primer did not prevent amplification in this family. Moreover, compared to data from the same samples obtained without the blocking primer , sardine (Sardina pilchardus), sprat (Sprattus sprattus) and mackerel (Scomber colias). Moreover, the results obtained from the 18S metabarcoding of stomach contents highlighted a high diversity of gelatinous taxa in the ABFT diet, which was stable across years; the importance of these taxa seemed to increase with ABFT weight and, thus, likely with age.Following the recommendation of Thunnus orientalis) consumes the same types of prey, with a diet dominated by fish, crustaceans, and cephalopods, including an age-based shift from crustaceans to more fish-based food during growth  in only one of the ABFT stomachs, with 5% of reads, and only three other detected taxa  most likely originated from left-over food from humans and could indicate either scavenging or even less target-oriented foraging then hitherto considered. Such opportunistic swallowing of potential food would, however, be in line with the extremely large amounts of different sized plastic debris found in ABFT stomachs  and other shrimp have been shown to be important food sources for ABFT, albeit more frequently reported in juveniles than adults  associated with generally low values (RRA ≤ 3%). In contrast, groups such as crustaceans showed considerable diversity associated with more frequent detection and a sometimes higher RRA (2.6% for COI and up to 8.5% for 18S). Their frequent presence, associated with a relatively high abundance even compared to fishes, is not surprising and has often been reported for ABFT juveniles, either through morphological or isotope analysis . Based on adults . Many ofn adults ); these n adults . Indeed,n adults , Table 3etection  and may Mnemiopsis leidyi observed in the Black Sea  to specifically target invertebrates . Like COlack Sea . In thisOur results also indicate that the use of stomach contents may be preferrable to faeces for ABFT because the morphology of the tuna stomach allows a very clear-cut separation of the stomach contents from the stomach wall, avoiding any significant contamination by the host DNA, which would be expected with faeces. The blocking primers developed in this study may, however, be useful for faeces analysis, as they showed effective blocking of tuna DNA during PCR, and the differences were not significant compared to data from the same stomachs obtained without blocking primers. Additionally, the use of blocking primers can hinder the detection of potential cannibalism, which has been observed towards ABFT larvae , and alsThe results presented here validate the use of metabarcoding to assess the diet of a top predator, Atlantic bluefin tuna (ABFT). In addition to providing a comprehensive list of prey taxa, our results unexpectedly showed a good match between semi-quantitative estimates (relative abundance) inferred from morphological and molecular (COI) inventories of stomach contents. They further confirmed the more opportunistic feeding behaviour of ABFT than hitherto indicated. Perhaps more importantly, the concomitant use of the 18S ribosomal barcode finally confirmed the importance of diverse gelatinous prey in the ABFT diet. The work presented here thus encourages the further use and improvement of molecular approaches to better understand trophic interactions and their predicted evolution in a changing environment.10.7717/peerj.11757/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.11757/supp-2Supplemental Information 2Click here for additional data file.


Necrotizing enterocolitis (NEC) is a typical disorder of preterm newborns, with a high mortality and morbidity rate. The therapeutic and nutritional management of disease depends on several factors. Its prognosis is linked, in addition to the severity of the disease and the need for surgery, to a correct enteral feeding in these patients. This study aims to identify the clinical characteristics of 18 patients with NEC, evaluating the different therapeutic paths undertaken, the type of formula used and the survival rate of this population. Average time of enteral nutrition before the NEC onset was 11,3 ± 11,6 days, with an average fasting period since the onset of 24 ± 18.9 days. 77.8% of patients received surgery and resumed enteral nutrition 17.7 ± 17.9 days after the intervention. The overall survival rate of our cohort was 55.5%. More prospective studies are needed to evaluate the long-term outcomes of survived children with NEC. Necrotizing enterocolitis (NEC) is a critical disease typical of premature infants. Although its incidence varies among different neonatal intensive care units, the average prevalence among very low birth weight infants (VLBW), defined as those with a first recorded weight < 1500 g, is 7% . The intThe Bell’s staging, introduced in 1978 and later modified by Kligeman and Walsh, stratifies the severity of this pathology and guide the treatment approach . In BellThe purpose of this study is to evaluate a population of patients affected by NEC, collecting their clinical data and analyzing the nutritional implications of disease.An observational prospective study was carried out through the collection of cases of NEC admitted to the Neonatology and Neonatal Intensive Care Unit of “Policlinico P. Giaccone”, Palermo between January 2015 and January 2019. We revised the medical records of these infants; data were extrapolated with a standardized table. The main outcome of this study was to evaluate the survival rate, distribution and characteristics of NEC patients in a single cohort, evaluating different approaches utilized and observing their possible clinical outcomes.Clinical data that have been collected were clinical condition and age at diagnosis, progression of disease, therapeutic nutritional and surgical management. For each patient a multidisciplinary evaluation has been carried out through joint visits, with the presence of pediatrician, surgeon, parents. Categorical variables were synthesized with frequencies, percentages and statistical distribution.The study was conducted according to the guidelines of the Declaration of Helsinki, and it has been approved by the ethical committee of the center involved.In total, 18 infants were included in the study: 8 males and 10 females .Table In our series, due to the prematurity, many infants started parenteral nutrition before NEC onset. It should be noted that in the event of death, the day of exitus was considered the day of parenteral suspension. On average, infants in our population practiced parenteral nutrition for 50 ± 28 days. Regarding the composition of the parenteral formula administered,12 newborns received olive oil lipid emulsions, 5 received soy-bean lipid emulsions and 1 received parenteral with lipid-free formula.4 patients did not have surgery (22.2%) and 14 had surgery (77.8%). Surgery was performed on average after 32.5 ± 27.1 days of life. Among operated infants, 5 had an intestinal resection with ileostomy placement (27.8%), 4 had only ileostomy packaging (22.2%), 1 had a colostomy, 2 had neither resection nor ileostomy. 2 patients were treated exclusively with an abdominal drainage. Within the group of operated infants, the reintroduction of enteral nutrition occurred on average after 17.7 ± 17.9 days from surgery.Iron, D and K vitamins supplementation is essential in preterm newborns  Our popuWe evaluated the administration of antacids and ursodeoxycholic acid (UDCA) in our population, trying to identify an indirect parameter that would give us an overview on main gastroenterological sequences of NEC (ex. gastric hypersecretion and liver disease). Many neonates with NEC, especially those who had jejunostomies, showed an increased gastric secretion that may require antacids . In our Many clinical and radiological signs were evaluated from the onset of NEC. Principal signs of food intolerance which brought to the interruption of enteral feeding were abdominal distension 83.2%), stagnation (33.3%) and intestinal bleeding (27.7%)  it is probably linked to the presence of a Pediatric Surgery Ward in the same departmental complex. Low birth weight and high prevalence of surgical NECs could explain the total high mortality rate in our sample 44.5%), which is higher than the mortality described in other reports [%, which favors the development of deficiencies in vitamins and nutrientsdetermines atrophy of the intestinal mucosapromotes bacterial overgrowthprolonged parenteral nutrition increasing its infectious and metabolic complications.Medical NEC therapy consists of the administration of broad-spectrum antibiotics, the fasting and the initiation of parenteral nutrition. The duration of fasting depends on clinical, laboratory and instrumental evaluation . AccordiClinicians should minimize fasting, reintroducing enteral nutrition as soon as there is a clinical improvement witnessed by the stability of vital signs, the abdominal physical examination, and the normalization of platelet count and radiologic signs The identification of biomarkers  , 22, capNeonates with NEC should start early parenteral nutrition with an adequate dose of amino acids (3.5–4 g/kg/day), in order to maintain a positive nitrogen balance, an improvement of weight growth and allowing the repair of damaged tissues , 23–25. Main metabolic complications of the parenteral nutrition are liver disease and intestinal failure (IFALD) . These cRecently, new lipid mixtures called Smoflipid have been developed. They are composed by soybean oil (30%), coconut oil (30%), olive oil (25%) and fish oil (15%).In children with cholestatic jaundice, there is an improvement in liver function after switching from soy-bean lipid emulsions to Smoflipid . SmoflipIn cases of NEC complicated by intestinal perforation, surgery is required. In our sample, 14 newborns underwent surgery. Among the 14 operated infants, 5 underwent intestinal resection with enterostomy packaging, 5 had enterostomy packaging only, 2 underwent a laparotomy without resection or enterostomy, and finally 2 were treated exclusively with the placement of an abdominal drainage. After extensive bowel resections intestinal failure can develop . This coIn our series, patients who received surgery resumed enteral nutrition 17.7 ± 17.9 days after the intervention. Figure n = 14), parenteral was practiced for 53.4 ± 29 days: 11/14 received olive oil lipid emulsions, 2/14 soybean lipid emulsions, finally 1/14 received lipid-free emulsions. Surgical NEC typically requires prolonged use of parenteral nutrition (> 21 days) [In patients of our series with NEC at stage III (21 days) –42. Pare21 days) . In clin21 days) .Ostomies result in losses of mineral salts and micronutrients, especially zinc. Intestinal resections alter the absorption of certain nutrients, depending on the intestinal tract resected. In case of jejunostomy, the absorption of iron and zinc can be impaired. In ileal resections and fasting, the absorption of vitamin B12 and the resorption of biliary salts is altered. In addition, infants who practice prolonged exclusive parenteral may develop carnitine deficiency. Prolonged parenteral can cause cholestasis that reduces the resorption of fat-soluble vitamins.Our report confirm that NEC may be considered a heterogeneous and nutritional disease, which poses a series of problems, mainly related to different patients with different risk factors related to different ages, with very different surgical approaches. For this reason, each patient has specific nutritional risks, and should be evaluated by nutritionists or neonatologists with specific knowledge to assess a proper management, a correct fasting period and how to manage parenteral nutrition.Breast milk represents the only real prevention of NEC, and it is also beneficial for newborns after surgery. It is ideal for re-nutrition both for digestibility and for the ability to stimulate intestinal trophism , 20, 45.The main challenge in the management of this disease is an early diagnosis, which could lead to a reduction in terms of mortality and morbidity. The identification of a specific biomarker could then allow early diagnosis or provide information on prognosis and severity.Regarding different nutritional strategies in newborns with NEC, an excessively prolonged fasting should be avoided because it could increase the duration of parenteral nutrition and promotes intestinal atrophy. Enteral nutrition should be introduced within 7 days in stage I and II cases of NEC, and within 14 days in patients on stage III NEC .Newborn with NEC have various risk factors for nutritional deficiencies: prematurity, intestinal damage, fasting, prolonged parenteral nutrition and possible presence of enterostomies . A macroIn cases of possible surgical therapy, it must be considered that it could cause malabsorption, ending up in a possible short bowel syndrome. Considering all these implications, in order to manage and limit the sequelae of the disease, the newborn with NEC needs for a periodic and frequent multidisciplinary evaluation made by a neonatologist, surgeon and nutritionist.Our study can be considered limited by the single center approach, although this decreases the homogeneity in the management approach of disease. Another limit of this study, can be the variability in weight and gestational age of preterms.Principal strength of this study, instead, is the number of patients for such a rare disease, which helped to identify the epidemiological characteristics of NEC patients and the different possible therapeutical management that can be performed.More studies are needed to identify the ideal nutritional strategy for these infants and how to identify specific pathology markers. Furthermore, a prospective evaluation of neonates with NEC may be useful to evaluate the quality of life of these patients in the long-term, considering the nutritional, auxological and neurological outcomes.


We recently described two outbred mouse lines that were selected for large litter size at first delivery. However, lifetime fecundity appears to be economically more important for the husbandry of many polytocous species for which mouse lines might serve as bona fide animal models (e.g. for pigs). In the present study, we compared the lifetime fecundities of two highly fertile mouse lines (FL1 and FL2: >20 offspring/litter at first delivery) with those of an unselected control line (ctrl) and two lines that were selected for high body weight (DU6) and high protein mass (DU6P) without selection pressure on fertility. We tested the hypothesis that selection for large litter size at first parturition would also increase lifetime fecundity in mice, and we observed very large differences between lines. Whereas FL1 and ctrl delivered up to nine and ten litters, none of the DU6 and DU6P females gave birth to more than five litters. In line with this observation, FL1 delivered the most pups per lifetime . FL2 females produced the largest average litter sizes (20.4 pups/litter) in the first four litters; however, they displayed a reduced number of litters. With the exception of ctrl, litter sizes declined from litter to litter. Repeated delivery of litters with high offspring numbers did not affect the general health of FL females. The presented data demonstrate that two biodiverse, highly fertile mouse lines selected for large litter size at first delivery show different lifetime reproductive fitness levels. Thus, these mouse lines might serve as valuable mouse models for investigating lifetime productivity and longevity in farm animals. Worldwide, more than 1000 mouse models show a specific fertility phenotype . Among tMouse models showing an increased fertility phenotype can be divided into two groups: (i) ‘classical’ transgenic or knockout inbred models and (ii) outbred mouse models. Only approximately a dozen transgenic or knockout inbred mouse models show an increased fertility phenotype. One example is the Bcl-2 knockout mouse. Due to the genetic inactivation of the Bcl-2 gene, affected females show decreased ovarian somatic cell apoptosis followed by enhanced folliculogenesis and a higher ovulation rate . A higheIn contrast to classical inbred mouse models, several attempts have been made to select for higher fertility through selective breeding, for example, in pigs and rabbits . These aAt the FBN Dummerstorf, the two high-fertility mouse lines  were developed by creating the unselected control line FZTDU (ctrl) as the initial population after crossing four inbred and four outbred founder mouse lines . To keepThe estimation of reproductive parameters (e.g. litter size or mating rate) in mouse lines often refers to the values at the first delivery. However, litter size at first delivery might not be the most interesting fertility parameter in economically important farm animal species  for which FLs might serve as bona fide animal models. For these species, increasing lifetime fecundity, including longevity, is more important and economically desired and ensures sustainable livestock farming. For various pig breeds, it has been described that litter sizes increase from litter to litter, reaching maximal levels at the 3rd to 4th delivery  environment with defined hygienic conditions at a temperature of 22.5°C, at least 45% humidity and a controlled light regime with a 12 h light:12 h darkness cycle. The mice were kept in polysulfone cages of 267 × 207 × 140 mm  or type II long cages (365 x 207 x 140 mm) for the larger line of animals and had free access to pellet concentrate and water. A standard breeding diet with 22% crude protein, 34% starch, 5% sugar, 4.5% crude fat, 3.9% crude fibre, 50.1% N free extracts and a 3.2% mineral mixture  was providedFL1 and FL2 lines were selected by the Dummerstorf fecundity index, which was calculated as fecundity index = 1.6 × litter size at birth + total litter birth weight (g) in primiparous females. Beginning with generations 174 (FL2) and 175 (FL1), selections were performed by BLUP (best linear unbiased prediction) breeding value estimation, focusing only on the number of pups in primiparous females.In 1975, selection on growth was started, creating the Dummerstorf growth line DU6 by selection for high body mass by sib/family selection  and ‘productive mating rate’ , we calculated the average of these characteristics over the last 22 generations in each of the mouse lines.Animals were randomly selected from the five purebred lines after the first successful delivery in the course of the standard breeding protocol. In summary, we used 30 females of DU6 and DU6P, 50 females of the ctrl and FL1 lines and 60 females of the fertility line FL2. The included dams from the ctrl, FL1 and FL2 lines came from two different successive groups, which was considered in the statistical model. Females were mated in a 1:1 female/male ratio with an adult buck from the same line. In Supplementary Fig. 1   and SAS software . Descriptive statistics and tests for normality were calculated with the UNIVARIATE procedure of Base SAS software . The litter size at birth count data was analysed with the GLIMMIX procedure of SAS/STAT software  using a Poisson model with the fixed effects line  and litter number  within each line and group. The length of the reproduction period per dam was analysed only as the remaining time in the experiment in days. For the analysis of the duration of the reproduction period per dam, we used the LIFETEST procedure of SAS/STAT software to compute nonparametric Kaplan–Meier (product-limit) estimates of the survivor functions for the five lines to compare the survival curves of these lines. In addition, least-squares means (LSMeans) and their We previously described two outbred mouse lines that were selected for high fertility via breeding for more than 45 years and 174 generations. The actual status over the last 22 generations of selection regarding the fertility traits ‘litter size at first parturition’ and the ‘percentage of productive mating’ are displayed in As shown in To correct these data, we analysed at which age the animals had to be removed from the experiment independent of the failure criterion. We observed that DU6, DU6P and FL2 females had to be removed from the experiment after 223, 267 and 269 days , whereas females of FL1 and ctrl remained in the breeding experiment for 385 and 393 days, respectively . In addiFurthermore, we observed several line-specific differences according to fertility and other physiological parameters. Females of the DU6 line require a longer period for gestation, especially in later matings  and were considerably heavier than females of the other lines .In the next experiment, we addressed the question of how many litters each dam could deliver. Here, we observed large differences between the different lines. Whereas DU6 females had an average of only 1.9 litters/animal, females of FL1 and ctrl had averages of 5.3 and 5.8 litters per animal, respectively .Figure These differences in fertility are partly reflected by the total number of offspring per lifetime and per female. On average, females of DU6 gave birth to 18.0 ± 1.6 offspring per lifetime, whereas females of FL2, ctrl and FL1 delivered 53.2 ± 3.0, 61.6 ± 3.1 and 85.7 ± 3.3 offspring per lifetime, respectively . In thisIn the next analysis, the question of how many animals of a particular line remained in the experiment at each round of mating was addressed. The number of dams able to produce subsequent litters after their first litter provides an indication of the female lifetime fertility for each line. As shown in In this experiment, we also counted the average litter size per female within the first four litters to eliminate the negative effect of declining litter sizes as the number of matings increased for the ctrl and FL1 lines. We observed the highest litter sizes per female in FL2 , with slightly lower litter sizes in FL1  and the other lines  . AlthougSince the productive mating rates in the purebred lines already indicate substantial differences , we analTo further compare the fertility characteristics of the investigated mouse lines, we integrated three fertility parameters into a comprehensive fecundity index according to We previously described two highly fertile mouse lines (FL1 and FL2). Based on selection over >45 years and 190 generations, these mouse lines almost doubled the number of offspring compared to a control line. From the very beginning of the selection process, both lines were developed separately and are now highly diverged mouse lines showing various differences in their physiological, behavioural, endocrine and molecular characteristics . Neverthwww.phenome.jax.org). Most recommendations include the following suggestions: (i) replace females after 60 days without producing a litter and/or (ii) replace females if the female produces pups but the pups do not survive for two to three litters . Based on these basic parameters, we set up the present experimental design. The protocol should include suckling of offspring to achieve a more comprehensive picture of the fertility of the mouse lines. Thus, females were mated together with a buck from the same line for 2 weeks followed by a 3-week observation period to determine whether the female conceived. In the case of an unsuccessful mating, the 2-week mating period followed by a 3-week observation period was repeated. Only those females that did not deliver offspring in this dual mating protocol were removed from the experiment due to impaired fertility. As mating partners, we selected adult bucks from the same line that were replaced after three to four rounds of mating. This protocol minimizes the impact of one particular buck since we recently described that the reproductive performance in Dummerstorf high-fertility mouse lines primarily depends on the female genotype in a two-factorial breeding experiment ; however, at least in the first three litters, the litter sizes were very large . This isFemales of FL1 and ctrl could deliver up to nine and ten litters  and B. IFrom the presented data, we cannot conclude that the frequent delivery of large litter sizes causes any health problems. The postpartum losses of newborns were not increased in the high-fertility lines compared to the other lines if the number of suckling pups was standardized to 10 immediately after birth . In thisFurthermore, for highly fertile dams, there was no increased incidence of being removed from the experiment due to health problems . MoreoveCombining all these data together in a fecundity index that includes (i) litter size, (ii) litters born per dam, and (iii) the productive mating rate (‘Silver fecundity index’ ), it becIn addition to the ‘Silver fecundity index’, one could also apply other fecundity indices, for example, the recently described ‘simplified method to measure mouse fertility’ (In view of the two high-fertility mouse lines FL1 and FL2 (high-fertility selection criterion: litter size of first delivery), it should be emphasized that they have alternative strategies to achieve their ‘high fertility’ phenotype. We have already noted differences in many physiological, behavioural, endocrine and molecular aspects in previous analyses, including a prolonged life expectancy in FL1  and the Leibniz Association  partly supported the research.M L, E W, M M, J S, N R and J M W conceived the study. M L, E W and N R were responsible for animal breeding. M L, E W, and A T conducted the statistical evaluation. M L, E W, M M and J M W analysed and interpreted the results and drafted the manuscript. All authors critically read the manuscript and approved the final draft.


Candidatus Liberibacter asiaticus’ (CLas) bacterium has caused significant financial damage to many citrus industries. Large-scale pathogen surveys are routinely conducted in California to detect CLas early in the disease cycle by lab-based qPCR assays. We have developed an improved reference gene for the sensitive detection of CLas from plants in diagnostic duplex qPCR and analytical digital droplet PCR (ddPCR) assays. The mitochondrial cytochrome oxidase gene (COX), widely used as a reference, is not ideal because its high copy number can inhibit amplification of small quantities of target genes. In ddPCRs, oversaturation of droplets complicates data normalization and quantification. The variable copy numbers of COX gene in metabolically active young tissue, greenhouse plants, and citrus relatives suggest the need for a non-variable, nuclear, low copy, universal reference gene for analysis of HLB hosts. The single-copy nuclear gene, malate dehydrogenase (MDH), developed here as a reference gene, is amenable to data normalization, suitable for duplex qPCR and ddPCR assays. The sequence of MDH fragment selected is conserved in most HLB hosts in the taxonomic group Aurantioideae. This study emphasizes the need to develop standard guidelines for reference genes in DNA-based PCR assays.Citrus huanglongbing (HLB) disease associated with the ‘ Candidatus Liberibacter asiaticus” (CLas), and vectored by Diaphorina citri Kuwayama  [Citrus huanglongbing (HLB or citrus greening disease) is associated with an alphaproteobacterium, “id; ACP) ,2,3. HLBid; ACP) . Since tIn-depth genetic analyses of CLas isolates characterized from many locations in CA show differences in sequence characteristics and suggest multiple introductions of the pathogen . HLB finStandard testing methods for CLas from citrus involve duplex real-time PCR assays (qPCR) for the simultaneous detection of a mitochondrial cytochrome oxidase gene (COX) and the 16S rDNA of CLas . Under cXanthomonas citri (associated with citrus canker) and Spiroplasma citri (causing citrus stubborn disease) by ddPCR have been described [Digital droplet PCR (ddPCR) is an absolute quantification assay that can estimate the number of target molecules in a sample . Detectiescribed ,16. ddPCescribed ,18. Howeescribed ,20 comprescribed .Citrus and other closely related genera. The goals of the current study were: (1) to identify an alternate reference gene that has a low copy number in citrus hosts; (2) to develop assays using a region of the reference gene highly conserved in members of subfamily Aurantioideae (family Rutaceae) so that all known natural plant hosts of HLB can be evaluated in diagnostic assays; (3) comparative analysis of the newly developed reference gene with COX gene in HLB diagnostic assays and; (4) to develop the reference gene suitable for data normalization in qPCR and ddPCR assays, while analyzing plant samples of In a previous study , we analWe selected a 92-bp fragment from the 900 bp region of the MDH gene to evaluate as internal control reference gene in qPCR assays. 9 copies/µL, was used to prepare a ten-fold serial dilution in 1× TE to determine the linearity and dynamic range of detection in qPCR and digital droplet PCR (ddPCR) assays. In qPCR, the standard dilutions utilized in the experiment were detected over a dynamic range of nine dilutions as expected: 10−1 to 10−9  were either absent or were detected in very low numbers. However, in dilutions 10−6 to 10−10, a sufficient number of negative droplets were recorded and, hence calculation of the target concentration was feasible  and digital PCR (for five dilutions). The coefficient of correlation was 0.9998 for qPCR and 0.9999 for digital PCR. A plasmid preparation containing CLas 16S rDNA cloned fragment, estimated to contain 1.2 × 10 to 10−9 A. The cyfeasible B. FigureLyophilized citrus samples were analyzed in duplicate assays by duplex qPCR (for CLas and COX genes) and by singleplex ddPCR (for CLas). Duplex ddPCR was not conducted since the droplets from COX reaction could not be counted unless the Ct values were 23 or higher. A total of 57 samples were positive by both qPCR and ddPCR. Results shown in Bergera and Murraya. When ten-fold dilutions of a CLas positive DNA were tested in duplex qPCR for CLas and COX or CLas and MDH in separate reactions, MDH was not detected in dilutions where the Ct value of COX was >29 . The duplex reactions for CLas 16S rDNA were carried out along with primers and probes for COX or MDH. Since the dilutions contained approximately equal amounts of host DNA, all dilutions showed a Ct of around 18 for COX and 26 for MDH. A plot of Ct values for CLas with either COX or MDH at different dilutions is shown in A similar set of reactions was carried out for detecting the RNR gene of CLas  using th2) was 0.9762 (MDH only); 0.9633 (MDH in duplex reaction); 0.9858 (CLas only), and 0.9977 (for CLas in duplex reaction). A CLas positive DNA preparation (Ct 24) was used to prepare ten-fold serial dilutions using TE buffer . ddPCR assays were conducted to detect internal control gene MDH alone, MDH in the presence of CLas, CLas alone, and CLas in the presence of MDH. The citrus industry in the USA has battled HLB for about 16 years, and management of the disease has proved to be challenging. After the first report of HLB in Brazil in 2004 ,31 and FIn most cases, CLas-positive ACP led to the detection of compromised citrus trees that were HLB-positive but usually non-symptomatic. In Southern California, about 2400 CLas positive trees have been reported as of August 2021. Commercial citrus groves have been free of HLB, and it may be possible to delay the onset of the HLB epidemic if proper disease control strategies are practiced.In California, monitoring for HLB is mainly done by qPCR assays of ACP and citrus samples. Neither citrus plants nor ACP carry the CLas bacterium throughout the year at high levels. ACP with presumably very low titers of CLas appear to cluster  and are The amount of starting material, sample acquisition and handling, the integrity of the sample types utilized, tissues selected for analysis, sample processing methods, PCR inhibitors, reactions efficiency, and final data analysis can vary significantly among different laboratories and between experiments conducted at various times. Appropriate normalization of the data requires including validated reference genes in the detection systems ,34. SampMurraya paniculata and Bergera koenigii that are highly preferred by ACP [As a mitochondrial gene, COX can be present in high copy numbers in plant cells. In metabolically active, growing tissues such as shoot tips, the number of mitochondria per cell will be much higher than in mature tissues. Acquisition of CLas from the young flush tissue and subsequent transmission can lead to rapid disease spread through ACP feeding . Detectid by ACP . HLB surd by ACP . The incIn most of our CLas assays using extractions from mature trees, Ct values for COX varied from about 20 to 23. However, in routine assays for CLas in extractions from greenhouse-grown plants and assays involving young tissue or bark, COX Ct values around 16–26 were documented. Excessive amounts of COX may overwhelm the PCR reaction and interfere with the detection of CLas. A comparison of COX and MDH as reference genes showed that when the Ct value of COX is 29 and above, there may not be enough nuclear DNA since the single-copy gene, MDH, is not detectable in parallel experiments. A cut-off value for COX at about Ct 27 may ensure that the extract used for CLas detection assays will have enough genomic DNA so that interpretation of the data will not be misleading. In this study, different taxa included in the analysis resulted in significant differences in Ct values between COX and MDH (about 8–16). DNA templates increase by one log every 3.322 cycles in optimal qPCR assays . When hiIdeally, the reference gene included in duplex assays should be present at an approximately similar level as the target gene . MitochoC. clementina, there are three genes coding for three different MDH enzymes: EC 1.1.1.37, EC 1.1.1.39, and EC 1.1.1.40. The 906 bp sequence selected in this study is from the gene that codes for EC 1.1.1.40. This enzyme catalyzes the oxidative decarboxylation of malate in the presence of NADP+ into pyruvate, carbon dioxide and NADPH (https://www.genome.jp/dbget-bin/www_bget?ec:1.1.1.40 accessed on 2 October 2021). While this gene is 4257 bp long with 18 exons and 17 introns, the 900 bp fragment selected for this study contains four exons , the selected region aligns to scaffold 00027: 654682-654773.In the genome of ur exons . The fraIn our early ddPCR assays, we carried out singleplex assays for CLas, because in duplex assays COX oversaturated the droplets, and the need for an appropriate reference gene was obvious. While qPCR is an assay amenable to high throughput and has a broad dynamic detection range, ddPCR assays with a higher degree of sensitivity are valuable for absolute quantification and confirm positive samples that are considered ambiguous by qPCR. Accordingly, the reference gene is helpful to measure the amount of host DNA, while the target gene quantifies the pathogen. A mitochondrial gene such as COX will have a variable number of copies per cell from hundreds to a few thousand ,20, and The linearity and dynamic range of sensitivity of duplex assays for CLas 16S rDNA, using either COX or MDH were analyzed using tenfold serial dilutions of DNA of a CLas positive sample. The dilutions were made using DNA from a CLas negative, healthy citrus extract. Dilutions in water or TE can be misleading since we are interested in detecting varying levels of CLas titer under natural conditions with about the same level of background host DNA. The use of MDH increased the sensitivity of detection of two target genes of CLas, 16S rDNA and RNR, by about 1–2 Ct values in most dilutions tested. Finally, we analyzed the linearity and sensitivity of both MDH and CLas (16S rDNA) in singleplex and duplex ddPCR assays. The results showed that the linearity and sensitivity of both assays were not affected by duplex reactions. Citrus clementina; NW_006263303.1) was analyzed from a wide variety of citrus cultivars and citrus relatives. A total of 197 plant accessions were sampled from the Citrus Variety Collection (http://www.citrusvariety.ucr.edu/citrus accessed on 2 October 2021), University of California, Riverside, CA. While most of these accessions belonged to the subfamily Aurantioideae to which Citrus belongs, a few accessions from other Rutaceae subfamilies were included in this study. Details of selected representative taxa are given in 4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 0.2 µM of each primer, and 1.25 units of Taq DNA polymerase . Thermal cycling consisted of one cycle of initial denaturation at 95 °C, 3′, 35 cycles of denaturation at 95 °C, 15″, annealing at 55 °C, 15″, extension at 72 °C, 60″, followed by a final extension at 72 °C, for 5′. The PCR products were electrophoresed on agarose gels, purified using QiaExII kit , and cloned into pCR4 TOPO vector . The plasmids were purified and sequenced at the University of California, Riverside IIGB core facility (Sanger sequencing using Applied Biosystems H37306l DNA sequencer) using T7 and T3 universal primers. Haplotypes were deduced by analyzing sequences of 8–16 clones per accession. The sequences were aligned using ClustalX [About 900 bp region from MDH  or an ABI ViiA7 real-time PCR system  for conducting qPCR assays. The general protocol used for qPCR was essentially as described earlier .DNA extractions from known CLas-positive trees from Fort Pierce, Florida were used to prepare ten-fold serial dilutions. DNA extractions from known CLas negative trees maintained in a greenhouse at the USDA facility in Riverside, CA were utilized for preparing serial dilutions for some experiments. Aliquots were prepared, stored at −20 °C and used as needed. All the DNA extractions were prepared using Plant MagAttract DNA extraction kit . These controls were used for qPCR and ddPCR assays, and as positive controls in all other diagnostic assays described in this study.Ten-fold serial dilutions of a CLas positive extract were prepared in healthy citrus DNA extractions (as described before) and analyzed by duplex qPCR for 16S rDNA, using both COX  and MDH Stored frozen DNA extractions from 36 accessions belonging to 12 diverse genera of Aurantioideae, retrieved from a previously conducted field trial in Fort Pierce, Florida were used for this study. These plants were planted in 2009, collected samples, and DNA extractions were tested for CLas multiple times over six years (Supplementary Table in ). DuplexThe samples used for this part of the study were a part of an evaluation panel prepared to compare different novel early detection technologies developed in CA for testing HLB-associated CLas from plants. Briefly, citrus samples were collected in Texas, lyophilized , ground by OPS Diagnostics LLC, Lebanon, NJ, USA, and shipped to various participating laboratories (including our laboratory) as blind samples . DNA extA 20 µL reaction was set up by mixing 10 µL of 2X ddPCR supermix (Bio-Rad), 900 nM of forward and reverse primers and 250 nM of FAM-labeled Taqman probe specific for 16S rDNA of CLas. ddPCRs were conducted using a Bio-Rad digital PCR system equipped with an automated droplet generator , as recommended by the manufacturer. Specialized DG32 cartridges and ddPCR oil designated for either Evagreen-based or, probe-based reactions (as appropriate) were loaded in the droplet generator. The reaction mix and oil were then mixed within the DG32 cartridges to create an oil-water emulsion containing up to 20,000 nanoliter-sized droplets. PCR plates containing the reaction mix were placed in the automated droplet generator, partitions were made through water-in-oil emulsion, and droplets were generated.In our experiments, approximately 14,000–15,000 droplets were generated in each well. The PCR plates were sealed using a PX1 PCR plate sealer, and PCR was conducted in a Bio-Rad C1000 thermal cycler with the following thermal profile: initial denaturation at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 30 s, annealing/extension at 60 °C for one minute with a ramp rate of 2 °C/s between steps. After completion of PCR, endpoint data collected using a Bio-Rad QX200 droplet reader was analyzed using the Bio-Rad QuantaSoft™ program. Fluorescence measurements were recorded in the appropriate channel in the reader. The reader counted the number of droplets containing the target sequence (positive) and droplets without the target (negative). Each positive droplet was assigned a value of one and negative droplets were assigned a value of zero. A Poisson correction was applied by the QuantaSoft™ program used for analysis so that the mean number of target sequences per partition can be estimated. Concentration reported after the analysis represents the number of target copies/uL of the template.We used COX or MDH as reference genes and 16s and RNR genes of CLas as test genes in duplex PCR reactions. The TaqMan probes consisted of fluorophores, FAM (for CLas probe), and HEX (for COX and MDH probes). Various concentrations of primers and probes were evaluated in standardization assays (data not shown). Fluorescence measurements of droplets were recorded in both channels in the reader for duplex assays. Ten-fold serial dilutions of selected samples were prepared and analyzed by qPCR, using MDH as an internal control in singleplex and duplex ddPCR reactions to analyze linearity in duplex reactions.We have developed an improved reference gene, MDH, suitable in duplex assays designed to detect CLas from plants. The detection system for the reference gene developed in this study is expected to be universal for all known HLB hosts in the subfamily, Aurantioideae. The Mitochondrial COX gene that is commonly used as a reference gene in duplex qPCRs is not adequate, since the copy number varies depending on the metabolic state of the tissue, growth conditions, and plant type. Using laboratory assays, we have demonstrated the linearity and sensitivity of detection in duplex qPCR and ddPCR assays when MDH is used as the reference gene. Data normalization requires a stable reference gene that does not overwhelm the PCR reaction due to its copy number. There is a need to develop standards for reference genes in both qPCR and ddPCR assays similar to MIQE guidelines, used mainly in gene expression studies. MDH will be useful as a reference gene in diagnostic qPCR assays used to detect many citrus pathogens.


Leather waste (BF) and high-density polyethylene (HDPE) were compounded in a lab scale internal mixer and processed by means of injection molding. In this study, leather waste and HDPE composites were characterized by instrumental techniques such as differential scanning calorimetry (DSC), thermo-gravimetric Analysis (TGA), and Fourier transform infrared spectroscopy (FTIR). Physical integrity of composites against chemical exposure and chromium-leaching properties of the composites were also investigated. This study shows that the incorporation of 30% leather waste fiber into HDPE composites decreases the thermal conductivity of the composite samples by 17% in comparison to that of neat HDPE samples. Composites showed no thermal degradation during processing cycle. Strong interfacial bonding between leather waste and polymer results in comparable low-leachate levels to maximum allowed concentration for nonhazardous waste, and good chemical resistance properties. The BF/HDPE composites could be a promising low-cost alternative in industrial application areas of HDPE, where high-mechanical strength and low-thermal conductivity is required. The recent increasing restrictions on traditional waste treatments and the growing concerns about overexploitation of natural resources, connected with severe environmental impacts, are promoting the implementation of a circular economy. The aim of a circular economy is to minimize resource use and the associated environmental pressures by improving waste management practices through recycling, reuse of products, avoiding waste, and turning unavoidable waste into a resource. The critical discussions about implementing a circular economy and a sustainable use of natural resources led industries to focus on developing a more resource-efficient approach in their production systems.Composite industry is one of the prominent growing sectors due to widened demand for high-performance materials and their vast usage in engineering applications, especially in the aerospace and automotive industries . The indIn the concept of efficient use of natural resources, the use of natural fibers derived from industrial and agricultural wastes as reinforcement material in thermoplastic polymer composites was a prominent direction of research, both in terms of valorization of agricultural wastes and recycling of industrial solid waste. Over the years, substantial research took place to develop and improve properties of natural fiber reinforced composites by using waste fibers from different plant sources  ,4,5,6. OLeather waste is one of the major sources of animal-based collagen fibers, which provide mechanical strength and flexibility to the skin. Throughout tanning process, collagen fibers are cross-linked with tanning agents and raw skins/hides are converted into leather. Only the 20% of the rawhide is transformed into leather final product, and the 45% of raw hide is discarded in the form of nontanned and tanned collagenous waste . Among tContrarily to other types of leather industry solid wastes, the treatment of BF is difficult and of great environmental concern. Exposure to its particles may cause respiratory tract irritation in humans, as they contain trivalent chromium along with organic and inorganic compounds ,15. BF wFor all the above, the present work aims at determining the impact of BF on the thermal properties of HDPE, including thermogravimetry, calorimetry and insulation, the chemical resistance and the Cr(VI) content of the leachate from composites after inducing the leaching process, which provides useful information of the end-of-life of the obtained materials.Leather buffing dust waste (BF) with a particle size of the leather fiber, in the range of (0.3 mm diameter and 0.09 mm length) was provided from a sheepskin processing leather factory in Turkey. Leather waste fibers were used without any additional or prior treatment such as milling, grinding, or chemical treatment. The characteristics of BF are listed in HDPE pellets and BF were dried at 80 °C, before processing, to assure very low water content. Composite samples with leather fiber content from 20 to 50 wt% were prepared by using a Brabender®  internal mixing machine. Materials were fed to mixing machine and compounded at 80 rpm speed for 10 min at 170 °C. Thereafter, the composite blends were granulated into pellets using a blade mill equipped with a 10 mm mesh strainer and kept in oven  at 80 °C until further use to prevent moisture absorption. The obtained pellets were injection molded using Meteor 40 injection-molding machine  to produce dog bone composite test specimens complying with ASTM D3641 specifications. The composite specimens were conditioned in a climatic chamber  at 23 °C and 50 % relative humidity for 48 h before the tests were performed, according to the ASTM D618-13 (2013) and ASTM D638-14 (2014) standards. Thermogravimetrical analysis (TGA) was performed using a Mettler Toledo SDTA851 thermobalance . The samples were heated from 30 °C to 700 °C at a heating rate of 10 °C/min. The test was performed in an inert atmosphere with a nitrogen flow rate of 40 mL/min.e thermal analyzer  to evaluate the influence of the incorporated leather fibers on the thermal transitions and on the crystallinity degree of matrix following ASTM E1269.01 standard specifications. The samples were initially heated from 30 to 220 °C for the first heating scan to erase their thermal history. Afterwards, cooling was performed, and the samples were heated again using the same temperature range. All runs were performed at heating or cooling rates of 10 °C/min under 40 mL/min flow of nitrogen atmosphere.Differential scanning calorimetry was performed using a Mettler Toledo DSC822Thermal conductivity of composite samples was analyzed by means of an insulated chamber equipped with four thermocouples and an electrical heating system  for air. This method is based on the UNE-EN 8990:1999, where the thermal transmission properties of samples are determined in steady state. The samples were positioned in the middle, dividing the chamber into two chambers , maintained at different temperatures in steady-state conditions. The thermocouples were placed at the hot chamber, and the thermal resistance of the samples was obtained by measuring the power required to keep the hot chamber at constant temperature and the temperature difference between the hot and the cold chambers. The temperature of hot side of the composite samples, the cold side of the composite samples, and the cold chamber was recorded (every minute) by means of a data acquisition system. λ is the thermal conductivity (W/m °C), e is the thickness of the composite sample (m), U is the global heat transfer coefficient (W/m2 °C), and Th, Tc, Ths, and Tcs are the temperatures of the hot chamber (Th), cold chamber (Tc), hot side (Ths), and cold side (Tcs), respectively, and all in °C. In addition to this, the U was calculated from Equation (2): Rsi is the inner surface thermal resistance (m2 °C /W) and accounts for 0.13 m2 °C /W, according to UNE-EN 8990:1999 for vertical enclosures. The temperatures of the two chambers are maintained constant and steady state conditions were kept.Thermal conductivity, was calculated using Equation (1): −1 using a Bruker Alpha FT-IR spectrometer (FT-IR) .Fourier Transformed infrared (FT-IR) measurements of BF waste, HDPE polymer and composites containing 30 wt% BF was carried out to determine the formation and changes in the functional groups of samples. IR spectra were obtained in the frequency range of 4000 and 400 cm2 pieces and placed into specimen jar bottles, and then immersed into various chemicals  such as acetone, methanol, sodium hydroxide solution , hydrogen peroxide , ethyl acetate, and acetonitrile, and left for 24 h to evaluate sample’s resistance to each chemical. Variation at weight and change of visual appearance was examined following 24 h of contact with chemicals. Composite samples were cut into 0.5 × 0.5 cmTo determine the extractable chromium leached from the composite sample, chromium leaching tests of composite samples incorporated with 30% of BF were performed according to ISO 17072. Extractable chromium in composite samples was determined by carrying out extraction with an acid artificial-perspiration solution and subsequent determination with inductively coupled plasma optical emission spectrometry. Solutions leached from composites samples were also analyzed for the determination of chromium (VI) content under defined conditions, in accordance with international standard ISO 17075-2.The thermal stability of neat HDPE, leather BF waste, and 20, 30, 40, and 50 wt% BF reinforced HDPE composites were analyzed with TGA. The results of the TGA assays and the first derivate (DTGA) are represented in The TGA figure  shows thThe BF weight loss at relatively low temperatures (T < 100 °C) is due to elimination of absorbed water by leather fibers. The BF waste is rich in collagenous, a protein which tend to have a high interaction with water. The change in the mass loss tendency and the DTGA confirms the apparition of another degradation process, probably related with the protein denaturalization. The weight loss that takes place between 150 and 300 °C, with the higher rate around 235 °C, can be attributed to volatile compounds such as oil and low molecular weight greases existing in the leather fibers ,27. The The TGA analysis of neat HDPE matrix shows a thermally stable material up to 380 °C. Then, HDPE presents a narrow degradation step between 400–490 °C. A total of 2 wt% residue is obtained at 700 °C. In the case of the composite material, the degradation steps of the leather BF became more appreciable as we increase the amount of leather BF in the composite materials. A small and fast degradation occurred around 150 °C, which could correspond to the first degradation step of BF (excluding the water loss). Besides, the mass loss in that process is small. Around 300 °C, a high loss of mass is observed, which coincides with the second and higher degradation step of BF. The next degradation of the BF, around 415 °C, seems to be overlapped with the HDPE degradation, and a single process is appreciated in composites materials. The onset degradation temperatures for neat HDPE and composites are given in Besides, and although the BF reduced the thermal stability of the composite materials, taking into consideration the processing temperature of neat HDPE, which is around 180–200 °C, composites filled with BF can be processed without confronting a thermal degradation in the processing machines. The 30 wt% reinforced composite maintained nearly 99% of its weight at this temperature; hence, no degradation is likely to take place during the short processing cycle of the composite.Although the thermal stability of the composite materials in the working and manufacture range seems to not be affected by the incorporation of BF as reinforcing filler, the main transitions temperatures of HDPE could be affected. The melting and crystallization behavior of the HDPE matrix and the BF reinforced composites were studied by means of DSC. Samples were subjected to a first heat to erase the thermal history of the materials. The thermograph obtained during the second heating of HDPE and its composites shows a single endothermic process corresponding to the melting of the HDPE matrix. An amplified thermograph of the process is shown in The intensity and the enthalpy of the peak is reduced during the melting process when the fiber load is increased in the composite. Nonetheless, it is due to the lower polymer content in the composite material. The results related with the filler quantity are shown in Regarding the cooling process, the HDPE and composites materials showed also one single exothermal process related with the crystallization process of the HDPE. The peak temperatures are shown in The DSC thermograph of the 1st and the 2nd heat of the BF is shown in 3) were reported between 0.024–0.026 W/m °C in the literature [Thermal conductivity of neat HDPE and 20, 30, 40, and 50% BF composite specimen were analyzed and compared. The thermal conductivity values of dry BF waste specimen  . On the 2 rocking (720 cm−1), CH2 bending (1460–1470 cm−1) and CH2 stretching (2850–2920 cm−1) [−1 is assigned to the characteristic of –OH and –NH stretching vibration of amino acid in leather waste, and in the literature on this band reported between 3000 and 3500 cm−1 [−1 and 1550–1570 cm−1 were related with amide I (C=O stretching of proteins) and amide II bands (C–N stretching and N–H bending of proteins) also to collagen respectively [−1), Cardona et al. (1650–1540 cm−1), Senthil et al (1643–1540 cm−1), and for chromium shaving waste by Mohamed et al. (1650–1540 cm−1), Li et al (1645–1544 cm−1), and Liu et al (1647–1545 cm−1) [The FT-IR spectra of BF, neat HDPE, and BF/HDPE composites are given in 20 cm−1) . Thus, t500 cm−1 ,35,36,37ectively . As prev45 cm−1) . −1 could be attributed to –CN and –CO groups of amino acids [−1 accounts for Cr (III) species, which is in line with previously published data by, Liu et al (662–556 cm−1), Cardona et al (650 cm−1) and Senthil et al (852 cm−1) [−1, 1,461 cm−1, and 718 cm−1, all three are related to HDPE. Nonetheless, the characteristic absorption of amide groups of leather slightly shifts to lower wave number and changes from 1548–1536 cm−1 and to a higher wave number from 1633–1643 cm−1 compared with that of the original leather BF. These changes could indicate interfacial interactions between HDPE and leather BF, which improve their compatibility.The weak band in region 1000–1250 cmno acids ,20,36,3952 cm−1) ,38. Thes2O2 50%) solution for 24 h, dissolved some of the leather fibers from composite samples, resulting in an approximate weight loss of 10% and changed physical integrity of samples  [The oxidation state of chromium is an important indicator of toxicity and potential mobility. Chromium in the hexavalent state is highly toxic and soluble, whereas the trivalent state is much less toxic and relatively insoluble. Analysis of chromium leachate was performed to determine the mobility and the amount of extractable chromium in composites. Cr0 mg/kg) . The str0 mg/kg) . The resHDPE and animal-based natural fibers provided from leather industry waste were compounded in a lab scale internal mixer and processed through injection molding processes. The effect of leather fiber incorporation on thermal and structural properties of composites were studied in terms of TGA, DSC, and FTIR analysis. Chemical resistance and chromium-leaching properties of the composites were also investigated.The experimental results showed that although the BF caused a slight reduction in thermal stability of the composite materials, composites filled with leather fibers from tannery waste can be processed without confronting a thermal degradation in the processing machines. The presence of the BF in composites does not have any impact on the melting process, and a negligible effect on crystallization process of composites due to slightly lower crystallization temperature. BF/HDPE composite showed all FT-IR characteristic peaks of HDPE and BF. On the other hand, incorporation of BF waste from chrome tanned leather into HDPE polymer matrix improved thermal insulation of final composite material by 17% due to thermal insulation capacity of leather fibers. Exposure of neat HDPE and composite samples to different chemicals did not exhibit any change in either visual appearance or weight of samples, except hydrogen peroxide (50%), which is a concentrated strong oxidizer. Further, the chromium leaching tests performed on composite samples provided comparable low leachate levels to maximum allowed concentration MAC for nonhazardous waste, and chromium VI is below detection limit in all the sample leachates.The main advantages of BF/HDPE composites are use of animal-based natural fiber from industrial waste as inexpensive filler, and there is no need for previous treatments for preparing composite samples. The BF/HDPE composites could be a promising low-cost and sustainable alternative in HDPE applications, where high-mechanical strength and heat insulation is needed. Further, the obtained results bring to the light the suitability of HDPE for encapsulating BF, which prevents the Cr(VI) leaching.


We estimated the biological state of seabed sedimentary habitats, with specified uncertainty, in 24 trawled regions worldwide. Seabed status differed greatly among regions ; 15 regions had average status > 0.9. Two-thirds of all assessed seabed area was untrawled with status = 1, 93% had status > 0.8, but 1.5% had status = 0. The total area swept by trawling was a strong driver of regional status, providing a relationship to predict status from the regional estimated total amount of trawling. Seabed status is high in regions where fisheries are exploited sustainably—emphasizing that good fishery management contributes to better ecosystem outcomes—and, conversely, low status highlights regions needing improved management. Bottom trawling is widespread globally and impacts seabed habitats. However, risks from trawling remain unquantified at large scales in most regions. We address these issues by synthesizing evidence on the impacts of different trawl-gear types, seabed recovery rates, and spatial distributions of trawling intensity in a quantitative indicator of biotic status (relative amount of pretrawling biota) for sedimentary habitats, where most bottom-trawling occurs, in 24 regions worldwide. Regional average status relative to an untrawled state (=1) was high (>0.9) in 15 regions, but <0.7 in three (European) regions and only 0.25 in the Adriatic Sea. Across all regions, 66% of seabed area was not trawled (status = 1), 1.5% was depleted (status = 0), and 93% had status > 0.8. These assessments are first order, based on parameters estimated with uncertainty from meta-analyses; we recommend regional analyses to refine parameters for local specificity. Nevertheless, our results are sufficiently robust to highlight regions needing more effective management to reduce exploitation and improve stock sustainability and seabed environmental status—while also showing seabed status was high (>0.95) in regions where catches of trawled fish stocks meet accepted benchmarks for sustainable exploitation, demonstrating that environmental benefits accrue from effective fisheries management. Furthermore, regional seabed status was related to the proportional area swept by trawling, enabling preliminary predictions of regional status when only the total amount of trawling is known. This research advances seascape-scale understanding of trawl impacts in regions around the world, enables quantitative assessment of sustainability risks, and facilitates implementation of an ecosystem approach to trawl fisheries management globally. Bottom-trawl fishing occurs worldwide and is the most extensive anthropogenic direct physical disturbance to seabed habitats , 2. Towi3Balancing fishery production and ecosystem sustainability, however, remains a globally challenging issue—partly because the required indicators of ecosystem state often are unavailable or cost prohibitive to acquire at management scales. Consequently, a common approach has been to consider the risks of fishing impacts using expert judgement and/or qualitative scoring approaches, which provide indicators of relative risk –15. In c132 worldwide, accounting for 18.9% of the 0- to 1,000-m depth range  habitats on continental shelves and slopes in 24 large regions covering 7.92 million kmth range  and 19.5th range  in a quath range , recentlth range . We focuth range ], most b2) in each region—after first updating the previous series of meta-analyses used to estimate depletion and recovery rates (Methods). We also estimate regional mean RBS as the average of grid cell values to provide a relative indicator of the overall state of regional seabeds.The RBS model estimates the eventual abundance of biota relative to their untrawled abundance as a function  and seabed penetration depths (PD) of different trawl gears, including some additional data and adding different sediment habitat types as a factor . Average depletion rates ranged from 0.047 to 0.261 depending on gear and habitat . Otter trawls caused the lowest depletion followed by beam trawls and towed dredges. Depletion rates were lower in sand than in gravel and mud.We estimated trawl depletion rates of benthic communities in mud, sand, and gravel habitats for each trawl gear type by remodeling the relationship  between SI Appendix, Fig. S3A and Table S4). Recovery rates were estimated . Slower recovery with increasing gravel reflects the greater proportions of longer-lived species found in more stable gravel habitats  as well as among regions . SAR was aggregated among cells at larger scales, but, at fine scales within small grid cells, most trawling tends to be distributed approximately randomly  of each grid cell, mapped for each region using available sediment data . We used these rates with the grid cell trawl SAR intensity values for each gear type in SI Appendix, Table S5) under current distributions of fishing. Grid cell RBS values range between 0 to 1; trawled cells have RBS < 1, and untrawled cells have RBS = 1. The mean RBS estimate represents a linear relative index of benthic state for sedimentary habitats, corresponding to effects on biota that have an average sensitivity to trawling . The lower CL of RBS is indicative of status for biota types having upper CL sensitivity. Thus, mean RBS = 0 does not imply that all biota are depleted; rather, that among the mix of biota present before trawling, those with average or greater sensitivity to trawling (the response indicated by RBS herein) would be entirely depleted, whereas more resilient types may remain.We assigned trawl depletion and recovery rates appropriate to the trawl gear and sediment type . The European regions had <50% untrawled area where RBS = 1; lowest were the North Sea (11%), west of Iberia (16%), the Adriatic Sea (17%), and the Irish Sea (18%). All non-European regions except Northern Benguela had an average RBS ≥ 0.95. Chile, Australasia, and Alaska had the highest average RBS and the largest untrawled areas (68 to 93%).Regional RBS was lower in most European regions and higher in most non-European regions  and cumulative seabed area  show theThe uncertainty intervals of RBS curves  arise frRBS with uncertainty intervals can be used to frame risk assessments for trawling impacts. For example, if, as part of regional environmental objectives, an appropriate threshold for acceptable seabed status is defined, then RBS curves with uncertainty intervals can estimate the probability of status being above or below that threshold, thus informing the risk of environmental objectives not being achieved. As an illustration, if an acceptable threshold is set at RBS > 0.8 for >80% of a regional area , then, i1SI Appendix, Fig. S4) and the distribution of trawling. Within regions, sand habitats typically have higher average RBS and smaller proportions of low RBS categories because of their lower sensitivity than mud or gravel. The biggest within-region differences among habitats are in the Irish Sea, where mud status is heavily reduced. Overall, eight regions have one or more habitats with average RBS < 0.8  , the average regional RBS is >0.95, suggesting that managing trawl fisheries for sustainable exploitation of fish stocks contributes substantially toward ensuring that seabed status is high.The total amount of trawling in a region is a strong driver of regional RBS A. For reMethods) B. There SI Appendix, Fig. S5A). This ranges from 0.2% of seabed area in southern Chile to 82% in the Adriatic Sea and is >20% for 10 regions . Areas for this metric are very similar to areas of regional “uniform” trawl footprints as estimated by Amoroso et al. . Where they do differ , the threshold SAR needed to categorize cells as trawled that yields an equivalent area as the uniform footprint remains indicative of extinction thresholds for highly sensitive biota . Thus, the area of the uniform footprint also corresponds closely to the area of regions where highly sensitive biota cannot persist. Furthermore, we also calculated that the percentage of each region where trawl SAR was <0.07, allowing highly sensitive biota to maintain a status >0.8, ranges from 18% of seabed area in the Adriatic to 98% in southern Chile  and is >80% for 10 non-European regions.While assessing the status of sedimentary habitats is critical to ensuring integrity of the majority area of seabed ecosystems at the broadest scales, perhaps more concern surrounds rarer, more sensitive biogenic habitat types. However, suitable high-resolution data on the distribution and sensitivity for such habitats were not available for all our 24 regions. Hence, although we took a precautionary approach by using recovery rates applicable to pretrawling seabed community compositions and considering the lower CI of RBS, we could not assess highly sensitive habitat-forming biota types that can characterize vulnerable marine ecosystems VMEs , 30]. VM. VM30)].o et al.  , national legislation , and susWe aimed to assess the status of sedimentary habitats because these habitat types comprise the majority of seabed area, contribute to the integrity of seabed ecosystems at the broadest scales, are where most bottom trawling occurs, and lack the quantitative status assessments that managers require. We used the RBS model developed by Pitcher et al.  to estimTrawl gear depletion rates per trawl pass were derived from trawl-impact estimates for four gear types provided by Hiddink et al. . We exteThe wide availability of sediment mapping data enabled assessment of sedimentary habitats, which is where the majority of bottom trawling occurs. For other habitat types highly sensitive to trawling, the lack of widely available distribution data precluded RBS assessment of status herein. Instead, we estimated the proportion of each region where highly sensitive, long-lived biota types could or could not persist because of chronic trawling.B), as a fraction of carrying capacity (K), can be estimated—implicitly accounting for dynamics—by the equilibrium solution of the Schaefer model:B/K represents the RBS of the seabed in the range 0 to 1, R is the proportional recovery rate per year, which varies according to habitat, D is the depletion rate per trawl, which depends on gear type and habitat, and F is the current chronic trawling intensity as SAR . The ratio D/R represents sensitivity to trawling, the time interval between trawls (years) that would cause local extinction of the biota (RBS = 0), and the ratio R/D is the corresponding critical F, the annual trawl SAR intensity at which a given sensitivity will have RBS = 0 (Fcrit). Estimating RBS requires only parameters for depletion and recovery rates and distribution maps of trawling intensity and of habitat types. These maps and the estimation of RBS within an assessed region should be determined for grid cells of size ∼1 to 3 km2—a scale at which the distribution of most individual trawls has been shown to be random ] ] .n = 152 records) of trawling impacts to estimate the proportional gear-scale depletion rate (d) of biota for each pass of trawls of different gear types. They used a linear mixed-effects model  to analyze the change in biota abundance (pooled relative biomass and numbers of epifauna and infauna) with time after experimental trawling, relative to the abundance before and/or in reference areas, as log-response ratio (lnRR). Their results for the immediate logE trawl impact values (i: the intercept of lnRR at time 0) are directly related to depletion (d = 1 − ei) and represent the mean estimates for each gear type across all habitat types . Here, we have also estimated the SEs on the natural scale and 95% CLs of the back-transformed d estimates for each gear type .Hiddink et al.  and ScibSI Appendix, Table S2 and Fig. S1).Hiddink et al.  also shoSI Appendix, Table S2 and Fig. S1).We propagated the uncertainty of the PD estimates for all gear components up to whole-gear estimates by taking 2,000 samples from the distributions of each gear-component mean, using SDs as given by the SEs of each mean reported by the fitted log(PD) model. The sampled estimates of each gear-component PD were aggregated up to whole gear-by-habitat estimates using the same procedure as for the means to provide 2,000 estimates of PD for all combinations of gear and habitat. The SDs of these estimates provide approximate SEs for PD, and the 2.5 and 97.5% quantiles provide approximate 95% CIs for PD . Furthermore, because the gear–mean d values estimated by Hiddink et al. , we calculated weighted mean PDs where the weights were the frequency of studies by habitat for each gear type in the experimental studies meta-analysis from which the four gear–mean d values were estimated . This was done so that the estimates of gear–mean PDs used to build the model  would correspond to the expected PDs of the mixed habitat types represented in the meta-analysis that provided the d estimates.We estimated trawl depletion rates k et al.  but usink et al.  were frod of benthic community abundance and the PD of trawl gear was significant  but with uncertainty. The uncertainty of the model is indicated by the 95% CIs of the fit and by the prediction intervals . Additional uncertainties arise from the input data used to build the model. These include the uncertainties for the gear–mean d-values  and uncertainties for gear-by-habitat PDs  plus additional uncertainty arising from calculating the weighted-mean gear PD across habitats. The additional uncertainties are presented in SI Appendix, Fig. S2, including the 95% CIs for each gear–mean d from the original experimental studies meta-analysis , approximate estimates of the 95% CIs for gear–mean PDs propagated using a sampling procedure (see next paragraph) , the 95% CIs for gear-by-habitat PDs , and approximate estimates of the 95% CIs for the predicted gear-by-habitat d-values , which were propagated from all sources of uncertainty using a sampling procedure (see next paragraph).The updated mean relationship between the depletion SI Appendix, Table S1). The 2.5 and 97.5% quantiles of these samples provide approximate 95% CIs for each gear–mean PD . These uncertainties and those for gear–mean d-values were propagated through the model by also sampling and back transforming 2,000 estimates of each gear–mean impact i from the distributions of each mean using the SDs given by the SEs of each mean reported by the fitted lnRR model . These two sets of 2,000 sampled estimates for each gear–mean d and PD were used to fit 2,000 regressions, and each regression was used to predict d corresponding to each gear-by-habitat mean PD . The SDs of these predictions provide approximate SEs for each gear-by-habitat d, and the corresponding 2.5 and 97.5% quantiles provide approximate 95% CIs .The uncertainties for each gear–mean PD were propagated by sampling the 2,000 estimates of each habitat PD for each gear type (generated as described in the previous section) in proportion to the frequency of studies by habitat for each gear type in the experimental studies meta-analysis  of seabed communities on gradients of trawling intensity (F) on trawl grounds. The slope of this relationship is D/R, and if D is known from experimental studies, then recovery R can be estimated for the biotic community on trawl grounds. This approach assumes that the sampled benthos populations are approximately in a balance between trawl impacts and recovery under chronic intensities of trawling, which are known without bias (including for untrawled sites) and reflect prior trawling on ecological timescales; that “space-for-time substitution” in the studies used to derive D and R appropriately compensated for lack of prefishing historical sampling data; and that random processes and other departures are captured by the uncertainty in the relationship. These assumptions are necessary given the scarcity of recovery information, and this approach represents a significant advance for estimating the grid–scale recovery rate R. Furthermore, they  ∼ bF, where the slope b of this relationship can be used to calculate R using a nonlinear function of D and F.In principle, experimental studies could also provide estimates of gear–scale recovery rates k et al.  observedre, they  noted thre, they , 54, henk et al.  found wan = 677 records) of trawl impacts on benthic invertebrate communities and used an analogous meta-analysis to estimate recovery. Data for the meta-analyses were collated from published studies following a systematic review protocol (B) since communities of benthic invertebrates comprise a combination of both biomass and numbers of a wide range of species present. Furthermore, Hiddink et al. (F) for sites sampled by two studies (Dataset S1) and revised the sediment gravel, sand, and mud fractions for these studies and seven others with information from Asch . Overall community relative abundance decreased with increasing trawling impact dF , with each unit increase in dF leading to a mean decrease in abundance of 88.1%. The rate of decrease was greater as the gravel content of the sediment increased relative to mud content.This variation of the model was fitted so that recovery rates of different sedimentary habitats could be estimated without confounding by trawl gear types. For each study, R was then estimated by equating B/K and solving for R, giving the following:b estimated by fitting model SI Appendix, Table S4 . To account for the nonlinearity of this relationship and to estimate recovery rates of pretrawling community compositions (which include larger/longer-lived sensitive biota) rather than higher R values associated with chronically trawled community compositions of more resilient biota, dF in −9). This approach implicitly accounts for the historical effects of prior trawling to the extent possible, and the associated uncertainty was characterized by using the SE of the slope b  to estimate 95% CIs of the mean recovery rates along sediment gradients .The grid–scale recovery rate 2 area each) divided by the area of those grid cells. For regions where collated data coverage was >70% of total trawl effort (24 of 31 regions), Amoroso et al. (F) that is an essential requirement for seabed status assessment. To avoid underestimating impacts, we scaled up cell F by 100/coverage% for each region and by gear type to approximate total trawl intensity and recalculated regional SARs and footprints . This scaling and recalculation assumed the collated data are representative of the spatial distribution of the total. In cases in which the unavailable data may have a different distribution, our assessment may slightly underestimate regional depletion but less so than without scaling up.Mapping trawl footprints requires detailed information about trawling location and effort ; however, in most countries, such information is confidential and not publicly available. Amoroso et al.  approacho et al.  used grio et al. . The griSI Appendix, Table S5). From these grain-size data , they classified broad seabed habitat types to provide a consistent definition of habitat across all regions. Here, we classified and mapped regional habitats as “gravel” if gravel% > 30, else “sand” if sand% > mud%, else “mud” to match the habitats for which depletion values were estimated by meta-analyses of experimental studies , depletion values , and predicted recovery values  for otter trawls, beam trawls, and towed dredges and for all possible combinations of percentage gravel, sand, and mud that constitute sedimentary habitats are presented as ternary plots in SI Appendix, Fig. S4. For PD and d, these are weighted habitat means by gear in which the weights are the percentage of gravel, sand, and mud fractions of the sediment ternary distribution—this, in effect, provides continuous estimates of PD and d. Recovery R for sediment gradients was estimated using SI Appendix, Table S4 (Model 2). The ratio d/R indicates that gravel habitats are more sensitive to trawling due to higher d-values and low R-values, sand habitats are less sensitive due to low d-values and intermediate R-values, and mud habitats have intermediate sensitivity because of high d-values and high R-values. All habitats are more sensitive to towed dredges and less sensitive to otter trawls. The ratio R/d gives the critical threshold trawling intensity (Fcrit) at which the estimated RBS of the average untrawled community composition would be zero .The predicted PD  along with grid cell SAR trawl intensity values F by gear type substituted into SI Appendix, Table S5). Trawl gear types included otter trawls, beam trawls, and towed dredges. Where more than one gear type had fished a given cell, the cumulative RBS was estimated by summing the depletion (Fd/R) due to the d and F values for each gear.For the 24 regions defined by Amoroso et al. , we usedb of R . The regional RBS distributions were calculated using the mean and lower CL for R to indicate the range of status for average to more sensitive compositions of biota, including larger/longer-lived types, typically present in sedimentary habitats prior to trawling. This was because of the higher level of concern for sensitive biota compared to more resilient biota that may be indicated by the upper CL for R.The region-wide status of sedimentary habitats, accounting for their different sensitivity and exposure to trawling by different gear types, was summarized by mapping the regional average of grid cell RBS values and by plotting the ordered distribution of grid cell RBS values (high to low) against the cumulative proportion of regional area. To capture a range of uncertainty in estimating regional RBS, the SE of the slope f relative to the fMSY at which fishery production is maximized over the long-term (MSY); fMSY is considered a limit reference point, and fishing exploitation rates are considered sustainable when the ratio f/fMSY < 1 , which corresponds to biota types with, for example, d∼0.6 and R∼0.2—or with d∼0.3 and R∼0.1—or any other d/R ratio of about 2.86. Assuming the longevity relationship shown in figure 3 of Hiddink et al. . We also calculated the percentage area of each region where sensitive biota, as defined, could persist with status > 0.8, which corresponds with where trawl SAR F was less than 0.07 .With this definition of highly sensitive biota, we calculated the percentage area of each region having trawl SAR F > 0.35 with the regional trawl footprints estimated by Amoroso et al. , which is indicative of a multiyear footprint. We also plotted the difference in the percentage of regional areas between these two calculations . Furthermore, for each region, we also calculated the threshold F that would be needed to define cells as “trawled” so that the total area of those grid cells corresponds to the area of the uniform footprint .We also examined the comparability of the area of regions where o et al.  (their “Supplementary FileSupplementary File