Population Genetic Structure of Anisakis simplex Infecting the European Hake from North East Atlantic Fishing Grounds

Ci a ion: Ramilo, A.; Rod íguez, H.; Pascual, S.; González, Á.F.; Abollo, E. Popula ion Gene ic S uc u e o Anisakis simplex In ec ing he Eu opean Hake om No h Eas A lan ic Fishing G ounds. Animals 2023,13, 197. h ps://doi.o g/ 10.3390/ani13020197 Academic Edi o s: Monica Ca a a and Pe la Tedesco Recei ed: 14 Decembe 2022 Re ised: 28 Decembe 2022 Accep ed: 29 Decembe 2022 Published: 4 Janua y 2023 Copy igh : © 2023 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). animals A icle Popula ion Gene ic S uc u e o Anisakis simplex In ec ing he Eu opean Hake om No h Eas A lan ic Fishing G ounds And ea Ramilo *, Helena Rod íguez , San iago Pascual, Ángel F. González and El i a Abollo Ins i u o de In es igaciones Ma inas, Consejo Supe io de In es igaciones Cien í icas, 36208 Vigo, Spain *Co espondence: and [email p o ec ed] Simple Summa y: The nema odes o he genus Anisakis a e among he mos p e alen pa asi es ound in ishes and ma ine mammals and hey a e he main cause o human anisakiasis. Gene ic s udies ha e desc ibed wo species, A. simplex and A. peg e ii, in Eu opean wa e s pa asi izing se e al hos s, being he Eu opean hake, by a , he ish wi h highe in ec ion alues. The aim o his s udy is o enhance he knowledge o he dis ibu ion and popula ion s uc u e o he Anisakis species in ec ing hakes om he majo Eu opean ishing a eas h ough he use o gene ic analysis. This s udy p o ides use ul in o ma ion abou he gene ic di e si y o hese pa asi es in he di e en ishing a eas and ish issues, aluable indings o unde s and he pa asi e specia ion o di e en hake issues and how hey a e s uc u ed along Eu opean wa e s. Abs ac : The Eu opean hake, one o he mos comme cially aluable species in ICES ishing a eas, is conside ed an impo an neglec ed sou ce o zoono ic isk by nema ode pa asi es belonging o he genus Anisakis.Me luccius me luccius is, by a , he mos impo an hos o Anisakis spp. a he Eu opean ishing g ounds, in e ms o demog aphic in ec ion alues, and ca ies he highes pa asi e bu den. These high pa asi e popula ion densi ies wi hin an indi idual ish hos o e a chance o explo e new sou ces o a ia ions o he gene ic s uc u e o Anisakis spp. popula ions. A o al o 873 Anisakis spp. hi d-s age la ae, o iginally sampled om isce a and muscula sec ions o hake collec ed a en ishing g ounds, we e p ima ily iden i ied using ITS DNA egion as molecula ma ke . A e ha , we used m DNA cox2 gene o e eal he high haplo ype di e si y and he lack o gene ic s uc u e o A. simplex. Dominan haplo ypes we e sha ed among he di e en ishing a eas and ish sec ions analyzed. Resul s indica e a clea connec ion o A. simplex om Eu opean hake along he No he n No h Sea o he Po uguese coas , cons i u ing a single gene ic popula ion bu e ealing a ce ain le el o gene ic sub-s uc u ing on he No hwes coas o Sco land. This s udy also p o ides use ul in o ma ion o ad ance he unde s anding o pa asi e specia ion o di e en ish hos issues o mic oen i onmen s. Keywo ds: Anisakis simplex;Anisakis peg e ii; hyb id geno ype; Eu opean hake; gene ic s uc u e 1. In oduc ion The Eu opean hake Me luccius me luccius (Linnaeus, 1758) (Gadi o mes, Me lucciidae) is one o he mos comme cially impo an species in he No h Eas A lan ic ishing a ea (FAO27). Nowadays, he In e na ional Council o Explo a ion o he Sea (ICES) conside s wo di e en s ocks in EU A lan ic wa e s sepa a ed by he Capb e on Canyon. The no he n hake s ock comp ises he No h Sea, Skage ak and Ka ega , he coas o UK, F ance and I eland, while he sou he n s ock is dis ibu ed along he A lan ic coas o Spain and Po ugal [ 1 , 2 ]. This deme sal p eda o is la gely o e exploi ed by indus ial ishe ies since i is a p oduc much p ized by Eu opean ma ke s, supplied ei he whole o ille ed. A he same ime, he Eu opean hake ops he lis o ish species wi h highes exposu e isk by zoono ic pa asi es o he genus Anisakis Duja din, 1845 (Rhabdi ida, Anisakidae) o sea ood consume s [ 3 – 5 ]. Fu he mo e, his no iceable high abundance o Anisakis Animals 2023,13, 197. h ps://doi.o g/10.3390/ani13020197 h ps://www.mdpi.com/jou nal/animals Animals 2023,13, 197 2 o 17 in hake ille s esul s in he ejec ion o pa asi ized ish p oduc s by o icial inspec o s in he indus y. Mo eo e , he p esence o Anisakis spp. in ish edible pa s gi es ise o he educ ion o he ma ke abili y o aes he ically una ac i e ish, wi h signi ican economic losses o Eu opean hake ma ke s [3,4,6]. The gene ic popula ion s uc u e s udies o pa asi es ha e con ibu ed o cla i y he axonomy o c yp ic species, mo phologically indis inguishable, which is common in nema odes o he genus Anisakis. To da e, nine species o his genus ha e been iden i ied, using di e en diagnos ic gene ic ma ke s, showing ha hey can be g ouped in ou dis inc clades. Two species o clade 1, A. simplex (s. s.) (Rudolphi, 1809) Duja din, 1845 and A. peg e ii Campana-Rouge & Bioca, 1955, ha e been epo ed up o now in se e al Eu opean ishing a eas and in se e al in e media e and de ini i e hos s [ 3 , 7 – 13 ]. A. simplex is he mos p e alen species in FAO27 and coexis s wi h A. peg e ii in Spanish and Po uguese coas s, e en hyb ids be ween hem ha e been ound in hese sympa ic a eas [8,14,15]. The ec ui men and accumula ion o Anisakis spp. la ae in Eu opean hakes is enhanced by hei key ophic ole in NE A lan ic ecosys ems. Hakes eed on mesozoo- plank on (euphausiids and amphipods), small zooplanc i o ous ish and la ge deme sal p ey, which a e all well known as in e media e and pa a enic hos s o Anisakis spp. [ 12 , 16 ]. Likewise, hake is an impo an p ey o he de ini i e hos o he pa asi e, pa icula ly dolphins, hus ein o cing he li e cycle o Anisakis spp. [ 7 , 11 ]. The e o e, he gene ic popu- la ion s uc u e o Anisakis spp. in any pa icula ish species can be a ec ed by changes in he ood-web s uc u e, including he ole o an h opogene ic impac s on he exploi ed ish s ocks and ecosys ems. Beyond he impac on human heal h and on ood indus y, he s udy o he gene ic popula ion s uc u e o pa asi es also p o ides in o ma ion abou hei dynamics o in ec- ion as hei hos -speci ici y, hei specia ion o di e en issues in a single hos o hei abili y o adap o local en i onmen s and o clima e change [ 17 – 19 ]. I has also been de- sc ibed ha he gene ic s uc u e o pa asi es could gi e in o ma ion abou he popula ion gene ic s uc u e o hei hos s, and hus, hey could be used as biological ags o s ock iden i ica ion o ish species [20]. This s udy has a double-aim: (1) o gene ically iden i y he Anisakis spp. la ae collec ed om Eu opean hake om he mos signi ican ishing g ounds o ICES a eas and (2) o es ablish he gene ic di e si y and he popula ion gene ic s uc u e o A. simplex in ela ion o ish o igin and si e o in ec ion. 2. Ma e ials and Me hods 2.1. Fish and Pa asi e Sampling A o al o 873 aceable high-quali y samples o Anisakis spp. hi d-s age la ae om Me luccius me luccius we e dona ed by he Technical Uni o he Ma ine Biobank a he Ins i u e o Ma ine Science (UTB-IIM-CSIC, Vigo). These samples consis ed o Anisakis specimens o iginally collec ed om en ICES di isions belonging o i e suba eas (Figu e 1): IVa (No he n No h Sea); VIa (No hwes Coas o Sco land and No h I eland); VIIb (Wes o I eland); VIIc (Po cupine Bank,); VIIh (Cel ic Sea/Sou h); VIIj (Sou hwes o I eland/Eas ); VIIIa (Bay o Biscay/No h); VIIIc (Bay o Biscay/Sou h); VIIId (Bay o Biscay/O sho e); and IXa (Po uguese Wa e s / Eas ). As a ule, o e 75 Anisakis ju eniles, L3 la ae, we e andomly selec ed om i e hakes, om each di ision sampled om 2018 o 2019 (15 pa asi es pe hos ), including specimens om he muscula sec ions: belly laps (BF) (N= 409), loins (LO) (N= 252) and ail (TA) (N= 72). Addi ionally, 140 isce al Anisakis la ae (VIS) we e chosen om i e hakes (o e 10 pa asi es pe hos ) caugh in suba eas VIIj, VIIIc and IXa in he pe iod 2013–2014. Animals 2023,13, 197 3 o 17 Animals 2023, 13, 197 3 o 18 Figu e 1. Sampling a eas (*) acco ding o ICES Di ision A eas: IVa (No he n No h Sea); VIa (No hwes Coas o Sco land and No h I eland); VIIb (Wes o I eland); VIIc (Po cupine Bank); VIIh (Cel ic Sea/Sou h); VIIj (Sou hwes o I eland/Eas ); VIIIa (Bay o Biscay/No h); VIIIc (Bay o Biscay/Sou h); VIIId (Bay o Biscay/ O sho e); and IXa (Po uguese Wa e s/Eas ). 2.2. Taxonomic Iden i ica ion o Anisakis spp. L3 La ae. Genomic DNA o each la a was ob ained employing he comme cial ki Wiza d Ge- nomic DNA Pu i ica ion Ki (P omega), acco ding o he manu ac u e ’s p o ocol. DNA quali y and quan i y was checked in a spec opho ome e Nanod op® ND-2000 (The mo Scien i ic). Gene ic iden i ica ion o Anisakis spp. was ca ied ou using ITS DNA egion as molecula ma ke . PCR assays o all Anisakis la ae we e pe o med in he obo ic wo ks a ion in o al olume o 25 μL con aining 1 μL o genomic DNA, PCR bu e a 1× concen a ion, 0·2 mM nucleo ides (The mo Scien i ic), 0·3 μM o each NC5/NC2 p ime s [21] and 0·025 U μL − 1 D eam Taq DNA polyme ase (The mo Scien i ic). A nega i e con- ol (no DNA) was used in each PCR assay. The PCR assays we e ca ied ou in a T g adi- en he mocycle (Biome a), unde he ollowing eac ion pa ame e s: 95°C o 5 min, 35 cycles a a mel ing empe a u e o 95°C o 30 s, an annealing empe a u e o 55°C o 45 s, an ex ension empe a u e o 72°C o 1 min, ollowed by a inal ex ension pe iod o 72°C o 7 min. The comple ed eac ions we e esol ed using 2% aga ose gel elec opho esis, Red Sa e s ained and isualized in Gel Doc™ XR Sys em (Bio Rad, He cules, Cali o nia, USA). PCR p oduc s we e cleaned o sequencing using ExoSap-I (The mo Fishe Scien- i ic, Massachuse s, USA) o 15 min a 37°C, ollowed by inac i a ion o 15 min a 80°C. Sequencing was pe o med by he company STABVIDA (Po ugal), and he ch oma o- g ams we e analyzed using Ch omasP o .1.41 (Technelysium P y L d., Sou h B isbane, aus alia). Two diagnos ic nucleo ide si es o ITS1 egion, 278 and 294, we e iden i ied in o de o di e en ia e A. simplex (T nucleo ide in bo h si es), A. peg e ii (C in bo h o hem) Figu e 1. Sampling a eas (*) acco ding o ICES Di ision A eas: IVa (No he n No h Sea); VIa (No hwes Coas o Sco land and No h I eland); VIIb (Wes o I eland); VIIc (Po cupine Bank); VIIh (Cel ic Sea/Sou h); VIIj (Sou hwes o I eland/Eas ); VIIIa (Bay o Biscay/No h); VIIIc (Bay o Biscay/Sou h); VIIId (Bay o Biscay/ O sho e); and IXa (Po uguese Wa e s/Eas ). 2.2. Taxonomic Iden i ica ion o Anisakis spp. L3 La ae Genomic DNA o each la a was ob ained employing he comme cial ki Wiza d Genomic DNA Pu i ica ion Ki (P omega), acco ding o he manu ac u e ’s p o ocol. DNA quali y and quan i y was checked in a spec opho ome e Nanod op ® ND-2000 (The mo Scien i ic, Wal ham, MA, USA). Gene ic iden i ica ion o Anisakis spp. was ca ied ou using ITS DNA egion as molecula ma ke . PCR assays o all Anisakis la ae we e pe o med in he obo ic wo ks a ion in o al olume o 25 µ L con aining 1 µ L o genomic DNA, PCR bu e a 1 × concen a ion, 0.2 mM nucleo ides (The mo Scien i ic), 0.3 µ M o each NC5/NC2 p ime s [ 21 ] and 0.025 U µ L − 1 D eam Taq DNA polyme ase (The mo Scien i ic). A nega i e con ol (no DNA) was used in each PCR assay. The PCR assays we e ca ied ou in a T g adien he mocycle (Biome a), unde he ollowing eac ion pa ame e s: 95 ◦ C o 5 min, 35 cycles a a mel ing empe a u e o 95 ◦ C o 30 s, an annealing empe a u e o 55 ◦ C o 45 s, an ex ension empe a u e o 72 ◦ C o 1 min, ollowed by a inal ex ension pe iod o 72 ◦ C o 7 min. The comple ed eac ions we e esol ed using 2% aga ose gel elec opho esis, Red Sa e s ained and isualized in Gel Doc ™ XR Sys em (Bio Rad, He cules, Cali o nia, USA). PCR p oduc s we e cleaned o sequencing using ExoSap-I (The mo Fishe Scien i ic, Massachuse s, USA) o 15 min a 37 ◦ C, ollowed by inac i a ion o 15 min a 80 ◦ C. Sequencing was pe o med by he company STABVIDA (Po ugal), and he ch oma og ams we e analyzed using Ch omasP o .1.41 (Technelysium P y L d., Sou h B isbane, Aus alia). Two diagnos ic nucleo ide si es o ITS1 egion, 278 and 294, we e iden i ied in o de o di e en ia e A. simplex (T nucleo ide Animals 2023,13, 197 4 o 17 in bo h si es), A. peg e ii (C in bo h o hem) and he e ozygo es ( wo o e lapping C/T peaks in bo h posi ions) [ 8 ]. All gene a ed sequences we e also assessed o simila i y agains known sequences using BLAST (Basic Local Alignmen Sea ch Tool) o he Na ional Cen e o Bio echnology In o ma ion (NCBI, Be hesda, MD, USA). 2.3. Gene ic Di e si y and Haplo ype Analysis Genomic DNA o all iden i ied A. simplex was ampli ied a he mi ochond ial cy- och ome oxidase 2 gene (m DNA cox2), using 211F/210R pai o p ime s desc ibed by Nadle and Hudspe h [ 22 ]. PCR eac ions we e pe o med as desc ibed abo e and unde he ollowing eac ion pa ame e s: 95 ◦ C o 5 min, 35 cycles a a mel ing empe a u e o 95 ◦ C o 30 s, an annealing empe a u e o 48 ◦ C o 45 s, an ex ension empe a u e o 72 ◦ C o 1 min, ollowed by a inal ex ension pe iod o 72 ◦ C o 7 min. PCR p oduc s we e also sequenced and analyzed as desc ibed abo e. Mul iple alignmen s o he sequences achie ed we e cons uc ed using MEGA 7 [ 23 ] and analyzed in he so wa e DnaSP 6 [ 24 ] in o de o know he gene ic di e si y o A. simplex popula ions. Th ee se s o m DNA cox2 sequences we e de ined in DnaSP 6 as h ee case s udies: (1) A. simplex sequences g ouped by he 10 ishing di isions sampled; (2) A. simplex sequences g ouped by he 3 muscula ish sec ions; (3) A. simplex sequences o muscula and isce a specimens om di isions VIIj, VIIIc and IXa. The numbe o haplo ypes (N h ), he haplo ype di e si y (H d ), nucleo ide di e si y (P i ), numbe o seg ega ing si es (S) and he a e age numbe o nucleo ide di e ences (K) we e calcula ed by DnaSP 6 o all de ined se s. Median-joining haplo ype ne wo ks [ 25 ] we e cons uc ed using PopART (h p://popa .o ago.ac.nz (accessed on 13 Decembe 2022)). Neu ali y es , Tajima’s D [ 26 ] and Fu’s Fs [ 27 ] we e pe o med in A lequin 3.5.2. so wa e [ 28 ] wi h 1000 simula ions o analyze he andomness o he DNA sequence e olu ion by he e i ica ion o he null hypo hesis o selec i e neu ali y (expec ed wi h popula ion expansion). In addi ion, he gene ic s uc u e o A. simplex popula ions was also e alua ed by a hie a chical analysis o molecula a iance (AMOVA). Pai wise compa isons o F s [ 29 ] alues be ween popula ions we e calcula ed wi h 1000 pe mu a ions. 3. Resul s 3.1. Gene ic Iden i ica ion o Anisakis spp. L3 La ae A agmen o 905 bp o ITS1-5.8S-ITS2 egion om 733 Anisakis la ae collec ed om ish muscula sec ions and 140 Anisakis om ish isce a we e ampli ied and success ully sequenced. Acco ding o he diagnos ic posi ions, a o al o 695 muscula specimens showed he homozygo e pa e n o A. simplex (T nucleo ide in 278 and 294 si es), 12 specimens showed he p o ile o A. peg e ii (C in bo h posi ions) and 26 Anisakis had a he e ozygo e geno ype (T/C in 278/294 si es). The gene ic iden i y o Anisakis specimens om isce a by sequencing o ITS egion showed he A. simplex pa e n in 76 cases, hose o A. peg e ii in 43 specimens and he e ozygo e p o ile in 21 la ae. Dis ibu ion o Anisakis species in ICES di isions. A. simplex was he p edominan species in hakes om all di isions sampled, ep esen ed he 94.82% o all specimens iden i ied, ollowed by hyb ids A. simplex xA. peg e ii (3.55%) and A. peg e ii (1.64%). A. simplex and hyb ids we e iden i ied in he en di isions whe eas h ee di isions showed o be sympa ic a eas o A. simplex,A. peg e ii and hyb ids: IXa (63 A. simplex, 7 A. peg e ii and 7 hyb ids); VIIIa (63 A. simplex, 3 A. peg e ii and 5 hyb ids) and VIIIc (71 A. simplex, 2 A. peg e ii and 3 hyb ids) (Table 1). Animals 2023,13, 197 5 o 17 Table 1. Taxonomic iden i ica ion o Anisakis la ae om muscula sec ions analyzed by sequencing o ITS DNA. ICES A ea As Ap Hyb To al IVa BF 62 0 3 65 LO 8 0 0 8 TA 1 0 0 1 To al 71 0 3 74 VIa BF 57 0 0 57 LO 13 0 1 14 TA 1 0 0 1 To al 71 0 1 72 VIIb BF 59 0 0 59 LO 11 0 1 12 TA 4 0 0 4 To al 74 0 1 75 VIIc BF 42 0 0 42 LO 23 0 1 24 TA 9 0 1 10 To al 74 0 2 76 VIIj BF 24 0 0 24 LO 31 0 2 33 TA 6 0 0 6 To al 61 0 2 63 VIIh BF 37 0 1 38 LO 35 0 0 35 TA 2 0 0 2 To al 74 0 1 75 VIIIa BF 2 0 0 2 LO 49 2 3 54 TA 12 1 2 15 To al 63 3 5 71 VIIIc BF 37 0 0 37 LO 28 2 3 33 TA 6 0 0 6 To al 71 2 3 76 VIIId BF 11 0 0 11 LO 37 0 1 38 TA 25 0 0 25 To al 73 0 1 74 IXa BF 61 6 7 74 LO 1 0 0 1 TA 1 1 0 2 To al 63 7 7 77 O e all 695 12 26 733 As: A. simplex; Ap: A. peg e ii: Hyb: hyb id geno ype be ween A. simplex and A. peg e ii. Anisakis species in ish sec ions. The p edominan species A. simplex in Eu opean wa e s ep esen ed simila pe cen ages 95.84% in belly laps, 93.65 % in loins and 93.06% in ails; A. peg e ii showed lowe pe cen ages in BF (1.47%), LO (1.59%) and TA (2.78%), simila o hose o hyb ids in BF (2.69%), LO (4.76%) and TA (4.16%) (Table 1). Anisakis la ae eco e ed om isce a om di isions VIIj, VIIIc and IXa showed di e en esul s (Table 2). Animals 2023,13, 197 6 o 17 Pa asi es om he sympa ic a ea IXa o A. simplex,A. peg e ii and hyb ids showed ha he p edominan species was A. peg e ii, doubled o A. simplex, whe eas A. simplex and A. peg e ii showed simila alues in he VIIIc di ision. Thus, 53.33% o pa asi es o isce a om di ision IXa we e A. peg e ii, he 22.22% A. simplex and 24.44% hyb ids o bo h species. The iden i ica ion o Anisakis species om VIIIc e ealed ha 43.14% was A. simplex; 37.25% was A. peg e ii; and 19.61% was hyb ids. The esul s ob ained o di ision VIIj showed he 100% o Anisakis iden i ied we e A. simplex. Table 2. Taxonomic iden i ica ion o Anisakis la ae om isce a analyzed by sequencing o ITS DNA. ICES A ea As Ap Hyb To al VIIj 44 0 0 44 VIIIc 22 19 10 51 IXa 10 24 11 45 O e all 76 43 21 140 As: A. simplex; Ap: A. peg e ii; Hyb: hyb id geno ype be ween A. simplex and A. peg e ii. 3.2. Gene ic Di e si y and Popula ion S uc u e o A. simplex 3.2.1. Case S udy 1: A. simplex om Di e en ICES Di isions A o al o 510 m DNA cox2 sequences we e ob ained om A. simplex collec ed in hakes om en ICES di isions. The alignmen o all A. simplex sequences (483 bp) con ained 115 a iable si es (S), which esul ed in 215 haplo ypes. Gene ic di e si y indices o all di isions a e shown in Table 3. The o e all alue o haplo ype di e si y (Hd) was 0.938, o nucleo ide di e si y (Pi) was 0.00723, and he alue o he a e age numbe o nucleo ide di e ences (K) was 3.49440. The gene ic di e si y indices calcula ed o A. simplex om each di ision showed a simila haplo ype di e si y o all o hem anged be ween 0.848 o di ision VIa o 0.970 o VIIIa and wi h pi alues anged be ween 0.00496 om VIa o 0.00948 om VIIc. Neu ali y es , Tajima’s D and Fu’s, showed nega i e alues s a is ically signi ican (p- alue < 0.05 and p- alue < 0.02, espec i ely) o all di isions, excep o VIIh whose Tajima’s D alue was no signi ican (P= 0.12800), ejec ing he null hypo hesis, i.e. he popula ion e ol es acco ding o he in ini e-si e model and all mu a ions a e selec i ely neu al (Table 3). Median-joining haplo ype ne wo k o he 510 m DNA cox2 sequences o A. simplex om he 10 di isions s udied (Figu e 2) was ep esen ed, showing he 215 haplo ypes ob ained by DNAsp. The haplo ype H3 was clea majo i y and he only haplo ype sha ed o all ishing di isions, including 120 sequences (23.53% o he o al sequences). I showed ela i e equency anging om 0.169 (di ision VIIIa) o 0.391 (di ision VIa). The o he mo e ep esen a i e haplo ypes ( hose including mo e han 10 sequences and wi h max- imum ela i e equencies o 0.0857) we e common only among some o di isions: H50 (20 sequences/3.92%) was common o all loca ions, excep o IVa and VIIh; H11 (20 se- quences/3.92%) was sha ed o all di isions excep VIIh and IXa; H5 (15 sequences/2.94%) was in all o hem, excep in VIIj and VIIh; H7 (12 sequences/2.35%) was common o di isions IVa, VIa, VIIb, VIIIc, VIIId and IXa; and H34 (10 sequences/1.96%) was sha ed o VIa, VIIb, VIIj, VIIIc, VIIId. Animals 2023,13, 197 7 o 17 Animals 2023, 13, 197 7 o 18 Figu e 2. Median-joining haplo ype ne wo k o A. simplex m DNA cox2 sequences ob ained om Eu opean hake om 10 ICES di ision a eas. Ci cles’ size ep esen s he equency o each haplo ype. Ha ch ma ks show he numbe o mu a ions dis inguishing he haplo ypes. Majo i y haplo ypes (including mo e han 10 sequences) a e epo ed. Black poin s indica e missing haplo ypes. Table 3. Gene ic di e si y indices and neu ali y es based on m DNA cox2 sequences o A. simplex pa asi izing Eu opean hakes om en ICES di isions. ICES a eas N Nh Nuh Pi Hd ± SD K S Tajima´s D Fu´s Fs D P Fs P IVa 44 27 13 0.00613 0.930 ± 0.030 2.96300 30 −1.93422 0.00800* −24.42734 0.00000* VIa 64 34 15 0.00496 0.848 ± 0.045 2.39633 30 −2.00076 0.00700* −26.84892 0.00000* VIIb 69 43 22 0.00718 0.937 ± 0.024 3.46974 41 −1.93982 0.00800* −26.18191 0.00000* VIIc 35 26 14 0.00948 0.965 ± 0.020 4.57815 44 −2.06327 0.00200* −19.69666 0.00000* VIIj 50 33 19 0.00740 0.953 ± 0.021 3.57633 34 −1.77822 0.01000* −26.07247 0.00000* VIIh 18 13 6 0.00755 0.902 ± 0.066 3.64706 17 −1.00685 0.12800 −6.41729 0.00100* Figu e 2. Median-joining haplo ype ne wo k o A. simplex m DNA cox2 sequences ob ained om Eu opean hake om 10 ICES di ision a eas. Ci cles’ size ep esen s he equency o each haplo ype. Ha ch ma ks show he numbe o mu a ions dis inguishing he haplo ypes. Majo i y haplo ypes (including mo e han 10 sequences) a e epo ed. Black poin s indica e missing haplo ypes. Table 3. Gene ic di e si y indices and neu ali y es based on m DNA cox2 sequences o A. simplex pa asi izing Eu opean hakes om en ICES di isions. ICES A eas N Nh Nuh Pi Hd ±SD K S Tajima’s D Fu’s Fs D P Fs P IVa 44 27 13 0.00613 0.930 ±0.030 2.96300 30 −1.93422 0.00800 * −24.42734 0.00000 * VIa 64 34 15 0.00496 0.848 ±0.045 2.39633 30 −2.00076 0.00700 * −26.84892 0.00000 * VIIb 69 43 22 0.00718 0.937 ±0.024 3.46974 41 −1.93982 0.00800 * −26.18191 0.00000 * VIIc 35 26 14 0.00948 0.965 ±0.020 4.57815 44 −2.06327 0.00200 * −19.69666 0.00000 * VIIj 50 33 19 0.00740 0.953 ±0.021 3.57633 34 −1.77822 0.01000 * −26.07247 0.00000 * VIIh 18 13 6 0.00755 0.902 ±0.066 3.64706 17 −1.00685 0.12800 −6.41729 0.00100 * VIIIa 59 45 25 0.00860 0.970 ±0.015 4.15546 57 −2.25027 0.00100 * −25.85918 0.00000 * VIIIc 60 42 19 0.00752 0.956 ±0.019 3.63277 36 −1.74418 0.01700 * −26.07312 0.00000 * VIIId 65 38 18 0.00675 0.938 ±0.022 3.25962 32 −1.66792 0.02500 * −26.27973 0.00000 * Animals 2023,13, 197 8 o 17 Table 3. Con . ICES A eas N Nh Nuh Pi Hd ±SD K S Tajima’s D Fu’s Fs D P Fs P IXa 46 33 17 0.00764 0.966 ±0.018 3.69082 32 −1.67002 0.02700 * −26.01534 0.00000 * O e all 510 215 - 0.00723 0.938 ±0.009 3.49440 115 −2.31355 0.00000 * −25.42466 0.00100 * Numbe o sequences analyzed (N), numbe o haplo ypes (Nh), numbe o unique haplo ypes (Nuh), nucleo ide di e si y (Pi), haplo ype di e si y (Hd) wi h hei ela i e s anda d de ia ion (SD), a e age numbe o nucleo ide di e ences (K), numbe o a iable si es (S), Tajima’s D (D) and Fu’s F (Fs) s a is ics wi h hei P- alues (D, signi icance le el 0.05 and Fs, signi icance le el 0.02). * Signi ican alues. A o al o 168 haplo ypes (78.15% o he o al 215 haplo ypes) we e unique o some o he 10 di isions analyzed and, in all cases, we e ep esen ed o only one sequence, ep esen ing mo e han 44.12% o o al haplo ypes o each di ision (Table 3, Figu e 2). The popula ion s uc u e o A. simplex om he di e en ishing di isions was explo ed wi h AMOVA, showing ha 99.75% o gene ic a ia ion was explained by di e ences wi hin popula ions and 0.25% o gene ic a iance by di e ences among popula ions. The ixa ion index F s o A. simplex among he 10 di isions was 0.00255 (P= 0.11241). F s alues (pai wise gene ic di e en ia ion) among A. simplex sequences o he 10 di isions sampled a e shown in Table 4. The highes F s alues appea ed be ween Via, and he o he nine di isions compa ed wi h i , anging be ween 0.01503 and 0.06270; all o hem we e signi i- can ly di e en (p< 0.05). In con as , he di e ences be ween he o he nine popula ions o A. simplex we e no s a is ically signi ican , and hei F s alues we e lowe han hose o VIa. Table 4. Popula ion pai wise F s alues om m DNA cox2 sequences among A. simplex om 10 ishing di isions. * signi ican alue (signi icance le el 0.05). IVa VIa VIIb VIIc VIIj VIIh VIIIa VIIIc VIIId IVa - VIa 0.02373 * - VIIb − 0.00631 0.02802 * - VIIc 0.00118 0.02149 * − 0.00203 - VIIj 0.01078 0.04431 * 0.00325 − 0.00585 - VIIh 0.00614 0.06270 * 0.00116 − 0.00112 0.01541 - VIIIa − 0.00411 0.01503 * − 0.00565 − 0.01071 − 0.00473 0.00953 - VIIIc 0.00025 0.04427 * 0.00058 0.00612 − 0.00073 0.00587 − 0.00036 - VIIId − 0.00296 0.02871 * − 0.00479 − 0.00732 − 0.00082 0.01133 − 0.00553 0.00177 - IXa − 0.00303 0.04545 * − 0.00188 0.00673 0.01137 − 0.01415 0.00173 − 0.00629 0.00031 3.2.2. Case S udy 2: A. simplex om Di e en Fish Muscula Sec ions A o al o 510 m DNA cox2 sequences om A. simplex desc ibed abo e we e g ouped in belly lap (BF), loin (LO) and ail (TA), depending on whe e i was aken om. The gene ic di e si y indices calcula ed o A. simplex om each muscula sec ion we e simila be ween hem and o hose ob ained o he 10 ishing di isions (Table 5). The Hd alue o BF was 0.942, o LO 0.932 and o TA 0.943, wi h Pi alues o 0.00696, 0.00751 and 0.00783, espec i ely. Neu ali y es , Tajima’s D and Fu’s also showed nega i e alues s a is ically signi ican (p< 0.05 and p< 0.02, espec i ely) o he h ee sec ions. Median-joining haplo ype ne wo k o he 510 m DNA cox2 sequences o A. simplex om he h ee muscula sec ions was ep esen ed (Figu e 3). Twel e haplo ypes we e sha ed o BF, LO and TA: H3, H50, H11, H5, H7, H34, H33, H24, H77, H9, H99 and H111, being he H3 haplo ype, majo i y in all ishing suba eas, he mos equen (64 sequences in BF, wi h a ela i e equency o 0.229), 44 in LO (0.250) and 22 in TA (0.222). O he sha ed haplo ypes showed lowe ela i e equencies, being highe in haplo ypes: H50 was ep esen ed in BF wi h 11 sequences ( ela i e equency o 0.0393), in LO wi h 8 sequences (0.0455) and in TA wi h 1 sequence (0.0185); H11 occu ed in BF wi h 7 sequences (0.025), in LO wi h 9 sequences (0.0511) and in TA wi h 4 (0.0741). The ne wo k e ealed 19 haplo ypes Animals 2023,13, 197 9 o 17 we e sha ed be ween BF and LO, 4 be ween BF and TA and only 3 we e common o LO and TA, wi h ela i e equencies anging om a maximum o 0.025 and a minimum o 0.00357. Animals 2023, 13, 197 10 o 18 Figu e 3. Median-joining haplo ype ne wo k o A. simplex m DNA cox2 sequences ob ained o mus- cula sec ions om Eu opean hake: belly laps (BF), loins (LO) and ails (TA). Ci cles’ size ep esen s he equency o each haplo ype. Ha ch ma ks show he numbe o mu a ions dis inguishing he haplo ypes. Haplo ypes sha ed o he h ee sec ions a e epo ed. Black poin s indica e missing haplo ypes. A o al o 177 haplo ypes (82.33% o he o al 215 haplo ypes) we e unique o some o he h ee muscula sec ions analyzed. Rema kably, 100 unique haplo ypes we e ound in BF, ep esen ed by be ween one and ou sequences o each o hem; 63 unique haplo- ypes in LO (including 1 o 2 sequences pe haplo ype) and 14 exclusi e haplo ypes in TA (wi h 1 sequence pe haplo ype) (Table 5, Figu e3). The AMOVA esul s showed ha 100 % o gene ic a ia ion was explained by di e ences wi hin h ee popula ions (BF, LO and Figu e 3. Median-joining haplo ype ne wo k o A. simplex m DNA cox2 sequences ob ained o muscula sec ions om Eu opean hake: belly laps (BF), loins (LO) and ails (TA). Ci cles’ size ep esen s he equency o each haplo ype. Ha ch ma ks show he numbe o mu a ions dis inguish- ing he haplo ypes. Haplo ypes sha ed o he h ee sec ions a e epo ed. Black poin s indica e missing haplo ypes. Animals 2023,13, 197 16 o 17 19. Gilabe , A.; Wasmu h, J.D. Un a elling pa asi ic nema ode na u al his o y using popula ion gene ics. T ends Pa asi ol. 2013,29, 438–448. [C ossRe ] 20. MacKenzie, K. 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[C ossRe ] Disclaime /Publishe ’s No e: The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .