Takifugu flavidus skin collagen. We found electrodialysis to have better economic and environmental outcomes than traditional dialysis as it significantly reduced the purification time and wastewater (~95%) while maintaining high extraction yield . SDS-PAGE, amino acid composition analysis, and spectrophotometric characterization indicated that electrodialysis treatment retained the physicochemical properties of T. flavidus collagen. Heavy metals and tetrodotoxin analyses indicated the safety of T. flavidus collagen. Notably, the collagen had similar thermal stability to calf skin collagen, with the maximum transition temperature and denaturation temperature of 41.8 ± 0.35 and 28.4 ± 2.5 °C, respectively. All evidence suggests that electrodialysis is a promising technique for extracting collagen in the fishing industry and that T. flavidus skin collagen could serve as an alternative source of collagen to meet the increasing demand from consumers.Collagen is widely used in drugs, biomaterials, foods, and cosmetics. By-products of the fishing industry are rich sources of collagen, which can be used as an alternative to collagen traditionally harvested from land mammals. However, commercial applications of fish-based collagen are limited by the low efficiency, low productivity, and low sustainability of the extraction process. This study applied a new technique  for the extraction of  Collagen is the major structural component of various extracellular matrices in mammalian connective tissues, such as the skin, corneas, cartilage, bone, and blood vessel . CollageFish, a popular dietary ingredient, is a great alternative source of commercial collagen. In addition to its worldwide acceptance by different sociocultural belief systems, fish is less likely to carry mammalian-borne diseases, thus requiring lower sanitary costs for industrial production of collagen . More inWhile promising, industrial production of fish collagen is limited by the low efficiency, low productivity, and low sustainability of the purification process. Crude fish collagen extract is mainly purified by dialysis through a passive diffusion process in the industry. This method usually takes 3–4 days to isolate pure collagen and often processes less than 1 L of samples ,13. MoreElectrodialysis, as an active diffusion technique, appears to be an excellent solution to improve the efficiency, productivity, and sustainability of fish collagen purification. This method can purify charged proteins/peptides by ion-exchange membranes through a stimulated diffusion process under the influence of electric potential difference . A recenTakifugu flavidus) skin. Considering that this technique has not been applied in the fishing industry, we hypothesized that the application of electrodialysis could not only improve economic and environmental outcomes of fish collagen production but also retain the physicochemical properties of T. flavidus collagen. Extraction efficiency, productivity, and wastewater production were also determined in this study, and the quality of T. flavidus collagen was systemically evaluated by electrophoresis, spectrophotometric characterization, thermal properties, and solubility.The aim of this study was to assess the practicality of electrodialysis for the isolation and purification of collagen derived from yellowbelly pufferfish (−1), Cd (0.042 ± 0.004 mg kg−1), As (0.075 ± 0.002 mg kg−1), and Cr (1.152 ± 0.067 mg kg−1), which are below the permissible limit recommended by the Chinese national standards (−1), Cd (0.023 ± 0.002 mg kg−1), As (0.052 ± 0.001 mg kg−1), and Cr (0.863 ± 0.072 mg kg−1), which are also below the permissible limit recommended by the Chinese national standards , tandards  [20].T. flavidus) skin contains 1.7 ± 0.5 MU/g of tetrodotoxin. According to Tani [T. flavidus) skin is safe for use in food, cosmetics, and pharmaceutical raw materials. Additionally, the content of tetrodotoxin in the collagen sample in this study was below the detection limit, indicating the safety of pufferfish collagen.The pufferfish , consistent with a previous study on T. rubripes skin collagen (170 residues/1000 amino acid residues) [T. flavidus collagen was higher than many fishes, including big eye snapper, grass carp, and tiger pufferfish (167–195 residues/1000 amino acid residues), but similar to those from their porcine counterparts (220 residues/1000 amino acid residues) [T. flavidus collagen might have nutritional values similar to those of common mammalian collagen and could therefore potentially be used as an alternative source of gelatin.Imino acids (Pro and Hyp) had the second highest amino acid content among all residues of esidues) . They haesidues) . They caesidues) . The conesidues) ,29. The esidues) . This suT. flavidus skin. In T. flavidus collagen and authentic type І collagen standard both have bell shape UV spectra, with the maximum absorption wavelength around 234 nm. This strong absorption can be attributed to peptide bond absorptions by n → π* transitions within C=O, COOH, and CONH2 groups of collagen peptide chains [T. flavidus collagen also absorbed weakly around 250 and 280 nm. The inability to absorb at higher UV regions is related to the deficiency of Tyr and Phe in T. flavidus collagen  spectrum of type I collagen. Five characteristic peaks were identified in both T. flavidus collagen and rat type I collagen: amide A, B, I, II, and III (−1) was lower than that of free N–H stretching vibration (3400–3440 cm−1) [T. flavidus collagen helices during the formation of triple-helical structures [2 stretching within collagen peptides [−1) similar to theoretical values. As an indicator of C=O stretching vibration [T. flavidus collagen (1645 cm−1) was modestly red-shifted from the calculated value (~1660 cm−1) toward lower wavenumber [−1 [T. flavidus collagen (1551 cm−1) was at the lower region of this range, further confirming the influence of interhelical hydrogen bonds. In addition, C–H stretching and N–H bending vibrations were detected in T. flavidus collagen [−1) absorption.Consistent with UV analysis,  and III C. The wa40 cm−1) . This repeptides , and it ibration , amide Ivenumber . This wamber [−1 . The amicollagen  with eviT. flavidus collagen was characterized by denaturation temperature (Td) and maximum transition temperature (Tmax). Td refers to the temperature at which the triple-helical collagen structure in solution is disintegrated into random coil [T. flavidus collagen  [T. flavidus collagen. Furthermore, Tmax refers to the temperature at which collagen fiber shrinks to one third of its length. The phase transition involving the conversion of a crystalline triple-helical collagen structure to an amorphous random coil form occurs during the shrinkage process [T. flavidus collagen confirmed this observation with a Tmax value of 41.8 ± 0.35 °C  [T. flavidus collagen tri-helices are more stable at higher temperature than other fishes. More interestingly, the Tmax value of T. flavidus collagen was similar to calf skin collagen (40.8 °C) [T. flavidus [T. flavidus could potentially be used as an alternative source of collagen.The thermal stability of dom coil . The vis16.8 °C) , indicat process . Differe 0.35 °C B. The di33.3 °C) ,16,24. I40.8 °C) . ConsideT. flavidus collagen was likely due to its imino acid content. Imino acids, especially Hyp, have been known for their ability to stabilize collagen tri-helices via intermolecular hydrogen bonds [T. flavidus collagen  [T. flavidus collagen tri-helices, therefore increasing the molecular stability through rising entropy [The superior thermal stability of en bonds . Our stuesidues) ,16,23,24 entropy .T. flavidus collagen  was identified in the DSC thermogram B. It wascollagen A, indicaT. flavidus collagen at a concentration of 0.16 mg/mL. Increments in NaCl level (≤3 g/100 mL) appeared to have a modest impact on the collagen solution, leading to a minor reduction in the relative solubility . This phenomenon is in agreement with an earlier study on catfish skin collagen, which significantly precipitates at high NaCl levels (≥4 g/100 mL) [T. flavidus collagen in other salt solutions.In the absence of NaCl, 0.5 mol/L acetic acid was able to fully dissolve /100 mL) . Conside/100 mL) , it is rT. flavidus collagen at pH 3.0 (0.04 mg BSAE/mL) was used to determine the relative solubility of the collagen among all pH conditions (p < 0.05), sharper reductions were observed between pH 3.0 and 5.0, with the lowest relative solubility being 37.9 ± 0.01%. This event was also reported by Singh and colleagues in a research involving catfish skin collagen [T. flavidus collagen solutions (0.50–0.65) were much lower than the threshold value (1.01) during pH adjustments, it might have had a minor impact on the relative solubility of the collagen.The protein content of nditions B. While collagen . As ioniT. flavidus collagen between pH 3.0 and 5.0 was most likely due to deprotonation of the charged amino acid residues. In acidic environments (pH ≤ 3.0), collagen behaved as a positively charged particle, with most charged amino acid protonated. The net positive charges of collagen assisted in forming hydrogen bonds with water molecules and preventing aggregation with surrounding proteins [The reduction in relative solubility of proteins . Howeverproteins , it is rT. flavidus collagen continuously decreased, with the lowest value found at pH 7.0 , the relative solubility of collagen . It coulThe greater the optical density (OD) value indicted, the greater is the number of cells. As shown in T. flavidus collagen could be produced from 100 g of dry skin, which is much higher than the yields of T. rubripes collagen  extracted by traditional dialysis [Takifugu species. It is also likely due to electrodialysis-mediated improvements of extraction efficiency as the extraction yield obtained from this study was significantly higher than those reported by previous dialysis research on tilapia (27.2 g/100 g DW) [To our knowledge, this is the first time electrodialysis was introduced as part of fish collagen purification. The application of electrodialysis significantly increased the extraction yield of fish collagen. Our experiments showed that 67.3 ± 1.3 g of dialysis ,30. This00 g DW)  and gras00 g DW) .This study showed that electrodialysis produced more than 10 kg of collagen samples in one purification effort by purifying 100 L of crude collagen solution. It also concentrated the purification step from a traditional 96-h dialysis ,14,15 inT. flavidus collagen, electrodialysis consumed 45.0 L of acetic acid and produced 600 L of waste water . Upon arrival, the skin was washed with chilled distilled water and then stored at −20 °C until use. Coomassie blue R-250, N,N,N′,N′-tetramethylethylenediamime and sodium dodecyl sulfate were purchased from Bio-Rad Laboratories . Type I collagen  was purchased from Sigma-Aldrich Inc. . Electrophoresis loading buffer was obtained from Sangon Biotech Co., Ltd. . Other reagents  were purchased from Xiamen Green Reagent Glass Instrument Co. Ltd. .T. flavidus skin  was incubated in 150 L of 0.1 mol/L sodium bicarbonate for 3 h with continuous stirring and then rinsed with cold water until a neutral pH was reached. The resulting material was hydrolyzed in 120 L of 0.5 mol/L acetic acid for 24 h and then centrifuged at 10,000× g for 15 min to remove non-collagenous proteins and pigments. Skin collagen was salted out from the supernatant in the presence of 0.5 mol/L NaCl, followed by 20 min of 10,000× g centrifugation. The resulting precipitate was dissolved in 0.5 mol/L acetic acid to produce 100 L of crude collagen solution, which was then purified by electrodialysis. The samples were loaded to a DSA-П electrodialyzer  equipped with both polyethylene cation-exchange membrane  and polyethylene anion-exchange membrane . The process conditions of electrodialysis were as follows: feed compartment, 150 L of 3% NaCl (w/v); concentrate compartment, 150 L of distilled water; electric potential, 80 V/cm; flow rate, ≤1 m3/h; electrodialysis duration, 2 h. The purification products were lyophilized and stored at −20 °C until analysis. Isolation and purification were all performed at 4 °C to prevent microbial growth (l growth . The extv/v) and then digested using a Milestone ETHOS A microwave digestion instrument . Inductively coupled plasma mass spectrometry  was used to identify these metals. Standard solutions for the calibration curve were prepared according to the dilutions of the toxic metals. Prepared solutions of toxic metals had levels of Pb, Cd, As, and Cr within the range of 1–50 mg/L.Toxic metals in the pufferfish skin and collagen sample were characterized based on a method described by Kosker et al.  with slig for 15 min. The resulting supernatants were collected and analyzed directly with an enzyme-linked immunosorbent assay (ELISA) test kit  for tetrodotoxin. As described in the manual, samples and enzyme-labeled MAb-TTX were added to wells of a microplate in which standard tetrodotoxin had been immobilized as a competitive agent. The absorbance  was measured at 490 nm. The ratio of absorbance  was used as the ordinate. The logarithm of the corresponding standard solution concentration was plotted as the abscissa, a standard curve was prepared, and the tetrodotoxin content of the sample was calculated.Tetrodotoxin in the pufferfish skin and collagen sample was characterized based on a method described by Wang et al.  with sliT. flavidus extract was mixed with 100 µL of loading buffer, incubated in boiling water for 3 min, cooled at room temperature, and then centrifuged at 8500× g for 5 min. Supernatant (5 µL/lane) was characterized by a polyacrylamide gel (4% stacking and 8% running) on a mini vertical electrophoresis system . After electrophoresis, the gel was incubated in fixing solution  for 30 min, stained by 0.1% Coomassie brilliant blue for 30 min, and then rinsed by 30% methanol containing 10% acetic acid for 30 min. Contents of the collagen subunits were estimated by Quantity One 4.6.0 .SDS-PAGE was conducted as described in our earlier study . BrieflyT. flavidus collagen extract (5 mg) was hydrolyzed with 0.50 mL of 6.0 M hydrochloric acid at 110 °C for 8 h. The hydrolysate was vaporized by a vacuum evaporator, dissolved in 25 mL 0.1 M HCl, and then analyzed by applying the hydrolysate to a HITACHI 835-50 amino acid analyzer . Contents of the amino acid residues were expressed as residues/1000 residues.Amino acid composition of sample was conducted as described in our earlier study . The T. T. flavidus collagen were determined using a method described by Wang et al. [T. flavidus collagen extract (1 mg/mL) was determined by a UV-1780 spectrophotometer against 0.5 M acetic acid (blank). FTIR (500–4000 cm−1) of the same extract was measured by a horizontal ATR trough plate crystal cell  coupled with a Bruker Model VERTEX 70 FTIR spectrometer . FTIR spectra in the rage of 500–4000 cm−1 with automatic signal gain were collected in 16 scans at a resolution of 4 cm−1 prior to analysis by OPUS 6.5 data collection software program . In addition, CD spectrum (190–260 nm) of the collagen extract (0.25 mg/mL) was produced by accumulating 100 scans at a speed of 100 nm/min with 1 nm interval on a Chirascan spectrometer .Spectra of UV, FTIR, and CD of the purified g et al.  with sliThe effects of pH and NaCl on sample solubility were characterized as described in our earlier study .T. flavidus collagen extract was determined in the presence of 0–6 g/100 mL NaCl. Briefly, 8 mL of 3 mg/mL collagen extract was mixed with 5 mL of NaCl solution containing 0.5 M acetic acid. The mixture was centrifuged at 20,000× g and 4 °C for 30 min. The protein concentration of the supernatant was measured according to the calibration curve for bovine serum albumin by Folin assay [The relative solubility of the in assay . The absT. flavidus collagen extract was also determined at varied pH values (1–10). The collagen extract  was adjusted to designed pH condition by 6.0 M HCl (or NaOH) with a final volume of 10 mL. The resulting solution was centrifuged at 20,000× g and 4 °C for 30 min. The protein concentration of the supernatant was determined by Folin assay as described above. The relative solubility was calculated with Equation (3) below by normalizing the protein concentrations collected at pH conditions with values obtained at pH 3.0.The relative solubility of the w/v), incubated at 4 °C for 2 days and then characterized using a DSC 2 calorimeter . Heat flow of the hydrated samples was measured between 5 and 75 °C at 1 °C/min using an empty aluminum pan as reference. Tmax was defined as the peak of the transition curve.The thermal properties of the sample were characterized by an established DSC method  with sliw/v) was prepared by hydrating the collagen sample in 0.05 M acetic acid at 4 °C for 2 days. An Ostwald’s viscometer  loaded with collagen dispersion was incubated in a 15 °C water bath for an extended period. Small temperature increments (2 °C) were then applied to the viscometer at 30-min intervals in a stepwise manner. Viscosity was recorded between 15 and 50 °C prior to each temperature change. Fractional viscosity was calculated with Equation (4). Td was defined as the temperature for fractional viscosity to reach 0.50.The viscosity of the sample was characterized based on a method described by Kittiphattanabawon et al.  with sliCell proliferation experiments were performed using a method described by Golser et al.  with sliAll data in this study are expressed as average ± standard deviation of at least three measurements. One-way ANOVAs coupled with Dunnett’s test was performed using SPSS 13.0 to determine the statistical difference at 95% confidence level.T. flavidus). This cost-effective technique was found to be superior to traditional dialysis with its advanced efficiency , large capacity , high extraction yield (67.3 ± 1.3 g/100 g DW), and better environmental sustainability (6 L waste water/L sample). SDS-PAGE, amino acid composition analysis, and spectrophotometric characterization demonstrated that T. flavidus collagen extracted by electrodialysis primarily consisted of type I collagen with a purity of 96.1 ± 1.3%. ICP-MS analysis demonstrated that the heavy metals of T. flavidus collagen were less than the Chinese national standards. ELISA analysis indicated the safety of T. flavidus collagen. Notably, the collagen appeared to have better thermal stability than other fish species, with Tmax and Td of 41.8 ± 0.35 and 28.4 ± 2.5 °C, respectively. This observation can be attributed to its relative higher imino acid content (246 ± 0.04 residues/1000 amino acid residues). Cell proliferation experiment demonstrated that the cell proliferation rates of the experimental groups were slightly higher than those of the negative control group, but there was no significant difference. In addition, both NaCl level and pH condition were found to affect the relative solubility of T. flavidus collagen. All evidence suggests that electrodialysis is a promising technique for fish collagen and that T. flavidus skin collagen could serve as an alternative source of collagen to meet the increasing demand from academia and industry.Our study, for the first time, introduced electrodialysis for the extraction of fish skin collagen (


Anilocra capensis Leach, 1818 is the only named species of Anilocra Leach, 1818 from South Africa. Anilocra is a large genus (> 40 species) with high levels of diversity reported from the Caribbean and Indo-West Pacific. Considering it is highly unlikely that all records of Anilocra from South Africa can be of a single species, the aim of this study was to better understand the diversity of Anilocra from this region and continent.A. capensis, the syntypes of A. capensis and specimens recorded as A. capensis from Africa were borrowed from the Natural History Museum, London, UK, and The iZiko South African Museum, Cape Town. Newly collected fresh samples of A. capensis were collected from off Cape Town, South Africa. Morphological redescriptions of the syntypes, and other museum and fresh material were conducted. Fresh samples were used to characterise molecularly A. capensis using the mitochondrial cytochrome c oxidase subunit 1 gene (cox1).To redescribe A. capensis there are six Anilocra species new to science from Africa: Anilocra ianhudsoni n. sp., Anilocra bunkleywilliamsae n. sp., Anilocra paulsikkeli n. sp., Anilocra jovanasi n. sp., Anilocra angeladaviesae n. sp. and Anilocra hadfieldae n. sp. Of the species under study, specimens of A. capensis appear to demonstrate the most individual variation, which occurs in pleonite width, pleotelson form and uropod length. We determined that African species of Anilocra can be primarily differentiated by the proportional shape and size of the full body in dorsal view and pereonites 1, 6 and 7. Other defining morphological traits include proportional shape and size of the pereopods, and the antenna and antennula peduncles. Lastly, the molecular characterisation of A. capensis is provided and the interspecific divergence with Mediterranean species is smaller than that with Caribbean species.Morphological analyses demonstrated that apart from A. capensis and the first molecular barcode for this organism. Six new species of Anilocra from Africa are described, establishing that the diversity of Anilocra in this region is greater than previously known. With this new understanding of species differences, we can accurately conduct detailed molecular and ecological analyses of Anilocra from Africa with certainty of the organism under study.The results of this study provide a detailed redescription of  Anilocra Leach, 1818, and it remains one of the best studied ecologically [Anilocra was the first genus of a marine fish parasite to be described from South Africa [Members of the isopod fish parasitic family Cymothoidae Leach, 1818 conspicuously infest the skin, buccal cavity, or gill chamber of their hosts. This family has a global distribution and there are more than 43 recognised genera [ogically –4. In fah Africa .Anilocra to accommodate three species. One of these species was Anilocra capensis Leach, 1818, collected in the Cape region of South Africa. In Schioedte & Meinert’s (1879) redescription of A. capensis, the authors provided only two dorsal view drawings of the female, and one dorsal view drawing of the male [A. capensis has been published. As a result, nearly all reports of Anilocra infestation on South African marine fishes have been recorded as A. capensis.Leach  erected nert’s 189 redescrAnilocra have been reported from tropical environments, e.g. [Anilocra diversity in South Africa should be far greater than the single recorded species. This should particularly be the case as South Africa’s coastline encompasses two oceans that span temperate to subtropical ecosystems. Moreover, these ecosystems are suitable for a diversity of potential fish hosts.The majority of ts, e.g. –10. AlthA. capensis based on the syntypes and all other available material was warranted. We seek to provide descriptions of species of Anilocra from South Africa and the African continent in order to clarify and describe the diversity of Anilocra from the eastern Atlantic and western Indian Oceans. Moreover, we aim to provide molecular data for A. capensis so that future taxonomic studies can at a minimum describe Anilocra from Africa as A. capensis or a different species of Anilocra.To test the aforementioned prediction, a review of Anilocra capensis with locality data from Africa, along with the syntypes of A. capensis were borrowed from the Natural History Museum, London, UK (NHM). We examined other specimens of Anilocra from outside South Africa from NHM to confirm or reject if they were A. capensis. In July 2017, all specimens labelled as Anilocra from The iZiko South African Museum, Cape Town (SAM) were borrowed. In April 2018, live A. capensis were collected from the Hottentot seabream, Pachymetopon blochii Valenciennes, at two localities: Cape Town Harbour wall and the False Bay Yacht Club harbour. Infested fishes were speared on SCUBA under the direction and permitting of Two Oceans Aquarium. The fresh material was preserved in 80% ethanol for later morphological analysis  was used. Polymerase chain reactions (PCR) were used to amplify a 710 bp fragment of the mitochondrial cytochrome c oxidase subunit gene (cox1) using the primer sets LCO 1490 and HCO 2198 [2), 1.25 μl of each primer, 1 μl DNA, and 9 μl of PCR-grade nuclease free water . Total volume per reaction was 25 μl, and PCR reactions were conducted using a ProFlex™ PCR thermal cycler (Applied Biosystems by Life Technologies). Reactions were amplified under the following PCR conditions: Stage 1, 94 °C for 5 min, Stage 2, 36 cycles of 94 °C for 30 s, 47 °C for 50 s, 72 °C for 2 min, and Stage 3, 72 °C for 10 min. PCR products were sent to a commercial sequencing company  Ltd, Pretoria, South Africa) for purification and sequencing in both directions. Obtained sequences were assembled, and chromatogram-based contigs were generated using Geneious Ver. 9.1. Sequences were aligned and trimmed to the length of the shortest sequence using MEGA 7 software program [Genomic DNA was extracted from 8 of the freshly collected HCO 2198 . PCR was program .http://www.ncbi.nlm. nih.gov/blast), the obtained sequences were verified as belonging to the Isopoda. Mean intraspecific divergence for A. capensis and interspecific divergence with all other Anilocra reported on GenBank were examined using p-distance and number of bp differences. We utilised the following sequences: Anilocra physodes Linneaus, 1758 (EF455817); Anilocra chromis Williams & Williams, 1981 (KY5622739); Anilocra brillae Welicky, Hadfield, Sikkel & Smit, 2017 (KY562744); Anilocra haemuli Williams & Williams, 1981 (KY562752); Anilocra prionuri Williams & Bunkley-Williams, 1986 (LC15954); and Anilocra clupei Williams & Bunkley-Williams, 1986 (LC160309). Newly-generated sequences for Anilocra capensis were deposited in the GenBank database under the accession numbers MK450443-MK450452.Using BLASTn . The interspecific divergence between A. capensis and A. physodes was 63–74 nt (15.5%). The interspecific divergence between A. capensis and Caribbean species of Anilocra were: A. chromis: 138–157 nt (24.9%), A. brillae: 138–157 nt (24.9%), A. haemuli: (KY562752) 136–154 (24.4%), A. prionuri: (LC159541) 136–157 (24.8%), and A. clupei: (LC160309) 132–144 (24.4%).Comparative sequence analysis indicated that the intraspecific divergence of GenusAnilocraLeach, 1818Diagnosis. Diagnosis and synonymy in Bruce [Type-species: Anilocra cuvieri Leach, 1818.in Bruce . Body doRemarksAnilocra cuvieri Leach, 1818, Anilocra mediterranea Leach, 1818 and Anilocra capensis. Anilocra cuvieri was designated as the type-species by Kussakin [Anilocra capensis syntypic specimens held at The Natural History Museum, London, presented by Leach from the Cape of Good Hope  [Leach  describeKussakin . There aamined’) .Anilocra accepted today were described in the 1980s, with many originating from the Indo-Pacific and Caribbean [Anilocra brillae [Anilocra haemuli [Anilocra spp., and the importance of combining morphological and molecular data whenever possible.Nearly half of the species of aribbean . Since 2aribbean , Chile [aribbean , Lebanonaribbean  and the aribbean . In 2017 brillae  from theAnilocra most closely resemble species of Renocila Miers, 1880 and Nerocila Leach, 1818, but generic level differences can be distinguished without dissection or stereoscopic microscope [Anilocra spp. pereopod 7 is longer than 6, but in Renocila spp. pereopod 6 is similar in length to 7. The rostrum is not ventrally folded between the antennae in Renocila spp., but it is in Anilocra spp. The cephalon of Nerocila spp. is posteriorly strongly trilobed, whereas the cephalon of Anilocra spp. is not tri-lobed to very weakly tri-lobed. Further comparisons among cymothoid genera and generic features are detailed in Welicky et al. [Of the skin-attaching cymothoids, species of croscope –30. Briey et al. .Anilocra capensisLeach, 1818Type-host: Probable host Pachymetopon blochii referred to as “Sargus Hottentotus” in Schiöedte & Meinert (1883) [t (1883) .Type-material: BMNH 1979.329:207b (♀ 43.0 × 18.0) (designated as the lectotype in the present study); BMNH 1979.329:207c (♀ 46.0 × 19.5); BMNH 1979.329:207a (♂ 35.0 × 10.0). Paralectotypes: BMNH 1979.329:207c (♀ 46.0 × 19.5), BMNH 1979.329:207a (♂ 35.0 × 10.0). Presented by W. Leach in 1818 from Cape of Good Hope.New material examined: From Cape Town Harbor Wall  SAMC-A091297: ♂ , non-ovigerous ♀ , ovigerous ♀ , damaged ♀ (46.6 × 19.5). From False Bay Yacht Club Marina  SAMC-A091298: ♂ , non-ovigerous ♀ , ovigerous ♀ , damaged ♀ . All specimens collected by R. L. Welicky, O. Kudlai, A. Greyling, D. van Rooyen, T. Beukes and Two Oceans Aquarium personnel on 28–29 April 2017 on the host P. blochii.Additional museum material examined: Collected by UCT Ecological Survey from Table Bay on host P. blochii and held at SAM as SAMA44882: ♀ . Collected from SAM and not catalogued: ♀ .Representative DNA sequences: MK450443-MK450452DescriptionFemale lectotype. [BMNH 1979.329:207b; Fig. Antennula approximately as stout as antenna, comprised of 8 articles; peduncle articles 1 and 2 distinct, articulated. Antenna comprised of 8 articles.Uropod more than half the length of pleotelson, peduncle 0.4 times as long as rami, peduncle lateral margin without setae or without medial short acute robust seta; rami extending beyond pleotelson, marginal setae absent, apices broadly rounded. Endopod apically rounded, 3.6 times as long as greatest width, lateral margin weakly convex, medial margin weakly convex. Exopod extending beyond end of endopod, 7.2 times as long as greatest width, apically rounded, lateral margin weakly convex, medial margin weakly convex, terminating without setae.Female paralectotype. [BMNH 1979.329:207c; Fig. Antennula approximately as stout as antenna; peduncle articles 1 and 2 distinct and articulated.Uropod more than half the length of pleotelson, peduncle 0.7 times as long as rami, peduncle lateral margin without setae; rami extending beyond pleotelson, marginal setae absent, apices broadly rounded. Endopod apically rounded, 3.8 times as long as greatest width, lateral margin weakly convex, medial margin weakly convex. Exopod extending beyond end of endopod, 7 times as long as greatest width, apically rounded, lateral margin weakly convex, medial margin weakly convex, terminating without setae.Male paralectotype. [BMNH 1979.329:207a; Fig. appendix masculina distally narrowly rounded.Female. [New material SAMC-A091297; Figs. Antennula approximately as stout as antenna, comprised of 8 articles; peduncle articles 1 and 2 distinct, articulated; article 2 0.8 times as long as article 1; article 3 0.7 times as long as wide, 0.4 times as long as combined lengths of articles 1 and 2; antennula flagellum with 5 articles, extending to middle of eye; peduncle article 3 0.8 times as long as wide, article 2 with plumose setae, or articles 3 and 6 with robust setae. Antenna comprised of 8 articles, peduncle article 3 1.4 times as long as article 2, article 4 1.3 times as long as wide, article 5 1.5 times as long as wide, 1.0 times as long as article 4; antenna flagellum with 3 articles, terminal article terminating in 1–5 short simple setae, extending to middle of pereonite 1. Mandibular molar process ending in an acute incisor, with 23 simple setae. Maxillula simple, with 4 terminal robust setae. Maxilla medial lobe partly fused to lateral lobe; 2 recurved robust setae; and 2 large recurved robust setae. Maxilliped weakly segmented, with lamellar oostegite lobe or second, smaller oostegite lobe on basal part of article, article 3 with 3 recurved robust setae.Pereopod 1 basis 0.4 times as long as greatest width; ischium 1.9 times as long as basis; merus proximal margin without bulbous protrusion; carpus with straight proximal margin; propodus 2.0 times as long as wide; dactylus stout, 2.4 times as long as basal width. Pereopod 2 propodus 1.8 as long as wide; dactylus 1.3 as long as propodus. Pereopods gradually increasing in size towards posterior. Pereopod 6 basis 2.6 times as long as greatest width, ischium 0.4 times as long as basis, propodus 1.3 as long as wide, dactylus 1.4 as long as propodus. Pereopod 7 basis 2.6 times as long as greatest width; ischium 0.6 as long as basis, without protrusions; merus 1.1 times as long as wide, 0.5 as long as ischium; carpus 1.3 times as long as wide, 1 as long as ischium, without bulbous protrusion; propodus 2.4 times as long as wide, 0.7 as long as ischium; dactylus moderately slender, 1.1 times as long as propodus, 2.9 times as long as basal width.Pleopods without setae or lobes, exopod larger than endopod. Pleopod 1 exopod 1.3 times as long as wide, lateral margin weakly convex, distally narrowly rounded, medial margin weakly oblique, medial margin weakly convex; endopod 1.2 times as long as wide, lateral margin weakly convex, distally narrowly rounded, medial margin slightly convex, peduncle 4.5 times as wide as long, without retinaculae. Pleopods 2–5 similar to pleopod 1. Pleopods 3–5 endopods proximal borders do not extend below exopod to peduncle. Peduncle lobes absent on pleopods 1–3, or present, increasing on pleopods 4–5.Uropod more than half the length of pleotelson, peduncle 0.5 times as long as rami, peduncle lateral margin without setae or without medial short acute robust seta; rami extending beyond pleotelson, marginal setae absent, apices broadly rounded. Endopod apically rounded, 3.5 times as long as greatest width, lateral margin weakly convex, medial margin weakly convex. Exopod extending beyond end of endopod, 6 times as long as greatest width, apically rounded, lateral margin weakly convex, medial margin weakly convex, terminating without setae.Male. [New material SAMC-A091297; Figs. appendix masculina with parallel margins, 0.9 times as long as endopod, distally narrowly rounded.RemarksAnilocra capensis has a proportionally large ovoid and vaulted body with distinctly flexed distolateral margins of the pereonite 7. The pleotelson is wider than long and broadly rounded and the distal-most article of the antenna peduncle has one convex lateral margin. This species has the greatest range in proportional size of adult females and shows the most intrinsic variation with regards to pleonite width and uropod length. This was evident in the syntypes as well as the new fresh material examined. The freshly fixed specimens can be grouped mainly into two categories: specimens where pleonite 3 is most narrow and 5 is widest, and specimens where pleonite 1 and 2 are widest. Within each of these groups, proportional size and uropod length is variable, but no general differences in variability can be discerned between the two groups. There also appears to be colour variation  in A. capensis, but all the other characters to this species are observed in both colour types. Given that varying pleon morphology is the only character that can create groups within A. capensis, this is not sufficient to determine that there are two species.Anilocra capensis was found attached to the skin of host fish. It was situated below the anterior portion of the dorsal fin, facing anteriorly, and above and not blocking the operculum. This attachment site was consistent among all the freshly collected material.In the present study Anilocra capensis can be differentiated from the other species described below based on body, pereonite, pereopod and/or antenna/antennule form. The body shape of A. capensis is not twisted, whereas Anilocra jovanasi n. sp. and Anilocra hadfieldae n. sp. have weakly twisted bodies. Compared to A. capensis, Anilocra paulsikkeli n. sp. is 0.86 times as wide at the widest point and has notable sub-parallel body margins, and Anilocra ianhudsoni n. sp. is strongly narrowed anteriorly. Anilocra capensis can be distinguished from Anilocra bunkleywilliamsae n. sp., as the latter is proportionally shorter, pereonite 1 is longer, the antenna and antennule are narrower, and it has chromatophores. Anilocra capensis can be differentiated from Anilocra angeladaviesae n. sp., as A. capensis has a pronounced curvature on the lateral margin of the propodus of pereopod 6 that A. angeladaviesae n. sp. lacks. There is an elongated lateral margin of article 3 of the antenna of A. angeladaviesae n. sp. that extends onto article 4, and this does not occur in A. capensis.We note that the 200-year-old female paralectotypes were similar in shape and form to the fresh material, but their poor and fragile condition, along with missing some articles made it possible to only include a few reliable drawings without further damage. The male paralectotype examined was significantly larger and likely in a different and later developmental stage than the freshly collected males. There was consistency in size and form of the fresh male material examined, but during our collections and those loaned from SAM, we found no male similar in size to Leach’s specimen.A. capensis is Cape Town, South Africa, and the host is a sparid. Species of Anilocra are regarded as typically host specific to the family or genus level, and some even to the species level. Given this specificity and the general spatial distribution of A. capensis [A. capensis. Thus, the reports of A. capensis from the Canary Islands and from hosts representing more than five families of fish are dubious [It is important to note that the type-locality for capensis , it is u dubious , 33.Anilocra ianhudsonin. sp.Type-host: Unknown.Type-locality: Nahoon River, East London, South Africa.Type-material: Holotype: ♀ (18.0 × 8.0), paratypes: ♀ , ♂  (SAMC-A091293). Collected by personnel on the ‘SS Pieter Faure’.Other-material: Transitional (11.5 × 3.0) (SAMC-A091293).ZooBank registration: The Life Science Identifier (LSID) for Anilocra ianhudsoni n. sp. is urn:lsid:zoobank.org:act:7AF506D2-5DD9-4B4A-8965-B1D224DE3650.Etymology: This species is named for the first authorʼs (RLW) nephew, Ian Hudson Catalano, as a gesture of her appreciation to him and his mother, LM Catalano, for understanding the importance of her living abroad to conduct cymothoid-related research and to drive Ian’s curiosity in the natural sciences.DescriptionFemale. [SAMC-A091293; Figs. Pleotelson 0.9 times as long as anterior width, dorsal surface smooth, lateral margins convex, posterior margin converging to weak caudomedial point.Antennula more stout than antenna; comprised of 7 articles; peduncle articles 1 and 2 distinct, articulated; article 2 0.6 times as long as article 1; article 3 1 times as long as wide, 0.5 times as long as combined lengths of articles 1 and 2; flagellum with 4 articles, extending to middle of eye, terminal article with 1 plumose seta. Antenna comprised of 9 articles; peduncle article 3 1.4 times as long as article 2; article 4 1.5 times as long as wide, 1.1 times as long as article 3; article 5 1.5 times as long as wide, 1.2 times as long as article 4; flagellum with 4 articles, terminal article terminating in 1–5 short simple setae, extending to middle of pereonite 1. Mandibular molar process ending in an acute incisor, with 13 simple setae. Maxillula simple with 4 terminal robust setae. Maxilla medial lobe partly fused to lateral lobe; 2 recurved robust setae; and 2 large recurved robust setae. Maxilliped weakly segmented, with lamellar oostegite lobe or second, smaller oostegite lobe on basal part of article, palp article 2 with 0 simple setae, article 3 with 3 recurved robust setae.Pereopod 1 basis 1.7 times as long as greatest width; ischium 0.7 times as long as basis; merus proximal margin without bulbous protrusion; carpus with straight proximal margin; propodus 1.8 times as long as wide; dactylus moderately slender, 1.2 as long as propodus, 2.7 times as long as basal width. Pereopod 2 propodus 1.2 as long as wide; dactylus 1.9 as long as propodus. Pereopods gradually increasing in size towards posterior. Pereopod 6 basis 2.3 times as long as greatest width, ischium 0.5 times as long as basis, propodus 1.2 as long as wide, dactylus 1.8 as long as propodus. Pereopod 7 basis 2.8 times as long as greatest width; ischium 0.6 as long as basis, without protrusions; merus proximal margin without bulbous protrusion, merus 1.5 times as long as wide, 0.6 as long as ischium; carpus 1.7 times as long as wide, 0.5 as long as ischium, without bulbous protrusion; propodus 3.1 times as long as wide, 0.9 as long as ischium; dactylus slender, 1.0 as long as propodus, 4.5 times as long as basal width.Pleopods without setae, exopod larger than endopod. Pleopod 1 exopod 0.7 times as long as wide, lateral margin weakly convex, distally narrowly rounded, medial margin weakly oblique, medial margin weakly convex; endopod 0.5 times as long as wide, lateral margin weakly convex, distally narrowly rounded, medial margin slightly convex, without retinaculae. Pleopods 2–5 similar to pleopod 1. Pleopods 3–5 endopods proximal borders do not extend below exopod to peduncle. Peduncle lobes absent.Uropod longer than pleotelson, peduncle 0.5 times as long as rami, peduncle lateral margin without setae; rami extending beyond pleotelson, marginal setae absent, apices narrowly rounded. Endopod apically rounded, 3.0 times as long as greatest width, lateral margin weakly convex, medial margin weakly convex. Exopod extending beyond end of endopod, 6.0 times as long as greatest width, apically rounded, lateral margin weakly convex, medial margin weakly convex, terminating without setae.Male. [SAMC-A091293; Fig. appendix masculina with parallel margins, distally narrowly rounded.RemarksAnilocra ianhudsoni n. sp. is among the smaller of the Anilocra species that occur in South Africa. The body shape is unique as it has a diamond-triangulate shape, such that pereonites 1–3 narrow strongly. Unlike the other African species, A. ianhudsoni n. sp. has no flexed pereonite posterolateral margins. The antennula has a plumose seta on the terminal article and a maxilla with simple setae or 3 recurved robust setae. Anilocra ianhudsoni n. sp. also has more slender pereopods and a flatter and broader antenna compared to the other species described herein.Anilocra ianhudsoni n. sp. has a produced curvature on the lateral margin of the propodus of pereopod 6, which is similar to A. capensis and A. bunkleywilliamsae n. sp. Compared to Anilocra ianhudsoni n. sp., A. capensis and A. bunkleywilliamsae n. sp. both have broader, more ovoid pereonites, and a pleotelson that is closer in width to total body width.Anilocra bunkleywilliamsaen. sp.Type-host: Rhabdosargus holubi Steindachner, 1881.Type-locality: Mawalana Estuary, South Africa.Type-material: Holotype ♀ (24.0 × 10.0) paratype ♀  (SAMC-A091294). Collected by T. Mqolombia, on 17 February 1998.Other material: Transitional (18.0 × 5.0).ZooBank registration: The Life Science Identifier (LSID) for Anilocra bunkleywilliamsae n. sp. is urn:lsid:zoobank.org:act:9DF125DA-624A-4712-BA07-D45CF4BC7C8F.Etymology: This species is named in honour of Dr Lucy Bunkley-Williams, a world expert on Anilocra who described nearly all of the known species of Anilocra from the Caribbean.DescriptionFemale. [SAMC-A091294; Figs. Antennula more stout than antenna; peduncle articles 1 and 2 distinct and articulated; article 2 0.8 times as long as article 1; article 3 1.0 times as long as wide, 0.4 times as long as combined lengths of articles 1 and 2; flagellum with 4 articles, extending to middle of eye, articles 3 and 6–8 with robust setae. Antenna comprised of 9 articles; peduncle article 3 1 times as long as article 2; article 4 0.7 times as long as wide, 0.9 times as long as article 3; article 5 1.6 times as long as wide, 1.5 times as long as article 4; flagellum with 4 articles, terminal article terminating in 6–10 short simple setae, extending to middle of pereonite 1. Mandibular molar process ending in an acute incisor, with 17 simple setae. Maxillula simple, with 4 terminal robust setae. Maxilla medial lobe partly fused to lateral lobe; lateral lobe with 0 simple setae, 2 recurved robust setae; medial lobe with 0 simple setae, and 2 large recurved robust setae. Maxilliped weakly segmented, with lamellar oostegite lobe or second, smaller oostegite lobe on basal part of article, palp article 2 with 0 simple setae, article 3 with 4 recurved robust setae. Oostegites margin covered in numerous plumose setae.Pereopod 1 basis 1.7 times as long as greatest width; ischium 0.7 times as long as basis; merus proximal margin without bulbous protrusion; carpus with straight proximal margin; propodus 1.2 times as long as wide; dactylus moderately slender, 1.2 as long as propodus, 2.6 times as long as basal width. Pereopod 2 propodus 1.9 as long as wide; dactylus 1.1 as long as propodus. Pereopods gradually increasing in size towards posterior. Pereopod 6 basis 2.5 times as long as greatest width, ischium 0.4 times as long as basis, propodus 1.3 as long as wide, dactylus 1.4 as long as propodus. Pereopod 7 basis 2.8 times as long as greatest width; ischium 0.5 as long as basis, without protrusions; merus proximal margin without bulbous protrusion, merus 1.6 times as long as wide, 0.6 as long as ischium; carpus 2.0 times as long as wide, 0.6 as long as ischium, without bulbous protrusion; propodus 2.8 times as long as wide, 1 as long as ischium; dactylus slender, 1 as long as propodus, 3.6 times as long as basal width. Dense chromatophores present on 1–7.Exopod larger than endopod. Pleopod 1 exopod 1.4 times as long as wide, lateral margin weakly convex, distally narrowly rounded, medial margin weakly oblique, medial margin weakly convex; endopod 1.6 times as long as wide, lateral margin weakly convex, distally narrowly rounded, medial margin slightly convex, peduncle 2.9 times as wide as long, without retinaculae. Pleopods 2–5 similar to pleopod 1. Pleopods 3–5 endopods proximal borders do not extend below exopod to peduncle. Peduncle lobes absent.Uropod more than half the length of pleotelson, peduncle 0.5 times as long as rami, peduncle lateral margin without setae; rami extending beyond pleotelson, marginal setae absent, apices narrowly rounded. Endopod apically rounded, 5 times as long as greatest width, lateral margin weakly convex, medial margin weakly convex, terminating with 0 setae. Exopod extending beyond end of endopod, 9.7 times as long as greatest width, apically rounded, lateral margin weakly convex, medial margin weakly convex, terminating without setae.RemarksAnilocra bunkleywilliamsae n. sp. is most readily distinguished from all other African species as it has the most anteriorly rounded and arched rostrum, and more setae on the antennula and antenna of the species described herein. It also has large and dense chromatophores on its pereopods. The chromatophores are denser on the left side when in dorsal view.A. capensis, the body of A. bunkleywilliamsae n. sp. is more ovoid, and pereopod 2 has robust setae present. Compared to A. ianhudsoni n. sp., A. bunkleywilliamsae n. sp. is generally smaller, its pleotelson converges more and is rounder, and its dactyls are shorter. The ovate and non-twisted body shape of A. bunkleywilliamsae n. sp. makes it distinguishable from A. paulsikkeli n. sp. which is long and narrow, and A. jovanasi n. sp. and A. hadfieldae n. sp. which are weakly twisted. Anilocra bunkleywilliamsae n. sp.is most similar in pereonite shape to A. angeladaviesae n. sp., but these species can be differentiated from each other as A. angeladaviesae n. sp. has no produced curvature on the lateral margin of the propodus of pereopod 6, its pereopods are more slender, and the general shape and form of the antenna and antennula are dissimilar.Compared to Anilocra paulsikkelin. sp.Type-host: Unknown.Type-locality: Delagoa Bay, Mozambique.Type-material: Holotype ♀ (32.0 × 8.5), paratypes: ♀ , ♂  (SAMC-A091295). Unknown collector.ZooBank registration: The Life Science Identifier (LSID) for Anilocra paulsikkeli n. sp. is urn:lsid:zoobank.org:act:AFEA08B1-D27B-49F6-A30E-B4E142EFA5D9.Etymology: This species is named in honour of Dr Paul C. Sikkel, the PhD supervisor of the first author (RLW) and research collaborator of the second author (NJS). Dr Sikkel’s devotion to furthering our knowledge on host-parasite interactions, strengthening the relationships between scientists and non-scientists, and rigorously mentoring future fish-parasite ecologists is hereby acknowledged.DescriptionFemale. [SAMC-A091295; Figs. Antennula approximately as stout as antenna, comprised of 9 articles; peduncle articles 1 and 2 distinct and articulated; article 2 1.3 times as long as article 1; article 3 0.9 times as long as wide, 0.6 times as long as combined lengths of articles 1 and 2; flagellum with 5 articles, terminal article with robust and simple setae, extending to posterior margin of eye. Antenna comprised of 9 articles; peduncle article 3 1.0 times as long as article 2; article 4 1.15 times as long as wide, 1.5 times as long as article 3; article 5 1.4 times as long as wide, 1.2 times as long as article 4; flagellum with 4 articles, terminal article terminating in no setae, extending to middle of pereonite 1. Mandibular molar process ending in an acute incisor, with 11 simple setae. Maxillula simple, with 2 terminal robust setae. Maxilla medial lobe partly fused to lateral lobe; 2 recurved robust setae and 2 large recurved robust setae. Maxilliped weakly segmented, with lamellar oostegite lobe or second, smaller oostegite lobe on basal part of article, article 3 with 3 recurved robust setae. Oostegites margin covered in numerous plumose setae.Pereopod 1 basis 2.5 times as long as greatest width; ischium 0.5 times as long as basis; merus proximal margin with slight bulbous protrusion; carpus with straight proximal margin; propodus 2.1 times as long as wide; dactylus stout, 1.2 as long as propodus, 3 times as long as basal width. Pereopod 2 propodus 2.5 as long as wide; dactylus 1.1 as long as propodus. Pereopods gradually increasing in size towards posterior. Pereopod 6 basis 2.7 times as long as greatest width, ischium 0.5 times as long as basis, propodus 2.6 as long as wide, dactylus 1 as long as propodus. Pereopod 7 basis 2.4 times as long as greatest width; ischium 0.7 as long as basis, without protrusions; merus proximal margin without bulbous protrusion, merus 1.7 times as long as wide, 0.6 as long as ischium; carpus 1.5 times as long as wide, 0.44 as long as ischium, without bulbous protrusion; propodus 4.3 times as long as wide, 1.1 as long as ischium; dactylus slender, 0.9 as long as propodus, 4.4 times as long as basal width.Pleopods without setae, exopod larger than endopod. Pleopod 1 exopod 2.1 times as long as wide, lateral margin weakly convex, distally narrowly rounded, medial margin weakly oblique, medial margin weakly convex; endopod 2.2 times as long as wide, lateral margin weakly convex, distally narrowly rounded, medial margin slightly convex, peduncle 2.0 times as wide as long, without retinaculae. Pleopods 3–5 endopods proximal borders do not extend below exopod to peduncle. Peduncle lobes absent. Uropod more than half the length of pleotelson, peduncle 0.7 times as long as rami, lateral margin without setae; rami extending to pleotelson apex, marginal setae absent, apices broadly rounded. Endopod apically rounded, 3.3 times as long as greatest width, lateral margin weakly convex, medial margin weakly convex, terminating with 0 setae. Exopod not extending to end of endopod, 6.7 times as long as greatest width, apically rounded, lateral margin weakly convex, medial margin weakly convex, terminating without setae.Male. [SAMC-A091295; Fig. appendix masculina with parallel margins, 0.8 times as long as endopod, distally narrowly rounded.RemarksAnilocra paulsikkeli n. sp. has a distinctive elongate, narrow body, with sub-parallel lateral margins. Pleonite 1 is partly covered by pereonite 7. The lateral margins of the pleotelson are sub-parallel and from the last third of the pleotelson there is a strong convergence to a rounded medial point. The eyes of A. paulsikkeli n. sp. are nearly one-quarter the width of the cephalon.Anilocra paulsikkeli n. sp. is similar to A. leptosoma, but there are multiple differences. Anilocra paulsikkeli n. sp. is larger (3.7 times as long as wide whereas A. leptosoma is 3.4 times as long as wide), the first antenna article 3 is not produced, pleonite 1 is more concealed, and the posterior margin of the pleonites are straighter. Aneesh et al. [A. leptosoma but did not examine the A. leptosoma lectotype material, and the drawings of Aneesh are in disagreement with Bruce 1987’s drawings of the lectotype. In Aneesh et al. [A. leptosoma, and are more similar to A. capensis, with respect to body shape and form in dorsal view. Aneesh et al. [A. leptosoma. To identify the species these authors report on, specimens should be further examined morphologically, and molecular data should be provided.h et al.  recentlyh et al. , Fig. 1dh et al.  likely dAnilocra paulsikkeli n. sp. is also similar to Anilocra caudata Bovallius, 1887 but can be distinguished from A. caudata, by the wider pleotelson, pleonite 1 not being covered by pereonite 7 and the lateral margins of pleonite 5 are more acute.A. paulsikkeli n. sp. and A. jovanasi n. sp. are most similar. Both species have nodules that occur midway on the lateral margins of the dactyls of pereopods 1 and 2, but the nodules are larger on A. paulsikkeli n. sp. Anilocra jovanasi n. sp. also has antennula peduncle article 3 more strongly produced than that of A. paulsikkel n. sp.Compared to the other species described herein, Anilocra jovanasin. sp.Type-host: Unknown.Type-locality: Delagoa Bay, Mozambique.Type-material: Holotype ♀  (SAMC-A091296). Unknown collector.ZooBank registration: The Life Science Identifier (LSID) for Anilocra jovanasi n. sp. is urn:lsid:zoobank.org:act:DA044D6D-CA90-4E20-9D76-1D6FD636569C.Etymology: This species is named for Professor Jo G. van As (1949–2018), the late aquatic parasitologist and PhD advisor of the second author (NJS). Professor van As revived the field of aquatic parasitology in South Africa in the 1970s and all current active aquatic parasitologists in South Africa are either his former students, or a student of one of his former students. His contribution to the field of aquatic parasitology in South Africa is hereby acknowledged.DescriptionFemale. [SAMC-A091296; Figs. Antennula approximately as stout as antenna; comprised of 8 articles; peduncle articles 1 and 2 distinct and articulated; article 2 1.1 times as long as article 1; article 3 0.6 times as long as wide, 0.4 times as long as combined lengths of articles 1 and 2; flagellum with 5 articles, articles 6–8 with robust setae, extending to middle of eye. Antenna comprised of 10 articles; peduncle article 3 1.7 times as long as article 2; article 4 1.4 times as long as wide, 1.4 times as long as article 3; article 5 2.6 times as long as wide, 1.5 times as long as article 4; flagellum with 5 articles, articles 8–9 with setae, terminal article terminating in 1–5 short simple setae, extending to middle of pereonite 1. Mandibular molar process present, with 20 simple setae. Maxilla medial lobe partly fused to lateral lobe; 2 recurved robust setae; and 2 large recurved robust setae. Maxilliped weakly segmented, with lamellar oostegite lobe, palp article 2 with 0 simple setae, article 3 with 3 recurved robust setae. Oostegites margin covered in numerous plumose setae.Pereopod 1 basis 2.7 times as long as greatest width; ischium 0.6 times as long as basis; merus proximal margin without bulbous protrusion; carpus with straight proximal margin; propodus 2 times as long as wide; dactylus slender, 1.2 as long as propodus, 3.2 times as long as basal width. Pereopod 2 propodus 2.15 as long as wide; dactylus 1 as long as propodus. Pereopods gradually increasing in size towards posterior. Pereopod 6 basis 2.91 times as long as greatest width, ischium 0.5 times as long as basis, propodus 2.6 as long as wide, dactylus 1.1 as long as propodus. Pereopod 7 basis 3.1 times as long as greatest width; ischium 0.6 as long as basis, without protrusions; merus proximal margin without bulbous protrusion, merus 2.0 times as long as wide, 0.7 as long as ischium; carpus 1.9 times as long as wide, 0.6 as long as ischium, without bulbous protrusion; propodus 3.2 times as long as wide, 0.9 as long as ischium; dactylus slender, 0.86 as long as propodus, 5.0 times as long as basal width.Pleopods without setae, exopod larger than endopod. Pleopod 1 exopod 1.6 times as long as wide, lateral margin weakly convex, distally narrowly rounded, medial margin weakly oblique, medial margin weakly convex; endopod 1.6 times as long as wide, lateral margin weakly convex, distally narrowly rounded, medial margin slightly convex, peduncle 2.4 times as wide as long, without retinaculae. Pleopods 2–5 similar to pleopod 1. Pleopods 3–5 endopods proximal borders do not extend below exopod to peduncle. Peduncle lobes present on 5.Uropod more than half the length of pleotelson, peduncle lateral margin without setae; rami not extending beyond pleotelson, marginal setae absent, apices narrowly rounded. Endopod apically rounded, lateral margin weakly convex, medial margin weakly convex. Apically rounded, lateral margin weakly convex, medial margin weakly convex.RemarksAnilocra jovanasi n. sp. is weakly twisted and narrowly ovoid. In dorsal view, the antennula appears geniculate due to the anterodistal lateral angle of the third article; pereopods 6 and 7 possess few robust setae. Anilocra jovanasi n. sp. is the only species described in southern Africa with the posterolateral margins of the fifth pleonite extending onto the dorsal portion of the pleotelson.The body Anilocra jovanasi n. sp. is most similar in body shape to A. leptosoma, and A. paulsikkeli n. sp. However, the body of A. leptosoma is not twisted and the pleotelson converges strongly to a distinct point, whereas in A. jovanasi n. sp., the body is straight and the pleotelson converges broadly to a very weak point. The pleotelsons of A. paulsikkeli n. sp. and A. jovanasi n. sp. are similar in shape until the last third, at which point the lateral margins of the pleotelson of A. jovanasi n. sp. converge broadly to a very weak point, and those of A. paulsikkeli n. sp. converge strongly to a broad medial point. Moreover, pleonite 1 of A. jovanasi n. sp. is substantially more visible than that of A. paulsikkeli n. sp. Whereas A. paulsikkeli n. sp. has distinct nodules on the dactyls of pereopods 1 and 2, this is present only on pereopod 1 of A. jovanasi n. sp. The propodus of pereopod 2 is without a curved produced curvature on the lateral margin, separating it from A. capensis and A. bunkleywilliamsae n. sp. Anilocra ianhudsoni n. sp. has a distinctly different body form and its pereopods 6 and 7 are more dense in robust setae. Anilocra angeladaviesae n. sp. is more robustly ovoid, with a broader pleotelson than A. jovanasi n. sp. Anilocra hadfieldae n. sp. also has a weakly twisted body, but compared to A. jovanasi n. sp., it has a more produced pointed lateral margin of pleonite 7 and a medial indent on the pleotelson.Anilocra angeladaviesaen. sp.Anilocra capensis Monod, 1924Syn. Type-host: Uncertain (see Remarks).Type-locality: Cape Blanc, Morocco.Type-material: Holotype ♀ (BMNH1924.5.30.6) (37.0 × 18.0), paratypes (BMNH1924.5.30.7–10): ♀ , ♂ . There are 6 accession numbers but 7 specimens in this lot. Collected by M. Theo Monod, 1923.ZooBank registration: The Life Science Identifier (LSID) for Anilocra angeladaviesae n. sp. is urn:lsid:zoobank.org:act:3381D22E-67B0-4847-A559-0899B20E9F7D.Etymology: This species is named after Professor Angela Josephine Davies (1947–2013), PhD promotor and postdoctoral advisor to the second author (NJS), to commemorate her contribution to the knowledge of crustacean parasites of fishes, as well as her singular dedication to- and enthusiasm for sharing this knowledge with all those she mentored.DescriptionFemale. [BMNH1924.5.30.6; Fig. Antennula more stout than antenna, comprised of 7 articles; peduncle articles 1 and 2 distinct and articulated; article 2 1.2 times as long as article 1; article 3 1.1 times as long as wide, 0.4 times as long as combined lengths of articles 1 and 2; flagellum with 4 articles, extending to middle of eye. Antenna comprised of 9 articles. Peduncle article 3 2.17 times as long as article 2; article 4 1.5 times as long as wide, 0.9 times as long as article 3; article 5 2.0 times as long as wide, 1.3 times as long as article 4. Antenna flagellum with 4 articles, extending to middle of pereonite 1.Pereopod 1 basis 2.8 times as long as greatest width; ischium 1.9 times as long as basis; merus proximal margin without bulbous protrusion; carpus with straight proximal margin; propodus 1.8 times as long as wide; dactylus stout, 1.1 as long as propodus, 2.3 times as long as basal width. Pereopod 2 propodus 2.3 as long as wide; dactylus 0.9 as long as propodus. Pereopods gradually increasing in size towards posterior. Pereopod 6 basis 3 times as long as greatest width, ischium 0.6 times as long as basis, propodus 1.2 as long as wide, dactylus 2 as long as propodus. Pereopod 7 basis 2.0 times as long as greatest width; ischium 0.7 as long as basis, without protrusions; merus proximal margin without bulbous protrusion, merus 1.3 times as long as wide, 0.6 as long as ischium; carpus 1.4 times as long as wide, 0.5 as long as ischium, without bulbous protrusion; propodus 2.1 times as long as wide, 0.7 as long as ischium; dactylus slender, 1 as long as propodus, 2.9 times as long as basal width.Pleopods without setae, exopod larger than endopod.Uropod more than half the length of pleotelson, peduncle lateral margin without setae; rami not extending beyond pleotelson, marginal setae absent, apices narrowly rounded. Endopod apically rounded, 3.7 times as long as greatest width, lateral margin weakly convex, medial margin weakly convex, terminating with 0 setae. Exopod extending beyond end of endopod, 6.0 times as long as greatest width, apically rounded, lateral margin weakly convex, medial margin weakly convex, terminating without setae.Male. [BMNH1924.5.30.7–10; Fig. appendix masculina with parallel margins, distally narrowly rounded.RemarksAnilocra angeladaviesae n. sp. can best be identified by the distinct elongate lateral margin on the third article of the antenna peduncle that appears to partially cover the lateral margin of the fourth article; the antennule is nearly half the length of the antenna; the body is ovoid and pereonites 1–3 narrow weakly towards the cephalon.A. capensis from Morocco [A. capensis from Morocco. Monod [Sama and Dentex, and the species, Morone punctata .It is likely that the specimens examined here are those mentioned by Monod as  Morocco  because o. Monod  referencA. physodes, the pereonite margins and pleotelson of A. angeladaviesae n. sp. are much straighter and less acute, respectively. Compared to A. capensis, A. angeladaviesae n. sp. has a narrower pleonite 1 in some specimens. Anilocra angeladaviesae n. sp. lacks the curved lateral margin of the pereopod 6 propodus, which is present on both A. capensis and A. bunkleywilliamsae n. sp. Anilocra bunkleywilliamsae n. sp. also has large dense chromatophores that are not present on A. angeladaviesae n. sp. Anilocra paulsikkeli n. sp. and A. jovanasi n. sp. are both more elongate and have nodules on pereopods 1 and 2 compared to A. angeladaviesae n. sp. Anilocra angeladaviesae n. sp. has a more rounded rostrum and posterior margin of pereonite 7 and more ovoid body compared to A. hadfieldae n. sp.Compared to Schioedte & Meinert’s  drawingsAnilocra hadfieldaen. sp.Type-host: Unknown.Type-locality: Cape Blanco, Gambia.Type-material: Holotype ♀ ; paratype ♂ . Collected by M. Routh, 1952.ZooBank registration: The Life Science Identifier (LSID) for Anilocra hadfieldae n. sp. is urn:lsid:zoobank.org:act:9744DBAC-6A5C-4C5A-A622-D96F0A1C9F0A.Etymology: This species is named for Dr Kerry Ann Hadfield in gratitude for her expertise in cymothoid taxonomy and for making RL Welicky’s postdoctoral tenure in South Africa a truly memorable and valuable experience to her both academically and personally.DescriptionFemale. [BMNH1952.9.9.29–30; Fig. Antennula approximately as stout as antenna, comprised of 8 articles; peduncle articles 1 and 2 distinct and articulated; article 2 1.3 times as long as article 1; article 3 1 times as long as wide, 0.4 times as long as combined lengths of articles 1 and 2; flagellum with 5 articles, extending to middle of eye. Antenna comprised of 9 articles; peduncle article 3 1.6 times as long as article 2; article 4 1.4 times as long as wide, 0.9 times as long as article 3; article 5 2.8 times as long as wide, 1.4 times as long as article 4; flagellum with 4 articles, extending to middle of pereonite 1.Pereopod 1 basis 2.4 times as long as greatest width; ischium 0.42 times as long as basis; merus proximal margin without bulbous protrusion; carpus with straight proximal margin; propodus 1.5 times as long as wide; dactylus stout, 1.4 as long as propodus, 2.4 times as long as basal width. Pereopod 2 propodus 1.7 as long as wide; dactylus 1.2 as long as propodus. Pereopods gradually increasing in size towards posterior. Pereopod 6 basis 3.2 times as long as greatest width, ischium 0.5 times as long as basis, propodus 1.4 as long as wide, dactylus 1.6 as long as propodus. Pereopod 7 basis 2.8 times as long as greatest width; ischium 0.6 as long as basis, without protrusions; 0.8 as long as ischium; carpus 1.6 times as long as wide, 0.7 as long as ischium, without bulbous protrusion; propodus 1.9 times as long as wide, 0.8 as long as ischium; dactylus stout, 0.9 as long as propodus, 2.9 times as long as basal width.Pleopods without setae, exopod larger than endopod.Uropod peduncle 0.5 times as long as rami, lateral margin without setae; rami not extending beyond pleotelson, marginal setae absent, apices narrowly rounded. Endopod apically rounded, 5 times as long as greatest width, lateral margin weakly convex, medial margin weakly convex, terminating with 0 setae. Exopod extending to end of endopod or extending beyond end of endopod.Male. [BMNH1952.9.9.29–30; Fig. appendix masculina with parallel margins, distally narrowed.RemarksAnilocra hadfieldae n. sp. has a distinct body shape. The body is more parallel than ovoid and the posterior lateral margins of pereonite 7 are substantially more acute than round and more obviously bent upward; pereopods 7 have small and light pigmentation and the pleotelson has a distinct medial indentation. The eyes are proportionally narrower than the other species described above.Anilocra hadfieldae n. sp. differ from that of A. capensis, the latter being stouter. The antenna peduncle articles of A. hadfieldae n. sp. have more parallel lateral margins than those of A. angeladaviesae n. sp. Anilocra hadfieldae n. sp. has no nodules or curved lateral produced margins of the pereopods, making it distinct from A. capensis, A. bunkleywilliamsae n. sp., A. paulsikkeli n. sp., and A. jovanasi n. sp.The antenna and antennula of Key to the African species ofAnilocra1a Body weakly twisted ……………………………………………………………………………………………………. 21b Body straight ……………………………………………………………………………………………………………… 3A. jovanasi n. sp.2a Posterolateral margins of pleonite 5 extended posteriorly, overlaying lateral margins of pleotelson …………………………………………………………………………………………….. A. hadfieldae n. sp.2b Posterolateral margins of pleonite 5 not extended posteriorly, not overlaying lateral margins of pleotelson, posterolateral margins of pereonite 7 acute, extending in an arc abruptly laterally and dorsally …………………………………………………………………………………………… 3a Body elongate, pleonite 1 concealed by pereonite 7 …………………………………………………………. 43b Body ovoid, pleonite 1 not concealed by pereonite 7 ……………………………………………………….. 5A. paulsikkeli n. sp.4a Pleonite 1 substantially concealed by pereonite 7; body 3.7 times as long as wide; pleotelson lateral margins subparallel A. leptosoma Bleeker, 18574b Pleonite 1 partly concealed by pereonite 7, body 3.5 times as long as wide, pleotelson lateral margins convex A. ianhudsoni n. sp.5a Pereonites 1–3 strongly narrowed ………………………………………………………… 5b Pereonites 1–3 weakly narrowed ……………………………………………………………………………………. 6A. angeladaviesae n. sp.6a Lateral margin on article 3 of antenna peduncle elongate, extending onto lateral margin of article 4; pereopod 6 propodus not inflated 6b Lateral margin on article 3 of antenna peduncle not elongate, not extending onto lateral margin of article 4; pereopod 6 propodus with produced curvature 7A. bunkleywilliamsae n. sp.7a Body 2.2 times as long as greatest width; article 3 of antennula peduncle 1.2 times as long as wide; dense chromatophores present on pereopods A. capensis Leach, 18187b Body 2.4–2.6 times as long as greatest width; article 3 of antennula peduncle article 0.8 times as long as wide; dense chromatophores absent from pereopods Anilocra capensis has been perhaps the most utilised (but not necessarily correct) name for any species of Anilocra in South Africa and likely in other areas of Africa, too. This is not surprising as only two drawings of adult female A. capensis existed before the present work. With few data on A. capensis and other African species, it was a logical pitfall to identify species of Anilocra collected from South Africa and elsewhere in Africa as A. capensis. This paper provides a detailed description of one of the original named species, and descriptions of six species new to science and thus will aid in the correct identification of these organisms. It is important to note that our molecular findings were unexpected as the nucleotide- and p-distance values between A. capensis and other Anilocra showed at levels more similar to differences observed between genera [n genera . In ordeA. capensis. We provide morphological drawings and quantitative and qualitative descriptions of this species, as well as relevant comparisons among the six new species of Anilocra from Africa. By providing the first molecular data for A. capensis, we also contribute to improving the identification of cryptic species via molecular analyses. We also demonstrate the value of morphological data as we found an ample number of morphological differences among the closely similar species described herein. In particular, our A. capensis redescription validates the name A. capensis, and provides critical baseline data to understand species differences of African species of Anilocra.The findings of this work should greatly reduce further misidentification of


Malus spp. (particularly cv. “Ola”) are rich in fatty acids such as linolenic (57.8%), α-linolenic (54.3%), and oleic (25.5%). The concentration of total tocopherols varied among the Malus species and dessert apples investigated, representing the range of 16.8–30.9 mg mL−1. The highest content of total tocopherols was found in M. Bernu prieks, followed by M. cv. “Ola”, and M. × Soulardii pomace oils. A significantly higher amount of δ-tocopherol was established in the oil of M. Bernu prieks, indicating that this species could be utilized as a natural and cheap source of bioactive molecules. β-Sitosterol was the prevalent compound determined in all tested pomace oils with a percentage distribution of 10.3–94.5%. The main triterpene identified in the oils was lupeol, which varied in the range of 0.1–66.3%. A targeted utilization of apple pomace would facilitate management of tons of by-products and benefit the environment and industry.The amplified production of fruit as well as burgeoning demand for plant-made food products have resulted in a sharp increase of waste. Currently, millions of tons of by-products are either being discarded or utilized rather ineffectively. However, these by-products may be processed and further incorporated as functional ingredients in making high-value food products with many physiological and biochemical effects. The chemical analysis of pomace oils using gas chromatography-mass spectrometry (GC/MS) and reversed-phase-liquid chromatography coupled with fluorescence detector (RP-HPLC/FLD) systems led to the identification and quantification of 56 individual lipophilic compounds including unsaturated, polyunsaturated and saturated fatty acids, as well as phytosterols and four homologs of tocopherol. The oils recovered from by-products of  Malus × domestica Borkh.) is one of the most important fruit crops worldwide. According to FAOSTAT (2018) [Domesticated apple  . The incT (2018) . In largT (2018) . These bT (2018) .Malus (Rosaceae family) and are currently used not only for ornamental purposes, but also are one of the most important source of seeds with an increased concentration of all four homologs of tocopherol  [M. baccata) not only contained moderate concentrations of polyphenols (phloretin and phloridzin), but also fatty acid molecules such as palmitic acid, ethyl palmitate, methyl petroselinate and linolein, which are well recognized for medicinal uses [Dessert apple fruits as well as their by-products have been studied in terms of their chemical composition ,8, alongγ and δ) , and hydγ and δ) . Food inγ and δ) . Crab apγ and δ) . Dadwal γ and δ)  found thnal uses . Moreovenal uses  reportedMalus genus have potential to be used in the production of therapeutic substances with a wide application in pharmaceutical and natural cosmetic industries, thus contributing to the reduction of waste generated during apple processing [Malus crab apple, reflects the growing interest in finding new approaches for the recovery of by-products generated during fruit processing.The most common phytosterols in the human diet are β-sitosterol, campesterol, and stigmasterol . In vitrocessing ,14,18. TThe composition of FA in oils recovered from the pomace of four crab and one dessert apples analyzed using GC-MS are shown in M. × domestica Borkh. cv. Bravo de Esmolfe apples.Seventeen FAs in the form of methyl esters (FAMEs) were identified and quantified, among which the dominance of unsaturated fatty acids, mostly linoleic acid (from 43.0 to 57.8%), followed by oleic acid 19.4–25.5%), was found . Among t.4–25.5%,Prunus avium kernel [M. bacata seed [Vitis spp. oils [Viburnum opulus lipophilic extracts [Considering the content of minor fatty acid molecules, α-linolenic and arachidic acids were found to be dominant, varying in the range of 1.8–4.7% and 0.7–3.9%, respectively. It must be noted, however, that the amounts of α-linolenic acid reported here are significantly higher than those observed in m kernel , M. bacaata seed , Vitis spp. oils , and Vibextracts .M. crab apples, varying in the ranges of 13.3–54.3% and 26.7–32.9%, respectively. Considering the content of oxygenated fatty acids in oils, 9,10-dihydroxystearate was the only representative, and found in small amounts . This low amount may be due to the relatively high content of Ts responsible for protecting the oil against oxidation [Prunus avium and Malus × domestica Borkh. cv. “Red Fuji” was also described by Peschel et al. [Only small amounts of less stable and therefore more prone to oxidation FFAs were found . Among txidation . Among tl et al. , respectMalus spp. especially cv. “Ola” are rich in fatty acids such as linoleic and oleic acids. A targeted utilization of apple pomace would facilitate management of dozens of by-products and benefit the environment and industry [The results of the aforementioned study show that the oils recovered from by-products of industry .M. Bernu prieks, α-T was the prevailing homolog present in apple pomace oils, with the content in the range of 4.4–13.9 mg mL−1 (percentage distribution of 14.1–60.0%). The second abundant homolog, after α-T, was δ-T with a percentage distribution of 13.5–70%. A relatively high amount of δ-T was found in M. Bernu prieks apples with a concentration of 21.5 mg mL−1. Moreover, this amount was 13.45- and 19.94-fold higher than reported earlier for Jatropha curcas L. and M. × domestica Borkh oil, respectively [Tocochromanols are a group of the major forms of vitamin E, consisting primarily of four  homologs of tocopherol and tocotrienol  that areectively .Malus species and dessert apples investigated, varying between 16.8 and 30.9 mg mL−1. The lowest content of total Ts was found in dessert apple pomace oil, while the highest value for M. Bernu prieks, followed by M. cv. “Ola”, and M. × Soulardii. The calculated weight ratios of four Ts  in three crab and dessert apple pomace oils (excluding M. Bernu prieks) were 5.2:1.6:2.2:1.9 and 4.7:1.4:0.6:1.8, respectively. Having regard to the high content of δ-T that has been found in the oil of M. Bernu prieks; the ratios were calculated individually for this sample, corresponding to 4.4:2.9:.2.1:21.5. Only a few studies have previously reported the presence of the Ts as constituents of crab and dessert apple seed oils [−1 oil) when compared to seed oils recovered from crab apples (13.0–20.3 mg mL−1). The average ratios of calculated amounts of Ts in crab and dessert apple seed oils were as follows: 4.1:2.7:1.6:1 and 2.6:2.5:1.1:1 [The first in vivo model assessing the antineoplastic activity of Ts has shown that δ-tocopherol to be the most active homolog compared with α- or γ-T in inhibiting tumor growth, perhaps through trapping reactive oxygen and nitrogen species and inducing apoptosis . The coneed oils . The aut.5:1.1:1 . The resPhytosterols are some of the compounds which are distributed among the tissues of plant . A meta-M. Bernu prieks, stigmasterol was the prevailing phytosterol present in this sample, showing a content of 111.0 mg mL−1 oil (87.8%), while in the other oil samples this compound was not found. The results of this study are in agreement with those published previously [Eleven biologically active compounds, five phytosterols  and six triterpenes , were successfully identified and quantified in the tested pomace oils. β-Sitosterol was the prevalent compound found in all tested pomace oils with a percentage distribution of 10.3–94.5%. The second dominant compound was campesterol, where the percentage distribution varied in the range 0.1–4.6%. With regard to the eviously ,31, wherM. crab apples, varying in the range of 0.1–66.3% and 0.2–68.9%, respectively. Recent reports indicate the presence of dietary lupeol as a constituent of vegetables and fruit including Brassica oleracea, Capsicum spp. Cucumis sativus, Solanum lycopersicum, and of fruits such as Olea spp. Ficus carica, Mangifera indica, Fragaria spp. and Vitis spp. [Prunus avium [Malus spp., especially M. Berzukroga dzeltenais and M. cv. “Ola” to be considered as a good source of lupeol which could be incorporated as ingredients in making of high-value functional food products with many of physiological and biochemical effects.Among the analyzed triterpenes, lupeol and uvaol were found to be dominant compounds of tis spp. ,34, whilus avium . It mustus avium . The recus avium . The samus avium , pointin−1, respectively  and rose hip (Rosa rugosa) oils were characterized as the strongest contributors to antioxidant activity using the DPPH• radicals. The results of scavenging capacity assay using the DPPH• radicals did not show correlation between the total bound FAs (FAMEs) (R2 = 0.2191) (2 = 0.0086) or carotenoids (R2 = 0.2306) in apple pomace oils. In addition, a strong negative correlation was found between FFAs (silyl derivatives) and DPPH (R2 = 0.7206), while a moderate positive correlation was found between FFAs and DPPH values (R2 = 0.6581). The opposite results were obtained in a study by Tuberoso et al. [• radical was R2 = 0.7000. The data from FRAP assay were significantly different (p < 0.05) and no correlation was found with the DPPH•. The results showed that the oil recovered from M. cv. “Ola” is more active compared with other oils. Generally, the only positive and strong correlation (R2 = 0.8732) was found between the bound FAs and FRAP  A, Ts  with each compound is an independent process, the overall success of which depends predominantly on the chemical structure of a particular biologically active compound, rather than on the concentration.The results of this study show that the different constituents, as well as testing systems, may affect the capacity of oils to quench different radicals. A similar result was reported previously by Wang et al. , pointins et al.  proposedMalus spp. crab apple was estimated in vitro using the method recommended by Balouiri et al. [MIC is defined as the lowest concentration of an antimicrobial mean that will suppress the visible growth of a microorganism after overnight incubation . In thisi et al. . MIC dat−1) for M. × domestica Borkh. cv. “Gita”, M. × soulardii, and M. Bernu prieks. However, the pomace oil of M. cv. “Ola” had a MIC value of 31.2 mg mL−1 for Gram-positive Staphylococcus aureus. Different activities of the tested pomace oils towards Gram-negative test bacteria was observed. The pomace oil of M. Bernu prieks, M. cv. “Ola”, and M. Berzukroga dzeltenais exhibited an inhibitory activity with MIC value 31.2 mg mL−1 against E. coli, while only pomace oil of M. cv. “Ola” and M. Berzukroga dzeltenais were able to suppress the growth of P. aeruginosa at a concentration of 31.2 mg mL−1. Our data show that tested pomace oils of M. spp. had differing antimicrobial activity against bacteria. Only the pomace oil of M. cv. “Ola” showed an inhibition of both Gram-positive and Gram-negative test cultures, while, to a lesser extent, the M. Berzukroga dzeltenais pomace oil had the ability to inhibit only Gram-negative pathogenic bacteria.The growth of Gram-positive test bacteria was inhibited only at the highest oil concentrations applied (MIC 125.0 and 61.2 mg mL5 CFU mL−1) [5 to ≥ 108 CFU mL−1 may cause a reduction in bactericidal activity of all the antimicrobial compounds against pathogenic bacteria [M. cv. “Ola”, and M. Berzukroga dzeltenais. Further in-depth research on evaluation of antimicrobial activity of lipophilic individual isolates from Malus spp. is highly recommended.In contrast to our data, some reports have shown significantly better susceptibility of pathogenic bacteria to antimicrobial agents recovered from different plant extracts, however these utilized different inoculum concentrations (in particular 10FU mL−1) . It was bacteria . The resbacteria , which wMalus species  were collected in Pūre Horticultural Research Centre Ltd. , GPS location: N: 57°02′23.30″; E: 22°54′48.1″, on 26 September, 2017  was obtained from Supelco Analytical . HPLC grade pyridine was purchased from Honeywell Riedel-de Haën GmbH, , HPLC grade methanol, n-hexane, petroleum ether , bis(trimethylsilyl) trifluoroacetamide (BSTFA) and boron trifluoride–methanol solution (BF3/MeOH) were purchased from Sigma-Aldrich Chemie Ltd., . The 2.2-diphenyl-1-picrylhydrazyl, 2-propanol (HPLC grade), resazurin sodium salt were obtained from Sigma-Aldrich Chemie GmbH . Deionized water was prepared using an Elix Advantage 3 water purification system .Standards of α, β, γ, and δ homologs of tocopherol (T) (purity >95%) were purchased from Merck Millipore . Methyl heptadecanoic acid , γ-linoleic acid , dodecanal , 1-octadecanol , n-tetracosane , and ergosterol  were purchased from Sigma-Aldrich Chemie GmbH , while n-paraffin analytical standard  and heated for a minimum of 1 h at 70 °C. Tubes were then allowed to cool for approximately 20 min. When tubes reached ambient temperature, 3.0 mL of double distilled water (DDW) and 3.0 mL of n-hexane were added to each tube followed by vortex-mixing for 15 s. After centrifugation at 3200× g for 4 min, the upper n-hexane layer was quantitatively transferred to a 15 mL plastic tube. Each sample was extracted thrice using the above-mentioned procedure. The supernatant fractions were hereafter flushed with nitrogen for ~5 min and dry residues stored in a low-temperature chamber “VTK 201 V”  at −18 ± 1 °C until analysis, a maximum of two weeks. Directly prior to GC/MS analysis, a 0.1 g of each oil sample (FAMEs) was reconstituted with 1 mL of pyridine.Methylation of polyfunctional compounds in oil samples was carried out using the method of Lelacheur et al.  with sli−1), then increased to 270 °C (rate of 4 °C min−1). Helium (ultra-high purity 5.0 grade—99.999%) was used as a carrier gas at the initial flow rate of 2.0 min−1 and then held constant at 1.0 min−1 with the split ratio 4:1. The total separation time was 35.75 min.The analysis of FAMEs was carried out on a Clarus 580 system PerkinElmer, Inc.  equipped with a quadrupole analyzer Clarus SQ 8 C mass-selective detector . All analyses of FAMEs were done using Omegawax 250  column with a stationary phase of intermediate polarity . The injector temperature has been set to +280 °C; automatic injection using an autosampler, injection volume 0.5 μL; split ration 4:1. The initial oven temperature was maintained at 75 °C for 2 min, then raised to 150 °C (rate of 20 °C min−1), and after at a rate of 4 °C min−1 increased to 310 °C, followed by an isothermal operation for 5 min. Helium (ultra-high purity 5.0 grade—99.999%) was used as a carrier gas at the initial flow rate of 2.0 min−1 for 0.5 min−1, and then followed by a constant flow at 1.0 min−1. The total separation time was 54.75 min.After silylation using the method of Lelacheur et al. , oils we−1), while from 0.5 min (0.50 scan s−1) for silyl derivatives; ion multiplier: 1700 V; and ion m/z interval: 42–550 for FAMEs and 35–750 for silyl derivatives.Detector mode: Electron impact ionization was at 70 eV; ion source temperature: +230 °C; inlet temperature was +290 and 280 °C for silyl and FAMEs derivatives, respectively; capture time for FAMEs starting from 3.5 min   . The pre8–C40) standard mixture. The components were identified by comparison of their RI and mass spectral data using an in-house built library and the NIST mass spectral library NIST MS 2.2.Retention indexes (RI) for temperature programmed GC analysis at a constant heating rate were calculated using the method of van Den Dool and Kratz  based on® nylon syringe filter with 0.22 µm pore size  into 1.5 mL glass vials and immediately analyzed using the HPLC system. The chromatographic separation was carried out using the Shimadzu HPLC system  consisting of a pump (LC-10ADvp), a degasser (DGU-14A), a low-pressure gradient unit (FCV-10ALvp), a system controller (SCL-10Avp), an auto injector (SIL-10AF), a column oven (CTO-10ASvp), a fluorescence detector (RF-10AXL) and a Luna PFP column  with a guard column (4 mm × 3 mm) . The analysis was done using the following conditions: mobile phase methanol:water ; flow (1.0 mL min−1); and column oven temperature (40 °C). The total separation time was 13.0 min. The identification and quantification of each of tocochromanols homolog was estimated using a fluorescence detector at an excitation wavelength of 295 nm and emission wavelength of 330 nm.Tocopherols (Ts) were determined using the previously validated method of Górnaś et al. . Crab anTotal carotenoids were estimated using the method of Biehler et al.  As reporHumans typically consume a wide variety of carotenoids in the diet; β-carotene, along with lycopene, lutein, zeaxanthin, β-cryptoxanthin, and α-carotene, account for 90% of circulating carotenoids . Therefo−1), the following equation was used:450 being the mean absorbance maximum (A450), d = 1 cm, and F a dilution factor adjusting for extractions, drying, and reconstitution processes. Using an average molar mass (g mol−1), results can also be expressed as g per L−1 and as mg per 100 g−1.To estimate the average carotenoid concentrations (mol L−1 (mg mL−1).A 0.1 g oil sample was 10 times diluted using petroleum ether in centrifuge tubes (15 mL), and the absorbance was measured immediately at 450 nm using a UV-1800—Visible Spectrophotometer SHIMADZU . Results were expressed as mg β-carotene equivalent per mL• assay was determined using the method of Li et al. [• solution (0.0039 g DPPH• in 100 mL methanol). Absorbance of the oil extracts was done at 515 nm using a spectrophotometer. The absorbance results were converted using a calibration curve of the standard and expressed as mmol Trolox equivalent antioxidant capacity per mL−1 of oil (mmol TEAC mL−1).The radical scavenging activity, using the free-radical DPPHi et al.  with sli−1; pH 3.6), 2,4,6-tripyridyl-s-triazine (TPTZ) solution in 40 mmol L−1 of HCl and FeCl3 (20 mmol L−1). The three solutions were mixed together at the ratio of 10:1:1 (v/v/v), respectively, and then warmed to 37 °C. Extracts and standard (FeSO4·7H2O) or double distilled water (DDW) for blanks (100 µL) were mixed with FRAP reagent (3.6 mL). The change in absorbance from red to blue was followed at 593 nm after 10 min. A Trolox calibration curve was done between 0.1 and 1.0 mg mL−1. The absorbance results were converted using a calibration curve of the standard and expressed as mmol Trolox equivalent antioxidant capacity per mL−1 of oil (mmol TEAC mL−1).Free reducing antioxidant power (FRAP) was determined using the method of Radenkovs et al. . The FRAPseudomonas aeruginosa ATCC10145; E. coli ATCC25922; Enterococcus faecalis ATCC29212; Streptococcus pyogenes ATCC19615; and Staphylococcus aureus ATCC65388/NCTC7447). Bacteria were grown on Nutrient agar  at 37 °C for 20–24 h.The minimum inhibitory concentration (MIC) was determined using the microdilution method in 96-well plates . Five st8 CFU mL−1 [v/v) of dimethyl sulfoxide (DMSO)  in DDW in the range from 0.9 to 125.0 mg mL−1. The negative control consisted of DMSO, MHB, and test cultures. For the broth microdilution test, 100 µL of each bacterial suspension in broth medium was added in each well already containing 100 µL of two-fold serially diluted plant extract. The final volume in each well was 300 µL. Viability of the bacterial cell was visualized using 30 μL/well of 0.01% resazurin aqueous solution. The plates were incubated at 37 ± 1 °C for 24 h. The MIC values were identified as the lowest concentration in which no viable bacterial were observed.Prior to analysis, the microorganism colonies were suspended in Mueller Hilton broth  and the suspension was adjusted using the DEN-1B McFarland Tube Densitometer , to the final turbidity of 0.5 McFarland units which corresponds to 10CFU mL−1 . Under an = 3). The p-value < 0.05 was used to denote significant differences between mean values determined using one-way analysis of variance (ANOVA) and the Duncan’s multiple range test done using the assistance of IBM® SPSS® Statistics program 20.0 .The results obtained are shown as means ± standard error of the mean from three replicates . Only small amounts of less stable free fatty acids were found, varying in the range of 3.0–26.5 mg mL−1. The oils recovered from by-products of Malus spp., especially M. Bernu prieks, followed by M. cv. “Ola”, and M. × Soulardii are rich in Ts. A relatively high amount of δ-T was found in M. Bernu prieks apples with a concentration of 21.5 mg mL−1. This amount was 13.45- and 19.94-fold higher than reported earlier for Jatropha curcas L. and M. × domestica Borkh oil, respectively. In total, 11 compounds including five phytosterols and six triterpenes were identified and quantified in the tested pomace oils. β-Sitosterol was the prevalent compound found in all tested pomace oils with a percentage distribution of 10.3–94.5%. Among the analyzed triterpenes, lupeol was found to be the dominant compounds in M. crab apples, varying in the range of 0.1–66.3%. Thus far, this is the first report providing the evidence on the presence of dietary lupeol in the M. crab apples. Moreover, the amount of lupeol reported here are 99-, 16-, and 2-fold higher than observed earlier for olive fruit, aloe leaves, and ginseng oil, respectively. The concentration of total carotenoids was found to be higher in oil recovered from the pomace of yellow crab apple Berzukroga dzeltenais, while the lowest in oil extracted from the pomace of Bernu prieks apple, corresponding to 14.5 and 5.1 mg mL−1, respectively. The results of scavenging capacity assay using the DPPH• radicals did not show correlation between the total bound FAs (R2 = 0.2191), tocopherols (R2 = 0.0086) or carotenoids (R2 = 0.2306) in apple pomace oils, which suggests that radical scavenging (DPPH•) with each compound is an independent process, the overall success of which depends predominantly on the chemical structure of a particular biologically active compound, rather than on the concentration. The only pomace oil from M. cv. “Ola” showed an inhibition of both Gram-positive and Gram-negative test cultures, while, to a lesser extent, the ability to inhibit only Gram-negative pathogenic bacteria was attributed to pomace oil from M. Berzukroga dzeltenais.The majority of the identified fatty acids in the oils recovered from by-products of