Histopathological Lesions Caused by a Digenean Trematode in a Pest Apple Snail, Pomacea canaliculata, in Its Native Geographic Distribution Area

Ci a ion: Ma inez, L.E.; Gila doni, C.; Medina, C.D.; C emon e, F.; E chegoin, J.A. His opa hological Lesions Caused by a Digenean T ema ode in a Pes Apple Snail, Pomacea canalicula a, in I s Na i e Geog aphic Dis ibu ion A ea. Animals 2024,14, 1191. h ps:// doi.o g/10.3390/ani14081191 Academic Edi o : Da id B uce Conn Recei ed: 15 Ma ch 2024 Re ised: 11 Ap il 2024 Accep ed: 13 Ap il 2024 Published: 16 Ap il 2024 Copy igh : © 2024 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 His opa hological Lesions Caused by a Digenean T ema ode in a Pes Apple Snail, Pomacea canalicula a, in I s Na i e Geog aphic Dis ibu ion A ea Lo ena E angelina Ma inez 1, Ca men Gila doni 2,3,* , Cin ia Débo a Medina 4, Flo encia C emon e 2 and Jo ge Alejand o E chegoin 1 1Ins i u o de In es igaciones en P oducción Sanidad y Ambien e (IIPROSAM), CONICET-Uni e sidad Nacional de Ma del Pla a, Cen o de Asociación Simple CIC-PBA, Juan B. Jus o 2550, Ma del Pla a 7600, A gen ina; lo [email p o ec ed] (L.E.M.); [email p o ec ed] (J.A.E.) 2Ins i u o de Biología de O ganismos Ma inos (CCT CONICET-CENPAT), Boule a d B own 2915, Pue o Mad yn 9120, A gen ina; c [email p o ec ed] 3Ins i u o de Ciencias del Ma (ICM-CSIC), Passeig Ma í im de la Ba celone a, 37–49, 08003 Ba celona, Spain 4Ins i u o de Di e sidad y E olución Aus al (CCT CONICET-CENPAT), Boule a d B own 2915, Pue o Mad yn 9120, A gen ina; [email p o ec ed] *Co espondence: gila [email p o ec ed] Simple Summa y: The apple snail is one o he mos dange ous in asi e species in eshwa e en i onmen s. Using molecula and mo phological ools, we e-desc ibe an echinos oma id digenean pa asi izing snails om wo si es in he Buenos Ai es P o ince, A gen ina. The wo s ages ound (i.e., ediae and me ace ca iae) demons a e ha he apple snail ac s as he i s and second in e media e hos in i s li e cycle. The p e alence o he pa asi e was highe a one o he sampling si es, p obably because he bi ds bea ing he adul s age a e mo e abundan in ha a ea. A his ological s udy showed ha his pa asi e quickly in ades mul iple o gans o he snail, which is di e en om mos digenean in ec ions, which only in ec he gonad and diges i e glands. Hea y de e io a ion o emale and male ep oduc i e s uc u es associa ed wi h he p esence o he pa asi e was also obse ed, which indica es cas a ion. Abs ac : Pomacea canalicula a is one o he mos dange ous in asi e species. Mo phological and molecula analyses ha e e ealed ha a digenean species belonging o he amily Echinos oma idae pa asi izes his snail a wo si es in Buenos Ai es P o ince, A gen ina, Sou h Ame ica. Molecula esul s con i med ha he species belongs o a genus closely ela ed o Pa agi e . Analysis o he 28S DNA showed ha he sequences o he ediae and me ace ca iae a e iden ical, indica ing ha he apple snail ac s as he i s and second in e media e hos . The ce ca iae may encys as me ace ca ia inside he edia and also eme ge and e-in ec he same snail o ano he snail. The p e alence o digeneans was highe in one o he sampling loca ions (15.1% s. 0.72%), p obably because he bi d species ha ac s as he de ini i e hos is mo e abundan in ha a ea. His opa hological examina ion showed ha he pa asi e quickly in ades mul iple hos o gans (gills, in es ines, albumen gland, lung, kidney, and man le bo de ) besides he gonad and diges i e gland, as is usual in digeneans. In addi ion, he pa ial o o al cas a ion o snails was obse ed in cases o mode a e and high in ec ion in ensi y. In males, he e was loss o in eg i y in es icula ubules, while in emales, he eplacemen o o a ian issue by ediae was ound. Keywo ds: mollusc; Ampulla idae; echinos oma id; his opa hology; hos esponse 1. In oduc ion The golden apple snail Pomacea canalicula a (Lama ck, 1822) (Ampulla iidae, Caenogas- opoda) is na i e o Sou h Ame ica bu has sp ead, colonizing se e al coun ies such as Animals 2024,14, 1191. h ps://doi.o g/10.3390/ani14081191 h ps://www.mdpi.com/jou nal/animals Animals 2024,14, 1191 2 o 16 he Uni ed S a es, Spain, China, and Taiwan [ 1 ]. Fu he mo e, i has been expanding i s o iginal dis ibu ion in A gen ina [ 2 ]. In ac , P. canalicula a is cu en ly included in he mos dange ous g oup o in asi e eshwa e snails; i causes habi a al e a ions, modi ies ben hic communi y s uc u es, and dec eases local di e si y [ 3 , 4 ]. Acco ding o he IUCN, i is lis ed as one o he op one hund ed wo s in asi e species [5]. In addi ion o being able o cause damage o local ecosys ems, P. canalicula a may ac as a hos o se e al pa asi e species, including ema ode digeneans [ 6 , 7 ]. These pa asi es ha e complex li e cycles wi h a leas wo hos s, including ee-li ing and pa asi ic s ages. In a- molluscan s ages (spo ocys s and/o ediae) p oli e a e asexually in he snail when i ac s as he i s in e media e hos ; hey mig a e h ough blood spaces un il eaching he issues, om which hey ob ain nu ien s [ 8 , 9 ]. These in a-molluscan s ages can cause his ological damage and s uc u al al e a ions ha may include he cas a ion o he snail hos [ 10 – 14 ]. Beyond p o iding ene gy o mul iplica ion, in ec ing he hos ’s gonad allows he pa asi es o imp o e condi ions o su i al wi hou causing se e e damage and o comple e hei li e cycles [ 15 – 17 ]. The digenean pa asi e–hos associa ion shows high speci ici y, mainly a he le el o he snail as i s i s in e media e hos . In hese cases, he pa asi e e ades he hos ’s immune esponse by a oiding ecogni ion and elimina ion (see [ 18 ]). This ac demons a es ha his s ong associa ion comes om a long co-e olu iona y his o y [19]. To da e, a leas eigh di e en species o digeneans ha e been epo ed o use he apple snail P. canalicula a as an in e media e hos [ 7 , 20 ]. None o hem ha e been iden i ied by molecula s udies, and his ological s udies ha e no been ca ied ou o assess he damage hey cause. In Los Talas, Buenos Ai es P o ince, Ma o elli [ 21 ] epo ed ha he species Echinos oma pa cespinosum Lu z, 1924 (Echinos oma idae) uses he apple snail as i s i s in e media e hos in i s na i e dis ibu ion a ea. In membe s o his genus, he ediae gi e ise o he swimming la al s age, ce ca iae, capable o eme ging om he snail in sea ch o a second in e media e hos . These ce ca iae may e-in ec he same snail hos species, becoming an encys ed me ace ca iae. The same species o digenean may ha e, as an al e na i e, an abb e ia ed li e cycle, which implies ha he ce ca iae do no lea e he snail and become encys ed me ace ca ia wi hin he ediae. When he de ini i e hos , usually a bi d, eeds on he snail, he me ace ca iae ma u e sexually in o an adul s age, comple ing he li e cycle [8,9]. This wo k aimed o desc ibe a digenean belonging o he amily Echinos oma idae in he in asi e apple snail, Pomacea canalicula a, in i s na i e ange dis ibu ion a ea by using mo phological and molecula me hods. Fu he mo e, ia his ology, we assessed he si es o in ec ion and lesions in lic ed on snail issues. 2. Ma e ials and Me hods 2.1. Sample Collec ion and Mo phological Desc ip ion Sampling was conduc ed du ing he summe pe iod o 2022–2023; 67 indi iduals o Pomacea canalicula a we e collec ed om a small lagoon in he Ha bo Na u al Rese e (R) ( 38◦01′S , 57 ◦ 31 ′ W) and 137 snails om Los Pad es Lagoon (LP) (37 ◦ 56 ′ S, 57 ◦ 44 ′ W), Buenos Ai es P o ince, A gen ina. Snails we e collec ed by hand, sea ching he bo om o sub- me ged and loa ing ege a ion along a ansec pa allel o he sho eline. Specimens we e kep in sepa a e small eshwa e con aine s unde a 12-12 ligh –da k pho ope iod o 48 h o s imula e he shedding o he digenean la ae (ce ca iae) and inspec ed wice daily unde a s e eomic oscope (Zeiss S emi508, Zeiss, Jena, Ge many) o eme ged ce ca iae. The la e we e s ained wi h i al s ain (neu al ed o Nile blue) and s udied in i o unde a ligh mi- c oscope (Leica DM2500, Leica Came a, We zla , Ge many). The ediae and me ace ca iae (n: 15) we e s udied and measu ed ali e; measu emen s o ce ca iae (n: 15) we e aken om hea -killed specimens. All measu emen s a e p o ided in mic ome e s ( µ m) wi h he mean ollowed by he ange in pa en heses. D awings we e made wi h he aid o a d awing ube, and he pho og aphs we e made using he Leica Applica ion Sui e so wa e e . 4.1.0.1264. Some specimens ( ouche s) we e deposi ed a he Pa asi ological Collec ion (CNP-Pa ) o he Ins i u o de Biología de O ganismos Ma inos, CCT CONICET-CENPAT, Pue o Animals 2024,14, 1191 3 o 16 Mad yn, Chubu P o ince, A gen ina. The p e alence o he pa asi e (P) was calcula ed as he numbe o in ec ed snails di ided by he o al numbe o snails examined a each si e, exp essed as a pe cen age. The numbe o me ace ca iae pe pa asi ized snail was coun ed, and he mean in ensi y was calcula ed as he o al numbe o me ace ca iae di ided by he numbe o pa asi ized snails (I). The in ensi y o in ec ion is p esen ed as he mean ollowed by he in e al in pa en hesis. The p e alence and in ensi y o me ace ca iae we e calcula ed only by coun ing me ace ca iae ou side he ediae (i.e., when ce ca iae lea e he hos and en e he same snail o ano he snail). 2.2. His ology A e emission obse a ions, all specimens we e dissec ed o see i hey had p e-pa en in ec ions, measu ed wi h a calipe om he apex o he end o he spi e, and subsequen ly p ocessed acco ding o he OIE- ecommended p o ocol (h ps://www.oie.in / ileadmin/ Home/eng/Heal h_s anda ds/aahm/cu en /chapi e_gene al_in o ma ion_2_4.pd , ac- cessed on 15 Janua y 2022), including eeze chilling ( − 20 ◦ C) o 10 min be o e dissec ion. A o al o 98 specimens om bo h si es (R = 31; LP = 67) we e p ocessed o his ology. Fo each snail, wo biopsy casse es we e used o include he ollowing o gans: gonad, diges i e gland, inhalan siphon, oo , lung, kidney, s omach, gills, man le, and albumen gland o penis. The casse es we e placed in Da idson’s ixa i e o 24 h and p ocessed wi h classical his ological p ocedu es [ 22 ]. One 5 µ m hick sec ion om each block was cu and s ained wi h hema oxylin and eosin (H&E). His ological sec ions we e examined unde a ligh mic oscope o s udy he in ec ion si es and damage o issues and o gans. Pho og aphs we e aken wi h a Leica DFC280 digi al came a and associa ed so wa e. The in ensi y o in ec ion was quali a i ely classi ied as ollows: ligh (cha ac e ized by ew ediae mainly con aining ge minal balls and unde eloped ce ca iae), mode a e (in e me- dia e numbe o ediae con aining de eloped and/o unde eloped ce ca iae), and high (nume ous ediae mainly con aining de eloped ce ca iae and some imes me ace ca iae). This ep esen s an assessmen o he o ma ion o he pa asi e in apopula ion based on a ime scale om he in oduc ion o mi acidium o he comple e coloniza ion o he mollusc body by ep oducing ediae. 2.3. DNA Ex ac ion, Ampli ica ion, and Sequencing Some ediae (N = 3) and me ace ca iae (N = 8) we e ixed in 96% e hanol and hen washed o 24 h in a TRIS (10 mM)–EDTA (1 mM) bu e solu ion. DNA was ex ac ed using he GenElu eTM Mammalian Genomic DNA Minip ep Ki (Sigma, S . Louis, MO, USA) acco ding o he manu ac u e ’s ins uc ions. The 28S egions o he DNA o he ediae and me ace ca iae we e ampli ied by PCR. PCRs we e pe o med in a o al olume o 50 µL con aining 10 × bu e (200 mM T is-HCl pH 8.4, 500 mM KCl), 0.2 mM o each dNTP, 1.5 mM o MgCl 2 , 0.4 µ M o each p ime , and 1 U o pla inum Taq poly- me ase. In o al, 2 µ L o genomic DNA was used as a empla e. The 28S egions we e ampli ied using 28S-28S: 5′′-GTGAATACCCGCTGAACTTAAGC-3′′ as a o wa d p ime , si ua ed 16 bp om he 3 ′′ end o he conse ed egion o he ls DNA, and 28S-28S: 5′′-TCTCCTTGGTCCGTGTTTCAA-3′′ as a e e se p ime , loca ed 868 bp om he 5 ′′ end o he conse ed egion o he ls DNA. These p ime s we e used ollowing sou ces such as C emon e e al. [ 23 ]. The cycling condi ions included an ini ial dena u a ion a 94 ◦C o 5 min, ollowed by 40 cycles o 30 s a 94 ◦ C, 30 s a 52 ◦ C (annealing), and 2 min a 72 ◦ C, wi h a inal ex ension s ep o 10 min a 72 ◦ C. Ampli ied PCR p oduc s we e elec opho e ically sepa a ed in a 1 % (w/ ) aga ose gel s ained wi h gel g een. Nega i e con ols o he PCR we e always un o con ol o con amina ion. Rele an bands we e sen o pu i ica ion and sequencing (Mac oGen, Seoul, Republic o Ko ea). All sequences ha e been deposi ed in GenBank. Animals 2024,14, 1191 4 o 16 2.4. Sequence Alignmen s and Phylogene ic Analysis The pa ial 28S DNA sequences gene a ed in his s udy we e aligned wi h sequences o ep esen a i es o he Echinos oma idae amily a ailable in GenBank (Table 1) and Fasciola hepa ica Linnaeus, 1758 (Fasciolidae) (AY222244), which was used as an ou g oup. The conca ena ed alignmen s we e pe o med using he MAFFT so wa e e . 7 (a ailable a h p://www.ebi.ac.uk/Tools/msa/ma /, accessed on 23 Feb ua y 2024) and MEGA X [ 24 ]. Phylogene ic and molecula e olu iona y analyses we e conduc ed on he aligned nucleo ide sequences o 28S and we e in e ed by bo h he maximum likelihood (ML) me hod using he W-IQTREE 1.0 online so wa e [ 25 ] and Bayesian in e ence (BI) using BEAST 1.8.0 [ 26 ]. Fo he ML ee, he bes - i model chosen acco ding o he Akaike in o ma ion c i e ion (AIC) was GTR + F + I + G4. Node suppo s we e e alua ed using a boo s ap ul a as es wi h 1000 eplica es [ 27 ]. The pe cen age o ees in which he associ- a ed axa clus e ed oge he is shown nex o he b anches. Fo he BI ee, o de e mine he e olu ion model ha bes i s ou da ase , he p og am jModel es 2.1.1 [ 28 ] was employed, wi h model selec ion based on AIC. The esul s indica ed ha GTR + G was he mos app op ia e. Ma ko Chain Mon e Ca lo (MCMC) chains we e un o 10,000 gene a ions , sampling e e y 10 gene a ions, wi h he i s 250 sampled ees disca ded as “bu n-in”. Finally, a 50 % majo i y ule consensus ee was cons uc ed. These analyses in ol ed 43 nucleo ide sequences wi h 995 posi ions in he inal da ase . Gene ic di e gences (p- dis ance) we e calcula ed o 19 nucleo ide sequences g ouped in he clade, which include he species s udied he e, using Mega X. Table 1. Summa y da a o 28S DNA sequences o echinos oma oid axa included in he molecula analyses. Abb e ia ions: ed ( edia), ce (ce ca ia), me a (me ace ca ia), ad (adul ), Pc (Pomacea canalicula a), R (Rese e), and LP (Los Pad es). Taxon Li e Cycle S age Hos Coun y GenBank N◦Re e ences Family Echinos oma idae Looss, 1899 Sub amily Echinos oma inae Looss, 1899 Echinos oma idae gen. e sp. ( ed_Pc_R) ed Pomacea canalicula a (Lama ck) A gen ina PP390560 P esen s udy Echinos oma idae gen. e sp. (me a_Pc_R)me a Pomacea canalicula a A gen ina PP391013 P esen s udy Echinos oma idae gen. e sp. ( ed_Pc_LP) ed Pomacea canalicula a A gen ina PP391016 P esen s udy Echinos oma idae gen. e sp. (me a_Pc_LP)me a Pomacea canalicula a A gen ina PP391021 P esen s udy Echinos oma miyagawai Ishii, 1932 ad Anas pla y hynchos L. Uk aine KT956916 [29] Echinos oma pa aulum Die z, 1909 ce Lymnaea s agnalis (L.) Ge many KP065604 [30] Echinos oma pa aensei Lie and Basch, 1967 ad “hams e ” USA EU025867 [31] Echinos oma i ol is (Co , 1914) ad Mesoc ice us au a us Wa e house (exp.) USA AY222246 [32] Echinos oma nasinco ae Fal ýnko áe al., 2015 ce Plano ba ius co neus (L.) Czech Republic KP065603 [30] Echinos oma sp. IG ce Radix au icula ia (L.) Ge many KP065606 [30] Echinos oma e olu um (F ölich, 1802) (sensu la o)ad Ay hya colla is (Dono an) USA KT956915 [29] Echinos oma e olu um (F ölich, 1802) (sensu s ic o) ad Ay hya uligula (L.) Czech Republic KP065596 [30] Echinos oma bolschewense (Ko o a, 1939) ce Vi ipa us ace osus (Bou guigna ) Slo akia KP065592 [30] Neoacan hopa yphium echina oides (de Filippi, 1854) ce Vi ipa us ace osus (Bou guigna ) Slo akia KT956922 [29] Moliniella anceps (Molin, 1859) me a Plano ba ius co neus (L.) Li huania KT956921 [29] Echinopa yphium aconia um Die z, 1909 ce Lymnaea s agnalis (L.) Czech Republic KT956912 [29] Echinopa yphium sp. ad Anas clypea a L. USA KT956914 [29] Echinopa yphium cinc um (Rudolphi, 1803) ad Anas pla y hynchos L. Uk aine AF184260 [33] Echinopa yphium ecu a um ( on Lins ow, 1873) ce Radix o a a (D apa naud) Slo akia KT956913 [29] Echinopa yphium ub um Co , 1914 ce Plano bella i ol is (Say) USA JX262943 [34] Hypode aeum conoideum (Bloch, 1782) ad Anas pla y hynchos L. Uk aine KT956918 [29] Hypode aeum conoideum (Bloch, 1782) ad Anas acu a L. USA KT956919 [29] Eupa yphium c . Mu inum Tubangui, 1931 ad Malacomys longipes Milne-Edwa ds Uganda KT956917 [29] Pe asige adia um Duja din, 1845 ad Phalac oco ax ca bo (L.) Uk aine KT956927 [29] Pe asige phalac oco acis (Yamagu i, 1939) ad Phalac oco ax ca bo (L.) Uk aine KT956926 [29] Pe asige exae e us Die z, 1909 ad Phalac oco ax ca bo (L.) Uk aine KT956923 [29] Animals 2024,14, 1191 5 o 16 Table 1. Con . Taxon Li e Cycle S age Hos Coun y GenBank N◦Re e ences D epanocephalus au i us Kudlai e al., 2015 ad Phalac oco ax au i us (Lesson) USA KP683124 [35] Pe asige sp. 1 ce Plano bis plano bis (L.) Czech Republic KM191800 [36] Pe asige sp. 2 ce Gy aulus albus (O. F. Mülle ) Ge many KM191802 [36] Pe asige sp. 3 ce Plano bis plano bis (L.) Ge many KM191804 [36] Pe asige sp. 4 me a Gas e os eus aculea us L. Canada KM191807 [36] Neope asige n. sp. ad Podiceps g isegena (Boddae ) USA KT956925 [29] Pe asige islandicus Kos adino a and Skí nisson, 2007 ad Aechmopho us occiden alis (Law ence) USA KT956924 [29] Sub amily Neph os ominae Mendheim, 1943 Pa agi e sp. 1 ce Biomphala ia sudanica (Ma lens) Kenia MK482507 [37] Pa agi e sp. 2 ce Biomphala ia p ei e i Kenia MK482497 [37] Pa agi e ioscai Lumsden, 1962 ad Eudocimus albus (L.) USA KT956946 [29] Pa agi e bilobus (Rudolphi, 1819) ad Plegadis alcinellus (L.) Uk aine KT956945 [29] Sub amily Himas hlinae Odhne , 1910 A y echinos omum su a y ex Lane, 1915 ad Sus sc o a dom. India KF781303 - Sub amily Chaunocephalinae T a assos, 1922 Chaunocephalus e ox (Rudolphi, 1795) ad Ciconia nig a (L.) Uk aine KT447522 - Family Psilos omidae Looss, 1900 Sub amily Ribei oiinae T a assos, 1951 Ribei oia onda ae (P ice, 1931) ad Pelecanus e y h o hynchos Gmelin USA KT956956 [29] Family Ca haemasiidae Fuh mann, 1928 Sub amily Ca haemasiinae Fuh mann, 1928 Ca haemasia hians (Rudolphi, 1809) ce Plano bis plano bis (L.) Czech Republic KT956947 [29] Family Rhopaliidae Looss, 1899 Rhopalias mac acan hus Chandle , 1932 ad Didelphis i giniana (Ke ) USA KT956959 [29] Family Fasciolidae Raillie , 1895 Sub amily Fasciolinae Raillie , 1895 Fasciola hepa ica Linnaeus, 1758 ad Cap a hi cus (L.) Saudi A abia AY222244 [32] 3. Resul s Table 2shows he apple snail da a o shell leng h and sex a io om he Ha bo Na u al Rese e (R) and Los Pad es Lagoon (LP). Fu he mo e, he p e alence and in ensi y o in ec ion o bo h s ages ound— ediae (wi h de eloping ce ca iae and/o me ace ca iae) and me ace ca iae—a e gi en. The p e alence was highe in he Rese e han in Los Pad es. Hal o he in ec ed snails in he Rese e had p e-pa en in ec ions (i.e., he snails did no shed ce ca iae), while he only in ec ed snail in LP was emi ing ce ca iae. Encys ed me ace ca iae we e ound by s e eoscopical examina ion in he pe ica dial ca i ies o he snails a he wo s udied si es (Table 2). Fu he mo e, encys ed me ace ca iae we e ound by obse ing he his ological sec ions in he albumen gland and kidney. The species s udied he ein we e iden i ied based on mo phology and gene ics as belonging o he Echinos oma idae amily. Rediae, ce ca iae, and me ace ca iae om R and LP a e e y simila mo phologically, bu hey di e in 28S DNA sequences. Table 2. Apple snail da a (shell leng h and sex a io) om he wo sampling si es in A gen ina wi h p e alence and mean in ensi y o in ec ion caused by he digenean ema ode Echinos oma idae gen. e . sp. P e alence and mean in ensi y a e ollowed by ange in pa en hesis. The i s pa o he able shows he esul s o he o al numbe o snails collec ed a e s e eomic oscopic examina ion; he second pa includes he esul s o he subsample p ocessed o his ology and examined unde a ligh mic oscope. Sampling Si es Ha bo Na u al Rese e Los Pad es Lagoon N o al (s e eoscope mic oscope examina ion) 67 137 Shell leng h mean (±SD) (cm) 4.37 (0.7) 4.87 (0.7) Sex a io ( emale/male) 41/25 83/54 P e alence o ediae (%) 15.1 0.72 P e alence o me ace ca iae (%) 74.2 9.5 Mean in ensi y o me ace ca iae 298 (17−1912) 41 (2−169) N (his ology subsample) 31 67 Shell leng h mean (±SD) (cm) 4.34 (0.8) 4.86 (0.8) Sex a io ( emale/male) 19/31 41/67 Animals 2024,14, 1191 6 o 16 3.1. Desc ip ion o Redia, Ce ca ia, and Me ace ca ia o Echinos oma idae gen. e sp. Conside ing he mo phological simila i ies obse ed be ween he in a-molluscan s ages o he digeneans collec ed om bo h sampling si es and he highes p e alence ound in he Rese e, desc ip ions o he de elopmen s ages we e made wi h samples collec ed a his si e. Rediae (Figu e 1A) Animals 2024, 14, x FOR PEER REVIEW 7 o 17 Me ace ca ial cys s a e usually sphe ical and 336.6 (300–376.6) in diame e . Cys wall: 11.3 (9–13). Taxonomic summa y Fi s and second in e media e hos s: Pomacea canalicula a (Lama ck, 1822) (Ampulla - iidae, Caenogas opoda). Si es o in ec ion: Rediae wi h ce ca iae and/o me ace ca ia in he gonad, diges i e gland, albumen gland, gills, in es ine, kidney, lung, and man le; me ace ca iae (ou side he ediae) mainly in he pe ica dial ca i y bu also in he albumen gland and kidneys. GenBank accession numbe s: PP390560 ( ediae om he Rese e), PP391013 (me a- ce ca iae om he Rese e), PP391016 ( ediae om Los Pad es), and PP391021 (me ace - ca iae om Los Pad es). Specimens deposi ed: Rediae wi h ce ca iae and me ace ca iae (CNP-Pa 226) and me ace ca iae (CNP-Pa 227) om he Rese e (e hanol ixed specimens). Figu e 1. De elopmen al s ages o Echinos oma idae gen. e . sp. (A) Redia, in i o, showing pha - ynx (ph), caecum (dc), ce ca ia (Ce), podium (p); (B) ce ca ial body, line d awing; (C) ail en al iew, line d awing; (D) de ail o head colla spines; (E) me ace ca ia om pe ica dial ca i y, in i o, showing he exc e o y g anules (eg) and spines (s). Ba s = 50 µm (D), 100 µm (B,E), 150 µm (C), and 200 µm (A). Figu e 1. De elopmen al s ages o Echinos oma idae gen. e . sp. (A) Redia, in i o , showing pha ynx (ph), caecum (dc), ce ca ia (Ce), podium (p); (B) ce ca ial body, line d awing; (C) ail en al iew, line d awing; (D) de ail o head colla spines; (E) me ace ca ia om pe ica dial ca i y, in i o , showing he exc e o y g anules (eg) and spines (s). Ba s = 50 µ m (D), 100 µ m (B,E), 150 µ m (C), and 200 µm (A). Body size: 2090 (1560–2600) long by 560 (490–620) wide. Muscula pha ynx: 8.3 (7.8–8.8) long by 7.7 (6.0–10) wide. Esophagus opening in o a sac-like caecum: 877 (850–1260) long. Tegumen a y colla behind he pha ynx. A pai o locomo o y appendages nea he pos e io end o he body. Bi h po e immedia ely pos e io o he egumen a y colla . Ma u e ediae con aining 1 o 3 de eloping ce ca iae o 1 o 3 me ace ca iae. Ce ca iae (Figu e 1B–D) Animals 2024,14, 1191 7 o 16 Body: 970 (750–1220) long by 420 (350–500) wide, wi hou spines. Colla wi h 31 spines , wi h ou co ne spines on each side and a single an e io ly unin e up ed ow o 23 spines. O al sucke sub e minal: 82 (79–90) long by 81 (67–90) wide. Ven al sucke : 127 (123–132) long by 131 (120–150) wide. P epha ynx: 35 (32–39) long. Pha ynx: 45 (39–50) long by 39 (31–42) wide. Esophagus: 260 (200–290) long by 18 (15–24) wide, consis ing o app oxima ely 14 o 16 cells, bi u ca es a 438 (336–480) om he an e io end o he body and caeca ex ending o he pos e io end o he body. Nume ous cys ogenous cells a e loca ed be ween he pha ynx and he pos e io ma gin o he body. Exc e o y sys em s enos oma e: The exc e o y esicle was di ided in o wo chambe s loca ed a he pos e io end o he body. The p ima y exc e o y ubes o igina ed in he an e io chambe o he exc e o y esicle and dila ed in he a ea be ween he en al sucke and he pha ynx o accommoda e 21–30 sphe ical e ac ile g anules 14 (11–18) in diame e . Flame cells we e di icul o see, wi h a leas 15 pai s. The caudal duc o he exc e o y sys em en e s he an e io po ion o he ail and bi u ca es in o wo b anches ha end a he la e al ma gins o he ail. Tail: 960 (840–1200) long by 91 (70–120) wide, wi h ou do so en al in- olds, wo an e io , wo pos e io , and one con inuous la e al in- old ha co e s bo h sides o he ail. Me ace ca iae (Figu e 1E) Me ace ca ial cys s a e usually sphe ical and 336.6 (300–376.6) in diame e . Cys wall: 11.3 (9–13). Taxonomic summa y Fi s and second in e media e hos s: Pomacea canalicula a (Lama ck, 1822) (Ampulla i- idae, Caenogas opoda). Si es o in ec ion: Rediae wi h ce ca iae and/o me ace ca ia in he gonad, diges i e gland, albumen gland, gills, in es ine, kidney, lung, and man le; me ace ca iae (ou side he ediae) mainly in he pe ica dial ca i y bu also in he albumen gland and kidneys. GenBank accession numbe s: PP390560 ( ediae om he Rese e), PP391013 (me ace - ca iae om he Rese e), PP391016 ( ediae om Los Pad es), and PP391021 (me ace ca iae om Los Pad es). Specimens deposi ed: Rediae wi h ce ca iae and me ace ca iae (CNP-Pa 226) and me ace ca iae (CNP-Pa 227) om he Rese e (e hanol ixed specimens). Taxonomic ema ks Acco ding o Pin o and de Melo ([ 38 ]) and Dambo enea e al. ([ 7 ]), wo species o echi- nos oma ids ha e been epo ed o pa asi ize P. canalicula a in Sou h Ame ica: Echinos oma pa cespinosum (in A gen ina and B azil) and Die ziella eg egia (Die z, 1909) (in A gen ina). Bo h species ha e a simila numbe o spines on he colla , 31 o 33 (E. pa cespinosum) and 31 (D. eg egia), which ag ees wi h he numbe o spines ound on he colla o he ce ca ia desc ibed he ein (31). Howe e , only a desc ip ion o he ce ca iae o E. pa cespinosum has been a ailable o mo phological compa isons since he li e cycle o his species was elucida ed by Ma o elli [ 21 ]. The ediae o his species ound in he Rese e a e la ge and wide bu mo phologically simila o hose o E. pa cespinosum. The ce ca iae ound in P. canalicula a a e dis inguished om he ce ca iae o E. pa cespinosum by ha ing a la ge body (750–1220 s. 820–960), sligh ly smalle o al and en al sucke s (67–90 s. 80–100, and 120–150 s. 170–180 in diame e , espec i ely) and in he numbe o in- olds in he ail. The ce ca iae om he Rese e had ou do so en al in- olds and one con inuous la e al in- old, while he ce ca iae o E. pa cespinosum ha e “a ail su ounded by a hin and anslucen in”. Rega ding he me ace ca iae, he diame e o he cys s o he wo species di e s sligh ly (330–350 s. 300–376.6). 3.2. Molecula Analyses A o al o ou sequences including a pa ial 28S o DNA was ob ained. Rediae and me ace ca iae om he Ha bo Na u al Rese e (R) p o ided p oduc s o 895 pb and 906 pb, espec i ely, and hey we e 100% iden ical. Rediae and me ace ca iae om Los Pad es Lagoon (LP) p o ided p oduc s o 917 pb and 871 pb, espec i ely, and hey Animals 2024,14, 1191 8 o 16 we e 100% iden ical oo. Sequences om R di e ed by 7 pb compa ed wi h sequences om LP. Phylog am ees buil wi h ML and BI p o ided simila opologies, and he BI analysis had highe nodal suppo han he ML analysis (Figu e 2). Species o he Echinos oma idae amily a e g ouped in o wo big clades; one o hem includes he gene a Echinos oma Rudolphi, 1809; Neoacan hopa yphium Yamagu i, 1958; Pa agi e Die z, 1909; A y echinos omum Lane, 1915; Moliniella Hübne , 1939; Hypode aeum Die z, 1909; and Echinopa yphium Die z, 1909. These gene a a e sepa a ed in o wo subclades: subclade 1 includes Echinos oma,Neoacan hopa yphium,Pa agi e , and A y echinos omum, and subclade 2 includes Moliniella,Hypode aeum, and Echinopa yphium. The species s udied he e is close o he Pa agi e clade, which is a monophyle ic clade ha includes ou species. Howe e , i seems o be a di e en genus composed o wo species (one om R and ano he om LP). The gene ic dis ance be ween he sequences om R and LP is 0.006, while he gene ic dis ance be ween Pa agi e spp. is 0.001–0.002, and he gene ic dis ance be ween Echinos oma spp. is 0.003–0.013 (Table 3). Animals 2024, 14, x FOR PEER REVIEW 10 o 17 Figu e 2. Phylog am esul ing om using Bayesian in e ence (BI) and maximum likelihood (ML) on he pa ial 28S DNA gene sequences o Echinos oma idae oo ed in Fasciola hepa ica. Pos e io p ob- abili y alues (BI) and boo s ap alues (ML) associa ed wi h he b anches a e shown as BI/ML; sup- po alues lowe han 0.70 (BI) and 50 (ML) a e no shown. The scale ba indica es he numbe o subs i u ions pe si e. Newly gene a ed sequences a e highligh ed in bold. 3.3. Si es o In ec ion and Hos Tissue Damage When P. canalicula a ac s as he i s in e media e hos , he ediae wi h de eloping ce ca iae inside a e ound mainly occupying he gonad and diges i e gland bu also he gills, in es ine, albumen gland, lung, kidney, man le bo de , and penis. Fi e o he eigh in ec ions s udied by his ology we e o mode a e o high in ensi y. In h ee cases, he in- ensi y o in ec ion was ligh , and in hese cases, al hough he ediae occupied he same o gans, hey we e conside ably less abundan . A double digenean in ec ion was ound only wice; spo ocys s belonging o ano he digenean species we e obse ed only occu- pying he diges i e gland, and he in ec ion in ensi y caused by he echinos oma id was ligh . In unin ec ed snails, he male gonad occupies he i s wo spi al u ns; in emales, he o a y is a h ead si ua ed wi hin he isce al mass a he inne apex o he spi al im (Figu e 3A,D). The pa ial o o al cas a ion o snails was obse ed in cases o mode a e and high in ec ion in ensi y, espec i ely. In he males, comple e lysis o gonadal issue and also hemocy ic in il a ion was obse ed due o he p esence o ediae (Figu e 3B). Fu he mo e, in one case, ediae we e ound in he penis shea h g oo e (Figu e 3C). In ligh o mode a e in ec ion in ensi y, ediae we e ound in he connec i e issue o he es is ubules; ne e heless, i was possible o obse e he p esence o some ubules. In emales wi h high in ec ion in ensi y, ediae in aded he connec i e issues o he o a ies, which almos comple ely a ophied; howe e , no hemocy ic in il a ion was obse ed (Figu e 3E). Mo eo e , in mode a e o high in ec ion in ensi y, ediae we e also ound in he sec e o y cells and duc s o he albumen gland (Figu e 3F). Figu e 2. Phylog am esul ing om using Bayesian in e ence (BI) and maximum likelihood (ML) on he pa ial 28S DNA gene sequences o Echinos oma idae oo ed in Fasciola hepa ica. Pos e io p obabili y alues (BI) and boo s ap alues (ML) associa ed wi h he b anches a e shown as BI/ML; suppo alues lowe han 0.70 (BI) and 50 (ML) a e no shown. The scale ba indica es he numbe o subs i u ions pe si e. Newly gene a ed sequences a e highligh ed in bold. 3.3. Si es o In ec ion and Hos Tissue Damage When P. canalicula a ac s as he i s in e media e hos , he ediae wi h de eloping ce ca iae inside a e ound mainly occupying he gonad and diges i e gland bu also he gills, in es ine, albumen gland, lung, kidney, man le bo de , and penis. Fi e o he eigh in ec ions s udied by his ology we e o mode a e o high in ensi y. In h ee cases, he in ensi y o in ec ion was ligh , and in hese cases, al hough he ediae occupied he same o gans, hey we e conside ably less abundan . A double digenean in ec ion was ound only wice; spo ocys s belonging o ano he digenean species we e obse ed only occupying he diges i e gland, and he in ec ion in ensi y caused by he echinos oma id was ligh . Animals 2024,14, 1191 9 o 16 Table 3. P-dis ance o 28S sequences o 19 species belonging o subclade 1 in he phylogene ic ee buil o his s udy. 1. Echino_ ed_Pc_R; 2. Echino_me a_Pc_R; 3. Echino_ ed_Pc_LP; 4. Echino_me a_Pc_LP; 5. Pa agi e sp. 1; 6. Pa agi e sp. 2; 7. Pa agi e bilobus; 8. Pa agi e ioscai; 9. Echinos oma nasinco ae; 10. Echinos oma miyagawai; 11. Echinos oma e olu um (sl); 12. Echinos oma i ol is ; 13. Echinos oma pa aensei; 14. Echinos oma pa aulum; 15. Echinos oma e olu um (s ); 16. Echinos oma bolschewense; 17. Echinos oma sp. IG; 18. A y echinos omum su a y ex; 19. Neoacan hopa yphium echina oide. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1. 2. 0.00000 3. 0.00600 0.00490 4. 0.00600 0.00490 0.00000 5. 0.01918 0.01838 0.01542 0.01501 6. 0.01799 0.01716 0.01423 0.01386 0.00115 7. 0.01679 0.01593 0.01305 0.01270 0.00231 0.00115 8. 0.01799 0.01716 0.01423 0.01386 0.00115 0.00000 0.00115 9. 0.01559 0.01593 0.01661 0.01617 0.02076 0.01961 0.01845 0.01961 10. 0.01799 0.01838 0.01898 0.01848 0.02307 0.02191 0.02076 0.02191 0.00461 11. 0.02638 0.02574 0.02728 0.02656 0.03114 0.02999 0.02884 0.02999 0.01269 0.01153 12. 0.02038 0.02083 0.02135 0.02194 0.02653 0.02537 0.02422 0.02537 0.00577 0.00807 0.01615 13. 0.01918 0.01961 0.02017 0.01963 0.02422 0.02307 0.02191 0.02307 0.00346 0.00577 0.01153 0.00461 14. 0.01687 0.01724 0.01788 0.01740 0.02202 0.02086 0.01970 0.02086 0.00348 0.00348 0.01159 0.00695 0.00463 15. 0.02558 0.02491 0.02651 0.02579 0.03044 0.02927 0.02810 0.02927 0.01171 0.01054 0.00468 0.01522 0.01288 0.01054 16. 0.02038 0.02083 0.02017 0.01963 0.02307 0.02191 0.02076 0.02191 0.01269 0.01038 0.02076 0.01384 0.01153 0.01159 0.01991 17. 0.02158 0.02206 0.02254 0.02194 0.02653 0.02537 0.02422 0.02537 0.01038 0.00923 0.01384 0.01384 0.01153 0.00927 0.01288 0.01499 18. 0.03234 0.03182 0.03081 0.02999 0.03114 0.02999 0.02884 0.02999 0.03345 0.03345 0.03922 0.03691 0.03460 0.03244 0.03864 0.03460 0.03460 19. 0.02278 0.02328 0.02135 0.02079 0.02540 0.02425 0.02309 0.02425 0.02425 0.02425 0.02771 0.02771 0.02309 0.02320 0.02931 0.02079 0.02656 0.03460 Animals 2024,14, 1191 16 o 16 37. Laidemi , M.R.; B an , S.V.; Mu uku, M.W.; Mkoji, G.M.; Loke , E.S. The di e se echinos omes om Eas A ica: Wi h a ocus on species ha use Biomphala ia and Bulinus as in e media e hos s. Ac a T op. 2019,193, 38–49. [C ossRe ] 38. Pin o, H.A.; De Melo, A.L. A checklis o ce ca iae (T ema oda: Digenea) in molluscs om B azil. Zoo axa 2013,3666, 449–475. [C ossRe ] 39. La e y, K.D. En i onmen al Pa asi ology: Wha can Pa asi es ell us abou Human Impac s on he En i onmen ? Pa asi ol. Today 1997,13, 251–255. [C ossRe ] 40. Whi ney, K.L.; Hechinge , R.F.; Ku is, A.M.; La e y, K.D. Endange ed ligh - oo ed clappe ail a ec s pa asi e communi y s uc u e in coas al we lands. Ecol. Appl. 2007,17, 1694–1702. [C ossRe ] 41. E chegoin, J.A.; Me lo, M.J.; Pa ie i, M. The ole o he in asi e polychae e Ficopoma us enigma icus (Fau el, 1923) (Se pulidae) as acili a o o pa asi e ansmission in Ma Chiqui a coas al lagoon (Buenos Ai es, A gen ina). Pa asi ology 2012,139, 1506–1512. [C ossRe ] 42. Digiani, M.C. Digeneans and ces odes pa asi ic in he whi e- aced ibis Plegadis chihi (A es: Th eskio ni hidae) om A gen ina. Folia Pa asi ol. 2000,47, 195–204. [C ossRe ] 43. Be ole o, A.; Na a o, J. A na i e bi d as a p eda o o he in asi e apple snail, a no el ice ield in ade in Eu ope. Aqua . Conse . Ma . F eshw. Ecosys . 2018,28, 1099–1104. [C ossRe ] 44. Lauckne , G. Diseases o mollusca: Bi al ia. In Diseases o Ma ine Animals; Biologische Ans al Helgoland: Hambu g, Ge many, 1983; Volume 2. 45. Choubisa, S.L.; Sheikh, Z.; Ja oli, V.J. His opa hological e ec s o la al ema odes on he diges i e gland o eshwa e snail species, Vi ipa a bengalensis and Lymnaea acumina a.J. Pa asi . Dis. 2012,36, 283–286. [C ossRe ] 46. Choubisa, S.L. His ological and his ochemical obse a ion on he diges i e gland o Melanoides ube cula us (Gas opoda) in ec ed wi h ce ain la al ema odes and ocus on hei mode o nu i ion. P oc. Indian Acad. Sci. 1988,97, 251–262. [C ossRe ] 47. C emon e, F.; Figue as, A.; Bu eson, E.M. A his opa hological su ey o some comme cially exploi ed bi al e molluscs in no he n Pa agonia, A gen ina. Aquacul u e 2005,249, 23–33. [C ossRe ] 48. Ra o, M.; Russel-Pin o, F.; Ba oso, C. Assessmen o digenean pa asi ism in Nassa ius e icula us (L.) Along he po uguese coas : E alua ion o possible impac s on ep oduc ion and imposex exp ession. J. Pa asi ol. 2009,95, 327–336. [C ossRe ] 49. Gama a-Luques, C.; Gi aud-Billoud, M.; Cas o-Vazquez, A. Rep oduc i e o ganogenesis in he apple snail Pomacea canalicula a (Lama ck, 1822), wi h e e ence o he e ec s o xenobio ics. J. Molluscan S ud. 2013,79, 147–162. [C ossRe ] 50. Wins ead, J.T.; Vole y, A.K.; G ego y, T.S. Pa asi ic and symbio ic auna in oys e s (C assos ea i ginica) collec ed om he Caloosaha chee Ri e and es ua y in Flo ida. J. Shell ish Res. 2004,23, 831–841. 51. La e y, K.D.; Ku is, A.M. Pa asi ic cas a ion: The e olu ion and ecology o body sna che s. T ends Pa asi ol. 2009,25, 564–572. [C ossRe ] [PubMed] 52. La uelle, F.M.; Daniel, P.; Roi man Vi ali, A. His ological analysis o ema odes in D eissena polymo pha: Thei loca ion, pa hogenic- i y, and dis inguishing mo phological cha ac e is ic. J. Pa asi ol. 2002,88, 856–863. [C ossRe ] [PubMed] 53. Demian, E.S. The espi a o y sys em and he mechanism o espi a ion in Ma isa co nua ie is (L.). A k. Fö Zool. 1965,17, 539–560. 54. San os, C.A.; Pen eado, C.H.; Mendes, E.G. The espi a o y esponses o an amphibious snail Pomacea linea a (Spix, 1827), o empe a u e and oxygen ension a ia ions. Comp. Biochem. Physiol. 1987,86, 409–415. [C ossRe ] 55. Rod iguez, C.; P ie o, G.I.; Vega, I.A.; Cas o-Vazquez, A. Assessmen o he kidney and lung as immune ba ie s and hema opoi- e ic si es in he in asi e apple snail Pomacea canalicula a.Pee J 2018,6, e5789. [C ossRe ] 56. Vazquez, N.; Glasino ich, N.; O iz, N.; Ges al, C.; Pon ones, J.; Gila doni, C.; C emon e, F. Hos -pa asi e ela ionship Oc opus ehuelchus—Agg ega a aldessensis in Pa agonian coas , sou hwes e n A lan ic Ocean. J. In e eb . Pa hol. 2023,201, 107993. [C ossRe ] 57. C ibb, T.H. Digenea (endopa asi ic lukes). In Ma ine Pa asi ology; Collingwood, K.R., Ed.; CSIRO Publishing (Commonweal h Scien i ic and Indus al Rsea ch O ganiza ion): Collingwood, VIC, Aus alia, 2005. 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 .