Unveiling the phylogeny of Contracaecum jorgei (Nematoda, Anisakidae) parasitizing Ardea cocoi (Aves, Ardeidae) in Argentina based on an integrative analysis

Unveiling the phylogeny of Contracaecum jorgei (Nematoda, Anisakidae) parasitizing Ardea cocoi (Aves, Ardeidae) in Argentina based on an integrative analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Unveiling the phylogeny of Contracaecum jorgei (Nematoda, Anisakidae) parasitizing Ardea cocoi (Aves, Ardeidae) in Argentina based on an integrative analysis Lucas Emiliano Garbin, Martín Miguel Montes, Nathalia Arredondo, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4450708/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Anisakidae nematodes of the Contracaecum genus are known for their wide host and geographic distribution. These parasites commonly infest aquatic organisms worldwide. The life cycles of Contracaecum species typically involve aquatic invertebrates and fish as intermediate and/or paratenic hosts, while piscivorous birds and mammals serve as definitive hosts. The aim of the present work is to identify the Contracaecum specimens parasitizing Ardea cocoi from the Magdalena city coast, Argentina, and to determine their phylogenetic relationships with its congeners based on both morphometric and phylogenetic analyses. One Cocoi Heron specimen was found dead at the Magdalena coast, Buenos Aires Province, and 53 nematodes were recovered from its esophagus and stomach. Some nematode specimens were cleared with lactophenol and studied using an optical microscope. Three males and females were examined in a scanning electron microscope. DNA extraction, PCR and sequencing of mtDNA cox2 , ITS1, ITS2, and SSrRNA genes were performed from three Contracaecum specimens. Both morphometric and phylogenetic analyses of the present studied specimens allowed us to recognize the species Contracaecum jorgei. In the constructed tree using SSrRNA sequences, a node aligns with C. microcephalum sequences. Conversely, ITS1 and ITS2 sequences respectively, establish a robust node positioning our sequence as the sister species to a node consisting of Contracaecum pyripapillatum , C. multipapillatum, C. gibsoni , and C. quadripapillatum . The mtDNA cox2 gene shows our sequences exhibiting concordance with C. jorgei . The p-distances calculated for the SSrRNA gene matrix displayed a distance of 14% from C. microcephalum and the present study specimens (PES) . For ITS1, the calculated distance is 16% from both C. pyripapillatum and C. multipapillatum . In the case of ITS2, the distances are 35% from C. multipapillatum, and 38% from C. pyripapillatum . Finally, the mtDNA cox2 gene displayed a distance of 1% observed for among our sequences and C. jorgei. According to the molecular analysis, PES clustered with the sister species C. microcephalum on the SSrRNA BI tree. Considering the BI analysis of ITS1-ITS2 genes, PES appear as a sister species to the node consisting of C. multipapillatum , C. pyripapillatum, C. quadripapillatum , and C. gibsoni . Finally, the topology of the mtDNA cox2 BI tree and the genetic distances between Contracaecum taxa supports the identification of the PES as C. jorgei . Contracaeum jorgei is closely related to C. multipapillatum . The record of C. jorgei parasitizing A. coccoi is the second report of an Anisakidae for this host species in Argentina, and also for any Ardeidae. Integrative molecular studies including morphological and molecular tools are important to know the real host and geographical distribution of parasite diversity and establish specific correspondences to determine phylogenetic relationships on the Contracaecum species. This work represents the second report of C. jorgei from Argentina based on morphological analysis conducted using optical and scanning electron microscopy. This study shed light on the limited information available regarding this conspicuous nematode and sets the stage for further investigations into its life cycles. Contracaecum Ardeidae SSrRNA gene ITS regions mtDNA cox2 gene Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Anisakidae nematodes, particularly those of the Contracaecum Railliet and Henry 1912, are known for their wide host and geographic distribution. These parasites commonly infest aquatic organisms worldwide (Rohde 2005). The life cycles of Contracaecum species typically involve aquatic invertebrates and fish as intermediate and/or paratenic hosts, while piscivorous birds and mammals serve as definitive hosts (Anderson 2000). Anisakidae nematodes are known to be causal agents of Anisakidosis disease. Third-stage Contracaecum spp. larvae have been reported in the stomach mucosa of humans causing a severe and painful condition after ingestion of raw or undercooked fish (Shamsi et al. 2019). In Argentina, only a few cases of Anisakidosis have been reported in Argentina so far (Degese et al. 2019; Menghi et al. 2020). Contracaecum larvae can only be identified to species level with the help of molecular markers. In all cases of Anisakidosis in humans, morphological identification of larvae has only been possible to genus level (Shamsi et al. 2019; Buchmann 2023; Caffara et al. 2023). Morphological identification of Contracaecum species can be challenging due to the presence of overlapping diagnostic characters. Traditional taxonomic approaches rely on morphological features such as cephalic, buccal, and pharyngeal structures, as well as cuticular details, male reproductive system characteristics (e.g. spicules), and the arrangement of papillae at the posterior end of males (Hartwich 1964; Fagerholm 1990; Mattiucci and Nascetti 2008). Recently, Garbin et al. (2024) proposed a differentiation of Contracaecum into three morphotypes based on the arrangement of papillae on the male tail. This classification scheme builds upon previous work by Fagerholm (1990) and provides a framework for distinguishing between species within the genus Contracaecum . The morphology of nematode specimens can be influenced by factors such as fixation methods and the duration of fixation, which may lead to deformation of the cuticle and potential misdiagnosis (Fagerholm 1990, Garbin 2009, Garbin et al. 2024). Additionally, taxonomists have encountered challenges in distinguishing between cryptic or sibling species, which appear morphologically similar but are genetically distinct (Mattiucci et al. 2008a, 2010; Shamsi et al. 2008; D’Amelio et al. 2012). To address these challenges, genetic-molecular studies have played a crucial role in clarify the classification of Contracaecum species (D’Amelio et al. 2007; Mattiucci et al. 2008b, Shamsi et al. 2019). Techniques such as allozyme markers and PCR of mitochondrial and ribosomal genes have been employed to identify species and elucidate the genetic relationships between larvae and adults (Kennedy and Harnett 2001; Mattiucci and Nascetti 2008; Mattiucci et al. 2003, 2010). Contracaecum jorgei Sardella, Mancini, Salinas, Simões & Luque, 2020, has been first described parasitizing the Neotropic Cormorant Nannopterum brasilianum (King 1828), and also reported in the fish Hoplias argentinensis Rosso, González-Castro, Bogan, Cardoso, Mabragaña, Delpiani, & Díaz de Astarloa, 2018, from a shallow Pampean lake in Córdoba, Argentina (Sardella et al. 2020). Several Contracaecum species have been documented parasitizing Ardeidae in South America (Table 1), with some records reported in Argentina (Garbin et al. 2023). Thus, Schuurmans-Stekhoven (1951) reported Contracaecum microcephalum (Rudolphi, 1809) Baylis, 1920, parasitizing the Great Egret Ardea (= Casmerodius ) alba Linnaeus, in the Tucumán province. Similarly, Boero and Led (1971) found C. microcephalum in the Cocoi Heron Ardea cocoi Linnaeus, and the Black-crowned Night Heron Nycticorax nycticorax Linnaeus, in the Buenos Aires province. Labriola and Suriano (1996) recorded Contracaecum philomultipapillatum Labriola & Suriano, 1996, infecting A. alba, the Snowy Egret Egretta thula (Molina), and the Western Cattle Egret Bubulcus ibis Linnaeus in the Buenos Aires Province. Navone et al. (2000) reported Contracaecum multipapillatum (Drasche, 1882) Lucker, 1941 parasitizing A. alba from Buenos Aires province and suggested that C. philomultipapillatum is a junior synonym of C. multipapillatum. The Cocoi Heron is a resident bird found in aquatic environments spanning from Colombia and Venezuela through most of South America to Chile and Argentina, with occasional sightings in Patagonia excluding the Andes cordillera. It is widely distributed and considered common throughout its range, typically inhabiting shorelines and shallow waters where it hunts for fish, amphibians, rodents, crustaceans, and insects (Ducommun and Beltzer 2010; Hayes et al. 2023). The aim of the present work is to identify the Contracaecum specimens parasitizing the Cocoi Heron A. cocoi from the Magdalena city, Buenos Aires province, Argentina, and to determine their phylogenetic relationships with its congeners based on both morphometric and phylogenetic analyses. Material and methods Sampling In September 2017, one Cocoi Heron specimen was found dead at the Magdalena city coast, Buenos Aires Province (34° 58′ 59.7″ S, 57° 44′ 9.05″ W). During necropsy, 53 nematode specimens were recovered from the oesophagus and stomach; some were fixed in formalin, and preserved in 70% ethanol for their morphological study. Other specimens were preserved in 96% ethanol for molecular and phylogenetic analysis. Morphological study Twenty nematode specimens − 10 males and 10 females- were cleared with lactophenol and studied using an optical microscope (OM) Olympus BX51. Three males and females were dehydrated in a graded ethanol series, and then dried using hexamethyldisilazane. Subsequently, they were mounted on stubs with carbon tape, coated with 40 nm of gold/palladium in a Thermo VG Scientific Polaron SC 7630, and examined in a Zeiss Supra 40 scanning electron microscope. Measurements of the parasite were recorded in millimeters (mm) -unless otherwise indicated- with mean values followed by the range in parentheses. Taxonomic identification was performed based on the diagnostic features of the family Anisakidae, following the classification criteria outlined by Fagerholm ( 1990 ) regarding the arrangement of papillae on the male tail, and by consulting relevant bibliography (Hartwich 1964 ; Garbin et al. 2019 , 2024 ). Based on the distribution pattern on the male papillae, three morphotypes were established (i.e. simple, intermediate and multiple) according to Garbin et al. ( 2024 ) based on Fagerholm ( 1990 ) (Fig. 1 ). Voucher specimens were deposited in the Helminthological Collection of the Museo de La Plata (MLP He xxxxxxx), Buenos Aires, Argentina. DNA extraction, amplification, and sequencing DNA extraction was performed using Wizard® Genomic DNA Purification Kit (Promega, USA) according to the manufacturer’s instructions. The mtDNA cox2 gene from three Contracaecum specimens was amplified according to the procedures as reported in Mattiucci et al. ( 2010 ) with the primers 211F (5’-TTT TCT AGT TAT ATA GAT TGR TTY AT-3’), and 210R (5’- CAC CAA CTC TTA AAA TTA TC-3’) from Nadler and Hudspeth (2000) spanning the mtDNA nucleotide position 10,639 − 11,248 as defined in Ascaris suum (Goeze, 1782) (GenBank X54253). Amplification was carried out in a volume of 25 µl containing 30 pmol of each primer, 2.5 mM MgCl2 (PB-L, Argentina), 1X PCR buffer (PB-L, Argentina), 0.2 mM each deoxynucleoside triphosphate (Promega, USA), 1 U Taq Pegasus DNA polymerase (PB-L, Argentina) and 1 µl of DNA. Amplification conditions included an initial step at 94°C for 3 min, followed by 34 cycles at 94°C for 30 sec, 46°C for 1 min, and 72°C for 1.5 min, then concluded by a step at 72°C for 10 min. The amplification of the ITS-1 region was carried out with the primers SS1 (5'-GTTTCCGTAGGTGAACCTGCG-3') and NC13R (5'-GCTGCGTTCTTCATCGAT-3') and the ITS-2 region with the primers SS2 (5'-TTGCAGACACATTGAGCACT-3') and NC2 (5'-TTAGTTTCTTTTCCTCCGCT − 3'), according to the procedure reported in Shamsi et al. ( 2008 , 2009 ). The PCR mixture (50 µl) contained 25 µl of Master Mix PCR 2X (PB-L, Argentina) 2.5 µl of each primer (10 µM), 18 µl of water, and 2 µl of extracted DNA. PCR conditions were: 94°C for 5 min, followed by 30 cycles at 94°C for 30 sec, at 55°C for 30 sec, at 72°C for 30 sec, and a final extension at 72°C for 5 min. The amplification of mitochondrial small subunit ribosomal gene SSrRNA was performed according to the procedures reported in D’Amelio et al. (2007) with the primers MH3 (5’-TTGTTCCAGAATAATCGGCTAGACTT-3’) and MH4.5 (5’TCTACTTTACTACAACTTACTCC-3’). The PCR mixture (50 µl) contained 25 µl of Master Mix PCR 2X (PB-L, Argentina) 2.5 µl of each primer (10 µM), 18 µl of water, and 2 µl of extracted DNA. PCR conditions included 10 min at 95°C, 35 cycles of 30 sec at 95°C, 30 sec at 55°C, 30 sec at 72°C, and a final extension step of 7 min at 72°C. The PCR was purified and sequenced by Macrogen (Seoul, South Korea) directly with the primers for amplification. The nucleotide sequences reported in the present study are available from the DDBJ/EMBL/GenBank databases under the accession numbers (xxxxxxxxx). Phylogenetic analysis Sequences were assembled using the Geneious program. The assembled sequences were checked for the presence of pseudogenes in the mtDNA cox2 gene by translating the amino acid sequences using the invertebrate mitochondrial genetic code within Geneious. Contracaecum spp. sequences were aligned using the online version of MAFFT 7 (Katoh and Standley 2013 ) with sequences of Anisakis spp. used as outgroups. The best partitioning scheme and substitution model for each DNA partition were chosen under the Bayesian Information Criterion (Schwarz 1978 ) using the “greedy” search strategy in Partition Finder v. 1.1.1 (Lanfear et al. 2012 ). Only the mtDNA cox2 fragment dataset was partitioned into first-, second-, and third-codon positions. The appropriate nucleotide substitution models implemented for each partition were HKY + I for the first and third codon position, and HKY + G for the second codon position. For the datasets of SSrRNA and ITS2 genes, the HKY + G was used, while K80 + G model was used for the ITS1 gene. Phylogenetic reconstruction was performed using Bayesian Inference (BI) through Mr. Bayes 3.2.3 (Ronquist et al. 2003). Phylogenetic trees were constructed using two parallel analyses of Metropolis-Coupled Markov Chain Monte Carlo (MCMC) for 20 million generations each, to estimate the posterior probability (PP) distribution. Topologies were sampled every 1,000 generations and the average standard deviation of split frequencies was observed to be less than 0.01 at the end of the run, as suggested by Ronquist et al. ( 2012 ). The robustness of the clades was assessed using Bayesian posterior probability (PP), where PP > 0.90 was considered strongly supported. A majority consensus tree with branch lengths was reconstructed for each run after discarding the first 25% trees sampled as “burn-in”. Additionally, uncorrected p-distance was calculated for each gen using MEGA X (Kumar et al. 2018 ) with the bootstrap method (1000 replicates) and nucleotide substitution (transition + transversions). A uniform rate was applied, and gaps/missing data were considered as complete deletion. Results Both morphometric and phylogenetic analyses of the present studied specimens allowed us to recognize the species: Contracaecum jorgei Sardella, Mancini, Salinas, Simões & Luque 2020 General morphology: (based on 10 males and 10 females): Body entirely transversally striated (Fig. 2a-f). Non-conspicuous cephalic collar, V-shaped lateral region without striations (Figs. a, b). Three smooth interlabia (Fig. 2a, b). Lips barely longer than interlabia with just longer with non-visible notches (Fig. 2a, b). Lips with two non-conspicuous and lobed auricles, no apparent tips (Fig. 2a, b). Notorious lip papillae, two on the dorsal lip, and one on each ventrolateral lip (Fig. 2a, b). Male : Simple postcloacal tail arrangement: two paracloacal papillae pairs, two subventral papillae pairs, two sublateral papillae pairs, and a phasmid pair (Fig. 2c, d). No apparent tail distal constriction (Fig. 2c, d). Short spicules approximately one-fifth the body length, and short spicule free-distal end (Fig. 2e, Table 2). Female : see Fig. 2f, Table 2. Taxonomic summary Type host: Nannopterum (= Phalacrocorax ) brasilianum (King 1828) (Pelecaniformes: Phalacrocoracidae). Type locality: Pampean shallow lakes, Unión Department, Córdoba province, Argentina (33°25′S, 62°54′W). New host: Ardea cocoi Linnaeus, 1766 (Pelecaniformes: Ardeidae). New locality: Magdalena city coast, Buenos Aires Province (34°58′59.7″S, 57°44′ 9.05″W). Infection site: stomach. Molecular characterization and phylogenetic analysis The obtained sequences include threes of mtDNAcox2, one SSrRNA, one ITS1 and one ITS2 with lengths ranging from 382 to 592 bp. The constructed matrix consist of 48 terminals and 618 bp for mtDNA cox2, 12 terminals and 460 bp for SSrRNA, 36 terminals and 383 bp for ITS1, and 34 terminals and 201 bp for ITS2. The phylogenetic trees constructed using these sequences reveal variable associations with species sequences archived in GenBank. In the constructed tree using SSrRNA sequences, a weakly supported node (PP = 0.79) aligns with C. microcephalum sequences (Fig. 3). Conversely, Figs. 4 and 5, based on ITS1 and ITS2 sequences respectively, establish a robust node (PP = 0.99) positioning our sequence as the sister species to a node consisting of Contracaecum pyripapillatum Shamsi, Gasser & Beveridge, 2008, C. multipapillatum, Contracaecum gibsoni Mattiucci , Paoletti, Consuegra-Solorzano & Nascetti, 2010, and Contracaecum quadripapillatum Saad, Younis & Rabei, 2018. In Fig. 6, the mtDNA cox2 gene shows our sequences exhibiting concordance with C. jorgei . A large clade of Contracaecum species with simple papillae arrangement on the male tail (Fig. 1) is observed at the top of the tree (Fig. 6). The p-distances calculated for the SSrRNA gene matrix displayed a distance of 14% from C. microcephalum and the specimens here studied (Table 3a) . For ITS1, the calculated distance is 16% from both C. pyripapillatum and C. multipapillatum (Table 3b). In the case of ITS2, the distances are 35% from C. multipapillatum and 38% from C. pyripapillatum (Table 3c). Finally, the mtDNA cox2 gene displayed a distance of 1% observed for among our sequences and C. jorgei from the original description (Sardella el at. 2020) (Table 3d). Remarks According to the morphometric analysis, the present study specimens (PES) displayed a morphological concordance with C. jorgei parasitizing N. brasilianum from the Córdoba province, Argentina (Sardella et al. 2020). However, a few slight morphometric differences can be observed between PES and those of C. jorgei from the original description. In this sense, PES are barely longer and thicker, and the spicules are slightly longer, but they do not constitute significant differences (Table 2). If we compare the SEM photos of the original description with the male of PES, we can observe a very noticeable distal constriction on the male tail. However, this might be due to the male individual on the original description is more mature adopting this pattern of striations. On the other hand, both sublateral papillae joining with the phasmid on the male tail are arranged in a straight line while a triangular arrangement can be observed in the PES. This arrangement may be variable and does not represent a significant difference as postulated by other authors (Fagerholm 1990; Mattiucci and Nascetti 2008). If we take into account other Contracaecum spp. parasitizing Ardeidae in Argentina, PES differ significantly from C. multipapillatum parasitizing A. alba they are smaller in length and thickness although their spicules are three times longer (Navone et al. 2000). This is this is reflected on the ratio total body length / spicule length (BL/EL= 24.11 vs. 5.24 PES) (Table 2). Something similar occurs with C. microcephalum parasitizing N. nicticorax , whose size is larger but its spicules are shorter reflected on the body length / spicule length ratio (BL/EL= 9.25 vs. 5.24 PES) (Boero and Led 1971). In addition, PES show a higher number of precloacal papillae (24-31 vs. 10 pairs) (Table 2). Although, the description of C. microcephalum by Schuurmans-Stekhoven (1951) parasitizing A. alba is brief, PES appear to be smaller in length and thinner, and its spicule free distal end is much longer. Considering the Contracaecum species parasitizing other birds from South America, PES are smaller in size than C. australe parasitizing the Neotropic cormorant –type host of C. jorgei - of Chile and Argentina. In addition, spicules of PES are much shorter than those of C. australe (Table 2). Present studied specimens’ auricle tips are less notorious and auricle notches are absent (Fig. 2) (Garbin et al. 2011; Biolé et al. 2012). Regarding Contracaecum species parasitizing other hosts from different parts of the world, PES are significantly smaller in length and thinner, and thus the internal digestive organs also show smaller dimensions than C. multipapillatum parasitic in the Australian pelican Pelecanus conspicillatus Temminck from Australia (Shamsi et al. 2008). Besides, PES own fewer precloacal papillae and possess spicules three times larger than those of C. multipapillatum . In addition, the papillae arrangement on the male tail is simple in PES, whereas in C. multipapillatum it is intermediate (Table 2, Fig. 1). Present study specimens show simple papillae arrangement on the male tail with respect to C. pyripapillatum parasitizing P. conspicillatus from Australia (Shamsi et al. 2008), and the spicules are twice in length (Table 2). Discussion Contracaecum jorgei was first described parasitizing N. brasilianum and Hoplias argentinensis (Sardella et al. 2020 ), a frequent item prey for A. cocoi (Ducommun and Beltzer 2010). Therefore, this fish species might facilitate the entry of C. jorgei third-stage larvae (L3) acting as an intermediate/paratenic host. Present report suggests an intermediate host-specificity of this nematode species parasitizing two different Pelecaniformes families. Montes (pers. obs.) found otoliths of several fish species in the Cocoi Heron from the coast of Magdalena, Río de la Plata, Buenos Aires Province. Therefore, this could indicate that there are more than one paratenic/intermediate host for C. jorgei L3 larvae. According to the molecular analysis conducted in this study, PES clustered with the sister species C. microcephalum on the SSrRNA BI tree (Fig. 3 ). As mentioned above, both species are morphologically similar and parasitize Ardeidae not only in South America but also in other parts from the world. However, the phylogenetic analysis results are restricted due the scarcity of sequences available in Genbank resulting in limited conclusions. Considering the BI analysis of ITS1-ITS2 genes, we can observe the topologies differ slightly each other (Figs. 4 , 5 ). Present study specimens appear as a sister species to the node consisting of C. multipapillatum , C. pyripapillatum, C. quadripapillatum , and C. gibsoni . As mentioned before, C. jorgei and C. multipapillatum would parasitize Ardeidae in Argentina (Navone et al. 2000 ; Sardella et al. 2020 )d pyripapillatum in Australia (Shamsi et al. 2019). But, C. quadripapillatum , and C. gibsoni parasitize Pelecanidae in Israel and Greece, respectively (Figs. 4 , 5 ). Therefore, this similarity should be explained from a parasite-host physiology point of view taking into account that both families belong to the Pelecaniformes order. Finally, the topology of the mtDNA cox2 BI tree and the genetic distances between Contracaecum taxa supports the identification of the PES as C. jorgei (Fig. 6 , Table 3 d). Contracaeum jorgei is closely related to C. multipapillatum , as shown in the BI analysis. However, despite their genetic similarity, the two species can be easily distinguished by their morphology. For instance, the arrangement of tail papillae in males varies significantly between the two species. While C. jorgei exhibits a simple pattern of postcloacal papillae (simple morphotype), C. multipapillatum is characterized by having a greater number of pre- and postcloacal papillae (intermediate morphotype), as shown in Fig. 1 (Navone et al. 2000 ; Garbin et al. 2024 ). The sequence MH044685 (unpublished data) identified as C. multipapillatum in GenBank groups with C. jorgei in the same branch. According to the information, this sequence belongs to a Contracaecum larvae specimen found parasitizing a catfish ( Rhamdia sp.) in Costa Rica and Guatemala (locality not specified). It is not correct to determine species with larval specimens as these do not develop secondary characters such as spicules and pre- and postcloacal papillae. Therefore, we believe that this sequence misidentified as C. multipapillatum belongs to C. jorgei . The genetic proximity between C. jorgei and C. multipapillatum can be explained from a parasite-host physiological point of view. This is because both Contracaecum species parasitize members of the Ardeidae family, specifically A. cocoi and A. alba . These birds have similar feeding habits and share a geographical distribution. The same can be observed for C. microcephalum , which is also a parasite of Ardeidae in Argentina (Table 2 ). The tree reflects these similarities, as the species that closely group with C. jorgei are those that use Ardeidae as hosts. The record of C. jorgei parasitizing A. coccoi is the second report of an Anisakidae for this host species in Argentina, and also for any Ardeidae. Integrative molecular studies including morphological and molecular tools are important to know the real host and geographical distribution of parasite diversity and establish specific correspondences to determine phylogenetic relationships on the Contracaecum species. In conclusion, our work represents the second report of C. jorgei from Argentina. Based on morphological analysis conducted using optical and scanning electron microscopes, we identified the species approximately 600 km away from its type collection site. Additionally, we correct the identification of the sequence deposited in Genbank MH044685 as C. jorgei . And therefore, the range of the species is extended to Central America. We have included new sequences of mtDNA cox2 and provided the firsts sequences of SSrRNA, ITS1 and ITS2 on C. jorgei . This study shed light on the limited information available regarding this conspicuous nematode and sets the stage for further investigations into its life cycles. We hope that new records, particularly those involving intermediate and definitive hosts, will contribute to elucidating the distribution of these parasites in the Americas, and potentially lead to the discovery of new species. Declarations Acknowledgments. We are grateful to the CEPAVE for providing laboratory space for processing and studying the specimens, and the CONICET and FONCYT for the grants assigned to researchers. Authors' contributions Lucas E. Garbin: Conceived and designed research. Wrote the main manuscript text, nematode specimen collection, morphometric and taxonomical nematode studies, phylogenetic analysis, photographs to scanning electron microscopy, preparation of figures 1, 2, and tables 1 and 2. Martín M. Montes: Conceived and designed research. Wrote the main manuscript text, bird digestive tract prospection, nematode specimen collection, phylogenetic analysis, preparation of figures 3, 4, 5 and 6, tables 3a-d. N. Arredondo: Made morphometric and taxonomical nematode studies, photographs to scanning electron microscopy, phylogenetic analysis. J. Barneche: Collected the Red-legged cormorant samples, bird digestive tract prospection. M. Ibáñez: DNA extractions and PCR assays. M. Moncada: DNA extractions and PCR assays. Julia I. Diaz: Morphometric and taxonomical nematode studies, images and figure preparation. Contributed to conceptualization and provide financial support. All authors reed and approved the final manuscript. Funding information This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CCT-CONICET-La Plata) (MMM., Grant number: 11220200101713CO); and Fondo para la Investigación Científica y Tecnológica (FONCyT) (M.M.M., Grant number: PICT-2020-SERIEA-01531; N.J.A. Grant number: PICT-2020- SERIEA-00660). Data availability statement The SEM-photographed Contracaecum jorgei specimens (3 males and 3 females) from Ardea cocoi are stored at the Servicio Nacional de Microscopía (SNM), Facultad de Ciencias Médicas, Universidad de Buenos Aires. All paratypes of Contracaecum jorgei mounted on lactophenol are preserved in 70% alcohol at the Helminthological Collection of CEPAVE and will be deposited in the Helminthological Collection of the La Plata Museum once the manuscript is accepted. The nucleotide sequences of both Contracaecum species reported in the present study are available from the DDBJ/EMBL/GenBank databases under the accession numbers (xxxxx). Ethical approval The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals. Consent to participate All the authors give their consent to participate in this work. Consent for publication All the authors give their consent for publication of this work. Competing Interests The authors declare that they have no conflicts of interest. 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Vidal-Martínez VM, Osorio-Sarabia D, Overstreet RM (1994) Experimental infection of Contracaecum multipapillatum (Nematoda: Anisakinae) from Mexico in the domestic cat. J Parasitol 80:576–579. https://doi.org/10.2307/3283194 Von Drasche R (1882) Helmlinthologische Notizen. Abhandlungen der K. K. Zool.-Botan. Gesellschaft Wien 32:139–142. Tables Table 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Table2.docx Table3a.docx Table3b.docx Table3c.docx Table3d.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4450708","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":306648159,"identity":"ce54dc01-acc2-43aa-8fc5-3166d8491118","order_by":0,"name":"Lucas Emiliano Garbin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYBACPiBmZjBgkEMIHSCghQ2qxZhULQwMiQ3Ea+Ff/OxzQcGd9P723oOfeSoY5PhuJDB+rsCnReKZ8ewZBs9yZ5w5lyzNc4bBWPJGArPkGbxaDhgz8xgczt0gkWMgObONIXHDjQQGyQa8Wo5/BmlJN5B/Y/xz5j+GeqAW5p94tfD3gG1JMJDgMZP42MCQYHAjgY2ALTzFzDMMDhvOOJOXZvHhmIThzDMP2yzxaeHnP76ZueDPYXn+9rOHbyTU2MjzHU8+fBOfFgaJBBiLB8wFYka8GoDWHEDRMgpGwSgYBaMAEwAAveFKwG2BuHIAAAAASUVORK5CYII=","orcid":"","institution":"Centro de Estudios Parasitológicos y de Vectores","correspondingAuthor":true,"prefix":"","firstName":"Lucas","middleName":"Emiliano","lastName":"Garbin","suffix":""},{"id":306648160,"identity":"5fecc82d-1307-40f3-a701-fecc2fb8700d","order_by":1,"name":"Martín Miguel Montes","email":"","orcid":"","institution":"Centro de Estudios Parasitológicos y de Vectores","correspondingAuthor":false,"prefix":"","firstName":"Martín","middleName":"Miguel","lastName":"Montes","suffix":""},{"id":306648161,"identity":"8744bb1c-43c0-4436-b455-aac84050dd5e","order_by":2,"name":"Nathalia Arredondo","email":"","orcid":"","institution":"Laboratorio de Sistemática y Biología de Parásitos de Organismos Acuáticos, Instituto de Biodiversidad y Biología Experimental y Aplicada","correspondingAuthor":false,"prefix":"","firstName":"Nathalia","middleName":"","lastName":"Arredondo","suffix":""},{"id":306648162,"identity":"d6b0a2d6-7d48-4ce6-807c-86302136368e","order_by":3,"name":"Jorge Barneche","email":"","orcid":"","institution":"Centro de Estudios Parasitológicos y de Vectores","correspondingAuthor":false,"prefix":"","firstName":"Jorge","middleName":"","lastName":"Barneche","suffix":""},{"id":306648163,"identity":"f4ec0a86-1149-4032-a655-6e900b7c9f30","order_by":4,"name":"Marina Ibáñez","email":"","orcid":"","institution":"Centro de Estudios Parasitológicos y de Vectores","correspondingAuthor":false,"prefix":"","firstName":"Marina","middleName":"","lastName":"Ibáñez","suffix":""},{"id":306648164,"identity":"fb7f4a51-7b9f-4f5a-ade9-7ff130022cff","order_by":5,"name":"Melisa Moncada","email":"","orcid":"","institution":"Centro de Estudios Parasitológicos y de Vectores","correspondingAuthor":false,"prefix":"","firstName":"Melisa","middleName":"","lastName":"Moncada","suffix":""},{"id":306648165,"identity":"7d051e72-4196-4c7c-9c6d-3ff82ffe4b5c","order_by":6,"name":"Julia Inés Diaz","email":"","orcid":"","institution":"Centro de Estudios Parasitológicos y de Vectores","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"Inés","lastName":"Diaz","suffix":""}],"badges":[],"createdAt":"2024-05-20 18:40:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4450708/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4450708/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57942784,"identity":"dc35f291-ebf7-406a-ab1b-67e34c1cc719","added_by":"auto","created_at":"2024-06-07 19:02:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":399462,"visible":true,"origin":"","legend":"\u003cp\u003eClassification criterion of the \u003cem\u003eContracaecum\u003c/em\u003e species into morphotypes according to the papillae arrangement on the mail tail based on Fagerholm (1990). Simple (A), composed of the following papillae: 2 sublateral pairs, 2 subventral pairs, 2 double paracloacals, one pair of phasmids (bold), and two single preanal rows. Intermediate (B), composed of 2 sublateral pairs, 2 subventral pairs, 2 double paracloacals, 4 adacloacal pairs, a pair of phasmids (bold), and two preanal single rows. Multiple (C), composed of one sublateral pair, 3 subventral pairs, 2 double paracloacals, 5 adacloacal pairs, a pair of phasmids (bold), and two double or triple preanal rows.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4450708/v1/3c1b30767937f884b607b273.jpg"},{"id":57942918,"identity":"9ecfa1df-0dcb-45a4-bb8b-f3d628b22a3d","added_by":"auto","created_at":"2024-06-07 19:02:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":23531831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eContracaecum jorgei\u003c/em\u003eparasitizing \u003cem\u003eArdea cocoi\u003c/em\u003e from the Magdalena coast, Buenos Aires province, Argentina. (a) Anterior end, lateral view; (b) Anterior end, detail of lateroventral lip, interlabia, cephalic labial papilla, lip auricles, amphid, lip auricle tip (arrow); (c) Male posterior end, precloacal papillae, paracloacal papillae (pcp), subventral postcloacal papillae (svp), sublateral postcloacal papillae (slp), phasmid (p); (d) Male posterior end, lateral view: precloacal papillae, paracloacal papillae (pcp), subventral postcloacal papillae (svp), sublateral postcloacal papillae (slp), phasmid (p); (e) Spicule free-distal end, lateral view; (f) Female posterior end. Scale bars expressed in micrometers.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4450708/v1/a17deb06879d524e1b29ccc5.jpg"},{"id":57942762,"identity":"024af466-5751-4546-8722-db5452093b41","added_by":"auto","created_at":"2024-06-07 19:01:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":767693,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic trees constructed by and Bayesian Inference (right) methods based on 11 sequences of SSrRNA mitochondrial gene under the substitution model HKY + G. Sequences are identified by taxon name, country, and GenBank accession number.\u003c/p\u003e","description":"","filename":"Fig31.png","url":"https://assets-eu.researchsquare.com/files/rs-4450708/v1/11bbd57481f2b6a62810a286.png"},{"id":57942924,"identity":"ec6ffada-6eb2-4417-91c1-c6d29f7ae0b8","added_by":"auto","created_at":"2024-06-07 19:02:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1787569,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic trees constructed by Bayesian Inference methods based on 15 sequences of concatenated ITS1 markers under the substitution model HKY + G. Sequences are identified by taxon name, country, and GenBank accession number.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4450708/v1/d3f99ca2e7737576115017b4.png"},{"id":57942921,"identity":"962338a7-e950-4fc2-ab0c-a590ef859cb0","added_by":"auto","created_at":"2024-06-07 19:02:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1797877,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic trees constructed by Bayesian Inference methods based on 15 sequences of concatenated ITS1 markers under the substitution model HKY + G. Sequences are identified by taxon name, country, and GenBank accession number.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4450708/v1/b5592b38afd9ac5db85c78c0.png"},{"id":57942782,"identity":"76001ff5-8688-4ded-b8ad-e0a3e44602e5","added_by":"auto","created_at":"2024-06-07 19:02:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2284407,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic trees constructed by Bayesian Inference (b) methods based on 33 sequences of a partial region of mtDNA \u003cem\u003ecox2\u003c/em\u003e gene under the substitution model TIM + F + I. Sequences are identified by taxon name, country, and GenBank accession number.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4450708/v1/1740b4ad05b6416d692e7a16.png"},{"id":58923349,"identity":"fb83adb6-aaf7-4b5e-b003-e5d7475218b7","added_by":"auto","created_at":"2024-06-24 07:41:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":37919403,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4450708/v1/65e31009-e692-45d2-8c60-d839b7faafb4.pdf"},{"id":57942765,"identity":"0e3999a1-b02a-4608-9982-8213758ca7a1","added_by":"auto","created_at":"2024-06-07 19:01:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25198,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4450708/v1/a14e4f22622828d92b34ced2.docx"},{"id":57942780,"identity":"07fbc847-f268-42c6-b32e-9156a3459bc8","added_by":"auto","created_at":"2024-06-07 19:02:05","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":26360,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4450708/v1/dd0b74ea7884cb824b916a86.docx"},{"id":57942770,"identity":"27ae99fc-bb14-4367-a46e-2513aa82462c","added_by":"auto","created_at":"2024-06-07 19:02:02","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14052,"visible":true,"origin":"","legend":"","description":"","filename":"Table3a.docx","url":"https://assets-eu.researchsquare.com/files/rs-4450708/v1/f03084bcf0c0190a77eef6f1.docx"},{"id":57942774,"identity":"7bdbd375-4510-4915-a70f-0eba145c3ce6","added_by":"auto","created_at":"2024-06-07 19:02:04","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":15410,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Table3b.docx","url":"https://assets-eu.researchsquare.com/files/rs-4450708/v1/93a7288eff5978062a82ff7b.docx"},{"id":57942758,"identity":"7859268a-f5e9-407f-864a-47f21f85a6df","added_by":"auto","created_at":"2024-06-07 19:01:56","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15277,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Table3c.docx","url":"https://assets-eu.researchsquare.com/files/rs-4450708/v1/d6687474402ec89a2f8fdce0.docx"},{"id":57942927,"identity":"6aef6734-f1c0-4899-8811-97fd198d3881","added_by":"auto","created_at":"2024-06-07 19:02:13","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":16590,"visible":true,"origin":"","legend":"","description":"","filename":"Table3d.docx","url":"https://assets-eu.researchsquare.com/files/rs-4450708/v1/d872d01abf40d5398fe2555e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Unveiling the phylogeny of Contracaecum jorgei (Nematoda, Anisakidae) parasitizing Ardea cocoi (Aves, Ardeidae) in Argentina based on an integrative analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnisakidae nematodes, particularly those of the \u003cem\u003eContracaecum\u003c/em\u003e Railliet and Henry 1912, are known for their wide host and geographic distribution. These parasites commonly infest aquatic organisms worldwide (Rohde 2005). The life cycles of \u003cem\u003eContracaecum\u003c/em\u003e species typically involve aquatic invertebrates and fish as intermediate and/or paratenic hosts, while piscivorous birds and mammals serve as definitive hosts (Anderson 2000).\u003c/p\u003e\n\u003cp\u003eAnisakidae nematodes are known to be causal agents of Anisakidosis disease. Third-stage \u003cem\u003eContracaecum\u003c/em\u003e spp. larvae have been reported in the stomach mucosa of humans causing a severe and painful condition after ingestion of raw or undercooked fish (Shamsi et al. 2019). In Argentina, only a few cases of Anisakidosis have been reported in Argentina so far (Degese et al. 2019; Menghi et al. 2020). \u003cem\u003eContracaecum\u003c/em\u003e larvae can only be identified to species level with the help of molecular markers. In all cases of Anisakidosis in humans, morphological identification of larvae has only been possible to genus level (Shamsi et al. 2019; Buchmann 2023; Caffara et al. 2023).\u003c/p\u003e\n\u003cp\u003eMorphological identification of \u003cem\u003eContracaecum\u003c/em\u003e species can be challenging due to the presence of overlapping diagnostic characters. Traditional taxonomic approaches rely on morphological features such as cephalic, buccal, and pharyngeal structures, as well as cuticular details, male reproductive system characteristics (e.g. spicules), and the arrangement of papillae at the posterior end of males (Hartwich 1964; Fagerholm 1990; Mattiucci and Nascetti 2008). Recently, Garbin et al. (2024) proposed a differentiation of \u003cem\u003eContracaecum\u003c/em\u003e into three morphotypes based on the arrangement of papillae on the male tail. This classification scheme builds upon previous work by Fagerholm (1990) and provides a framework for distinguishing between species within the genus \u003cem\u003eContracaecum\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe morphology of nematode specimens can be influenced by factors such as fixation methods and the duration of fixation, which may lead to deformation of the cuticle and potential misdiagnosis (Fagerholm 1990, Garbin 2009, Garbin et al. 2024). Additionally, taxonomists have encountered challenges in distinguishing between cryptic or sibling species, which appear morphologically similar but are genetically distinct (Mattiucci et al. 2008a, 2010; Shamsi et al. 2008; D\u0026rsquo;Amelio et al. 2012). To address these challenges, genetic-molecular studies have played a crucial role in clarify the classification of \u003cem\u003eContracaecum\u003c/em\u003e species (D\u0026rsquo;Amelio et al. 2007; Mattiucci et al. 2008b, Shamsi et al. 2019). Techniques such as allozyme markers and PCR of mitochondrial and ribosomal genes have been employed to identify species and elucidate the genetic relationships between larvae and adults (Kennedy and Harnett 2001; Mattiucci and Nascetti 2008; Mattiucci et al. 2003, 2010).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eContracaecum jorgei\u003c/em\u003e Sardella, Mancini, Salinas, Sim\u0026otilde;es \u0026amp; Luque, 2020, has been first described parasitizing the Neotropic Cormorant \u003cem\u003eNannopterum brasilianum\u003c/em\u003e (King 1828), and also reported in the fish \u003cem\u003eHoplias argentinensis\u003c/em\u003e Rosso, Gonz\u0026aacute;lez-Castro, Bogan, Cardoso, Mabraga\u0026ntilde;a, Delpiani, \u0026amp; D\u0026iacute;az de Astarloa, 2018,\u003cem\u003e\u0026nbsp;\u003c/em\u003efrom a shallow Pampean lake\u003cem\u003e\u0026nbsp;\u003c/em\u003ein C\u0026oacute;rdoba, Argentina (Sardella et al. 2020).\u003c/p\u003e\n\u003cp\u003eSeveral \u003cem\u003eContracaecum\u003c/em\u003e species have been documented parasitizing Ardeidae in South America (Table 1), with some records reported in Argentina (Garbin et al. 2023). Thus, Schuurmans-Stekhoven (1951) reported \u003cem\u003eContracaecum microcephalum\u003c/em\u003e (Rudolphi, 1809) Baylis, 1920, parasitizing the Great Egret\u0026nbsp;\u003cem\u003eArdea\u003c/em\u003e (=\u003cem\u003eCasmerodius\u003c/em\u003e) \u003cem\u003ealba\u003c/em\u003e Linnaeus, in the Tucum\u0026aacute;n province. Similarly, Boero and Led (1971) found \u003cem\u003eC.\u003c/em\u003e \u003cem\u003emicrocephalum\u0026nbsp;\u003c/em\u003ein the Cocoi Heron \u003cem\u003eArdea cocoi\u0026nbsp;\u003c/em\u003eLinnaeus,\u003cem\u003e\u0026nbsp;\u003c/em\u003eand the Black-crowned Night Heron\u003cem\u003e\u0026nbsp;Nycticorax nycticorax\u0026nbsp;\u003c/em\u003eLinnaeus, in the Buenos Aires province. Labriola and Suriano (1996) recorded \u003cem\u003eContracaecum philomultipapillatum\u003c/em\u003e Labriola \u0026amp; Suriano, 1996, infecting\u003cem\u003e\u0026nbsp;A. alba,\u0026nbsp;\u003c/em\u003ethe Snowy Egret\u003cem\u003e\u0026nbsp;Egretta thula\u0026nbsp;\u003c/em\u003e(Molina), and the Western Cattle Egret\u003cem\u003e\u0026nbsp;Bubulcus ibis\u003c/em\u003e Linnaeus\u003cem\u003e\u0026nbsp;\u003c/em\u003ein the Buenos Aires Province. Navone et al. (2000) reported \u003cem\u003eContracaecum multipapillatum\u003c/em\u003e (Drasche, 1882) Lucker, 1941 parasitizing \u003cem\u003eA. alba\u003c/em\u003e from Buenos Aires province and suggested that \u003cem\u003eC. philomultipapillatum\u003c/em\u003e is a junior synonym of \u003cem\u003eC. multipapillatum.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe Cocoi Heron is a resident bird found in aquatic environments spanning from Colombia and Venezuela through most of South America to Chile and Argentina, with occasional sightings in Patagonia excluding the Andes cordillera. It is widely distributed and considered common throughout its range, typically inhabiting shorelines and shallow waters where it hunts for\u0026nbsp;fish, amphibians, rodents, crustaceans, and insects\u0026nbsp;(Ducommun and Beltzer 2010; Hayes et al. 2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe aim of the present work is to identify the \u003cem\u003eContracaecum\u003c/em\u003e specimens parasitizing the Cocoi Heron \u003cem\u003eA. cocoi\u003c/em\u003e from the Magdalena city, Buenos Aires province, Argentina, and to determine their phylogenetic relationships with its congeners based on both morphometric and phylogenetic analyses.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSampling\u003c/h2\u003e \u003cp\u003eIn September 2017, one Cocoi Heron specimen was found dead at the Magdalena city coast, Buenos Aires Province (34\u0026deg; 58\u0026prime; 59.7\u0026Prime; S, 57\u0026deg; 44\u0026prime; 9.05\u0026Prime; W). During necropsy, 53 nematode specimens were recovered from the oesophagus and stomach; some were fixed in formalin, and preserved in 70% ethanol for their morphological study. Other specimens were preserved in 96% ethanol for molecular and phylogenetic analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMorphological study\u003c/h2\u003e \u003cp\u003eTwenty nematode specimens \u0026minus;\u0026thinsp;10 males and 10 females- were cleared with lactophenol and studied using an optical microscope (OM) Olympus BX51. Three males and females were dehydrated in a graded ethanol series, and then dried using hexamethyldisilazane. Subsequently, they were mounted on stubs with carbon tape, coated with 40 nm of gold/palladium in a Thermo VG Scientific Polaron SC 7630, and examined in a Zeiss Supra 40 scanning electron microscope. Measurements of the parasite were recorded in millimeters (mm) -unless otherwise indicated- with mean values followed by the range in parentheses. Taxonomic identification was performed based on the diagnostic features of the family Anisakidae, following the classification criteria outlined by Fagerholm (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) regarding the arrangement of papillae on the male tail, and by consulting relevant bibliography (Hartwich \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1964\u003c/span\u003e; Garbin et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on the distribution pattern on the male papillae, three morphotypes were established (i.e. simple, intermediate and multiple) according to Garbin et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) based on Fagerholm (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Voucher specimens were deposited in the Helminthological Collection of the Museo de La Plata (MLP He xxxxxxx), Buenos Aires, Argentina.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction, amplification, and sequencing\u003c/h2\u003e \u003cp\u003eDNA extraction was performed using Wizard\u0026reg; Genomic DNA Purification Kit (Promega, USA) according to the manufacturer\u0026rsquo;s instructions. The mtDNA \u003cem\u003ecox2\u003c/em\u003e gene from three \u003cem\u003eContracaecum\u003c/em\u003e specimens was amplified according to the procedures as reported in Mattiucci et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) with the primers 211F (5\u0026rsquo;-TTT TCT AGT TAT ATA GAT TGR TTY AT-3\u0026rsquo;), and 210R (5\u0026rsquo;- CAC CAA CTC TTA AAA TTA TC-3\u0026rsquo;) from Nadler and Hudspeth (2000) spanning the mtDNA nucleotide position 10,639\u0026thinsp;\u0026minus;\u0026thinsp;11,248 as defined in \u003cem\u003eAscaris suum\u003c/em\u003e (Goeze, 1782) (GenBank X54253). Amplification was carried out in a volume of 25 \u0026micro;l containing 30 pmol of each primer, 2.5 mM MgCl2 (PB-L, Argentina), 1X PCR buffer (PB-L, Argentina), 0.2 mM each deoxynucleoside triphosphate (Promega, USA), 1 U Taq Pegasus DNA polymerase (PB-L, Argentina) and 1 \u0026micro;l of DNA. Amplification conditions included an initial step at 94\u0026deg;C for 3 min, followed by 34 cycles at 94\u0026deg;C for 30 sec, 46\u0026deg;C for 1 min, and 72\u0026deg;C for 1.5 min, then concluded by a step at 72\u0026deg;C for 10 min.\u003c/p\u003e \u003cp\u003eThe amplification of the ITS-1 region was carried out with the primers SS1 (5'-GTTTCCGTAGGTGAACCTGCG-3') and NC13R (5'-GCTGCGTTCTTCATCGAT-3') and the ITS-2 region with the primers SS2 (5'-TTGCAGACACATTGAGCACT-3') and NC2 (5'-TTAGTTTCTTTTCCTCCGCT \u0026minus;\u0026thinsp;3'), according to the procedure reported in Shamsi et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The PCR mixture (50 \u0026micro;l) contained 25 \u0026micro;l of Master Mix PCR 2X (PB-L, Argentina) 2.5 \u0026micro;l of each primer (10 \u0026micro;M), 18 \u0026micro;l of water, and 2 \u0026micro;l of extracted DNA. PCR conditions were: 94\u0026deg;C for 5 min, followed by 30 cycles at 94\u0026deg;C for 30 sec, at 55\u0026deg;C for 30 sec, at 72\u0026deg;C for 30 sec, and a final extension at 72\u0026deg;C for 5 min.\u003c/p\u003e \u003cp\u003eThe amplification of mitochondrial small subunit ribosomal gene SSrRNA was performed according to the procedures reported in D\u0026rsquo;Amelio et al. (2007) with the primers MH3 (5\u0026rsquo;-TTGTTCCAGAATAATCGGCTAGACTT-3\u0026rsquo;) and MH4.5 (5\u0026rsquo;TCTACTTTACTACAACTTACTCC-3\u0026rsquo;). The PCR mixture (50 \u0026micro;l) contained 25 \u0026micro;l of Master Mix PCR 2X (PB-L, Argentina) 2.5 \u0026micro;l of each primer (10 \u0026micro;M), 18 \u0026micro;l of water, and 2 \u0026micro;l of extracted DNA. PCR conditions included 10 min at 95\u0026deg;C, 35 cycles of 30 sec at 95\u0026deg;C, 30 sec at 55\u0026deg;C, 30 sec at 72\u0026deg;C, and a final extension step of 7 min at 72\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe PCR was purified and sequenced by Macrogen (Seoul, South Korea) directly with the primers for amplification. The nucleotide sequences reported in the present study are available from the DDBJ/EMBL/GenBank databases under the accession numbers (xxxxxxxxx).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eSequences were assembled using the Geneious program. The assembled sequences were checked for the presence of pseudogenes in the mtDNA \u003cem\u003ecox2\u003c/em\u003e gene by translating the amino acid sequences using the invertebrate mitochondrial genetic code within Geneious. \u003cem\u003eContracaecum\u003c/em\u003e spp. sequences were aligned using the online version of MAFFT 7 (Katoh and Standley \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) with sequences of \u003cem\u003eAnisakis\u003c/em\u003e spp. used as outgroups. The best partitioning scheme and substitution model for each DNA partition were chosen under the Bayesian Information Criterion (Schwarz \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1978\u003c/span\u003e) using the \u0026ldquo;greedy\u0026rdquo; search strategy in Partition Finder v. 1.1.1 (Lanfear et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Only the mtDNA \u003cem\u003ecox2\u003c/em\u003e fragment dataset was partitioned into first-, second-, and third-codon positions. The appropriate nucleotide substitution models implemented for each partition were HKY\u0026thinsp;+\u0026thinsp;I for the first and third codon position, and HKY\u0026thinsp;+\u0026thinsp;G for the second codon position. For the datasets of SSrRNA and ITS2 genes, the HKY\u0026thinsp;+\u0026thinsp;G was used, while K80\u0026thinsp;+\u0026thinsp;G model was used for the ITS1 gene.\u003c/p\u003e \u003cp\u003ePhylogenetic reconstruction was performed using Bayesian Inference (BI) through Mr. Bayes 3.2.3 (Ronquist et al. 2003). Phylogenetic trees were constructed using two parallel analyses of Metropolis-Coupled Markov Chain Monte Carlo (MCMC) for 20\u0026nbsp;million generations each, to estimate the posterior probability (PP) distribution. Topologies were sampled every 1,000 generations and the average standard deviation of split frequencies was observed to be less than 0.01 at the end of the run, as suggested by Ronquist et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The robustness of the clades was assessed using Bayesian posterior probability (PP), where PP\u0026thinsp;\u0026gt;\u0026thinsp;0.90 was considered strongly supported. A majority consensus tree with branch lengths was reconstructed for each run after discarding the first 25% trees sampled as \u0026ldquo;burn-in\u0026rdquo;.\u003c/p\u003e \u003cp\u003eAdditionally, uncorrected p-distance was calculated for each gen using MEGA X (Kumar et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) with the bootstrap method (1000 replicates) and nucleotide substitution (transition\u0026thinsp;+\u0026thinsp;transversions). A uniform rate was applied, and gaps/missing data were considered as complete deletion.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eBoth morphometric and phylogenetic analyses of the present studied specimens allowed us to recognize the species:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eContracaecum jorgei\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eSardella, Mancini, Salinas, Sim\u0026otilde;es \u0026amp; Luque 2020\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGeneral morphology:\u003c/em\u003e (based on 10 males and 10 females): Body entirely transversally striated (Fig. 2a-f). Non-conspicuous cephalic collar, V-shaped lateral region without striations (Figs. a, b). Three smooth interlabia (Fig. 2a, b). Lips barely longer than interlabia with just longer with non-visible notches (Fig. 2a, b). Lips with two non-conspicuous and lobed auricles, no apparent tips (Fig. 2a, b). Notorious lip papillae, two on the dorsal lip, and one on each ventrolateral lip (Fig. 2a, b).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMale\u003c/em\u003e: Simple postcloacal tail arrangement: two paracloacal papillae pairs, two subventral papillae pairs, two sublateral papillae pairs, and a phasmid pair (Fig. 2c, d). No apparent tail distal constriction (Fig. 2c, d). Short spicules approximately one-fifth the body length, and short spicule free-distal end (Fig. 2e, Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFemale\u003c/em\u003e: see Fig. 2f, Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTaxonomic summary\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eType host: \u003cem\u003eNannopterum\u003c/em\u003e (=\u003cem\u003ePhalacrocorax\u003c/em\u003e)\u003cem\u003e\u0026nbsp;brasilianum\u003c/em\u003e (King 1828) (Pelecaniformes: Phalacrocoracidae).\u003c/p\u003e\n\u003cp\u003eType locality: Pampean shallow lakes, Uni\u0026oacute;n Department, C\u0026oacute;rdoba province, Argentina (33\u0026deg;25\u0026prime;S, 62\u0026deg;54\u0026prime;W).\u003c/p\u003e\n\u003cp\u003eNew host: \u003cem\u003eArdea cocoi\u003c/em\u003e Linnaeus, 1766 (Pelecaniformes: Ardeidae).\u003c/p\u003e\n\u003cp\u003eNew locality: Magdalena city coast, Buenos Aires Province (34\u0026deg;58\u0026prime;59.7\u0026Prime;S, 57\u0026deg;44\u0026prime; 9.05\u0026Prime;W).\u003c/p\u003e\n\u003cp\u003eInfection site: stomach.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eMolecular characterization and phylogenetic analysis\u003c/u\u003e\u003c/em\u003e\u003cu\u003e\u0026nbsp;\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe obtained sequences include threes of mtDNAcox2, one SSrRNA, one ITS1 and one ITS2 with lengths ranging from 382 to 592 bp. The constructed matrix consist of 48 terminals and 618 bp for mtDNA cox2, 12 terminals and 460 bp for SSrRNA, 36 terminals and 383 bp for ITS1, and 34 terminals and 201 bp for ITS2.\u003c/p\u003e\n\u003cp\u003eThe phylogenetic trees constructed using these sequences reveal variable associations with species sequences archived in GenBank. In the constructed tree using SSrRNA sequences, a weakly supported node (PP = 0.79) aligns with \u003cem\u003eC. microcephalum\u003c/em\u003e sequences (Fig. 3). Conversely, Figs. 4 and 5, based on ITS1 and ITS2 sequences respectively, establish a robust node (PP = 0.99) positioning our sequence as the sister species to a node consisting of \u003cem\u003eContracaecum pyripapillatum\u003c/em\u003e Shamsi, Gasser \u0026amp; Beveridge, 2008, \u003cem\u003eC. multipapillatum, Contracaecum gibsoni\u0026nbsp;\u003c/em\u003eMattiucci , Paoletti, Consuegra-Solorzano \u0026amp; Nascetti, 2010, and\u003cem\u003e\u0026nbsp;Contracaecum quadripapillatum\u003c/em\u003e Saad, Younis \u0026amp; Rabei, 2018. In Fig. 6, the mtDNA \u003cem\u003ecox2\u003c/em\u003e gene shows our sequences exhibiting concordance with \u003cem\u003eC. jorgei\u003c/em\u003e. A large clade of \u003cem\u003eContracaecum\u003c/em\u003e species with simple papillae arrangement on the male tail (Fig. 1) is observed at the top of the tree (Fig. 6).\u003c/p\u003e\n\u003cp\u003eThe p-distances calculated for the SSrRNA gene matrix displayed a distance of 14% from \u003cem\u003eC. microcephalum\u0026nbsp;\u003c/em\u003eand the specimens here studied (Table 3a)\u003cem\u003e.\u003c/em\u003e For ITS1, the calculated distance is 16% from both \u003cem\u003eC. pyripapillatum\u003c/em\u003e and \u003cem\u003eC. multipapillatum\u0026nbsp;\u003c/em\u003e(Table 3b). In the case of ITS2, the distances are 35% from \u003cem\u003eC. multipapillatum\u003c/em\u003e and 38% from \u003cem\u003eC. pyripapillatum\u0026nbsp;\u003c/em\u003e(Table 3c). Finally, the mtDNA \u003cem\u003ecox2\u003c/em\u003e gene displayed a distance of 1% observed for among our sequences and \u003cem\u003eC. jorgei\u0026nbsp;\u003c/em\u003efrom the original description (Sardella el at. 2020) (Table 3d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRemarks\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the morphometric analysis, the present study specimens (PES) displayed a morphological concordance with \u003cem\u003eC. jorgei\u003c/em\u003e parasitizing \u003cem\u003eN. brasilianum\u0026nbsp;\u003c/em\u003efrom the C\u0026oacute;rdoba province, Argentina (Sardella et al. 2020). However, a few slight morphometric differences can be observed between PES and those of \u003cem\u003eC. jorgei\u003c/em\u003e from the original description. In this sense, PES are barely longer and thicker, and the spicules are slightly longer, but they do not constitute significant differences (Table 2). If we compare the SEM photos of the original description with the male of PES, we can observe a very noticeable distal constriction on the male tail. However, this might be due to the male individual on the original description is more mature adopting this pattern of striations. On the other hand, both sublateral papillae joining with the phasmid on the male tail are arranged in a straight line while a triangular arrangement can be observed in the PES. This arrangement may be variable and does not represent a significant difference as postulated by other authors (Fagerholm 1990; Mattiucci and Nascetti 2008).\u003c/p\u003e\n\u003cp\u003eIf we take into account other \u003cem\u003eContracaecum\u003c/em\u003e spp. parasitizing Ardeidae in Argentina, PES differ significantly from \u003cem\u003eC. multipapillatum\u003c/em\u003e parasitizing \u003cem\u003eA. alba\u0026nbsp;\u003c/em\u003e they are smaller in length and thickness although their spicules are three times longer (Navone et al. 2000). This is this is reflected on the ratio total body length / spicule length (BL/EL= 24.11 vs. 5.24 PES) (Table 2). Something similar occurs with \u003cem\u003eC. microcephalum\u003c/em\u003e parasitizing \u003cem\u003eN. nicticorax\u003c/em\u003e, whose size is larger but its spicules are shorter reflected on the body length / spicule length ratio (BL/EL= 9.25 vs. 5.24 PES) (Boero and Led 1971). In addition, PES show a higher number of precloacal papillae (24-31 vs. 10 pairs) (Table 2). Although, the description of \u003cem\u003eC. microcephalum\u003c/em\u003e by Schuurmans-Stekhoven (1951) parasitizing \u003cem\u003eA. alba\u003c/em\u003e is brief, PES appear to be smaller in length and thinner, and its spicule free distal end is much longer.\u003c/p\u003e\n\u003cp\u003eConsidering the \u003cem\u003eContracaecum\u003c/em\u003e species parasitizing other birds from South America, PES are smaller in size than \u003cem\u003eC. australe\u003c/em\u003e parasitizing the Neotropic cormorant \u0026ndash;type host of \u003cem\u003eC. jorgei\u003c/em\u003e- of Chile and Argentina. In addition, spicules of PES are much shorter than those of \u003cem\u003eC. australe\u0026nbsp;\u003c/em\u003e(Table 2). Present studied specimens\u0026rsquo; auricle tips are less notorious and auricle notches are absent (Fig. 2) (Garbin et al. 2011; Biol\u0026eacute; et al. 2012).\u003c/p\u003e\n\u003cp\u003eRegarding \u003cem\u003eContracaecum\u003c/em\u003e species parasitizing other hosts from different parts of the world, PES are significantly smaller in length and thinner, and thus the internal digestive organs also show smaller dimensions than \u003cem\u003eC. multipapillatum\u003c/em\u003e parasitic in the Australian pelican \u003cem\u003ePelecanus conspicillatus\u003c/em\u003e Temminck from Australia (Shamsi et al. 2008). Besides, PES own fewer precloacal papillae and possess spicules three times larger than those of \u003cem\u003eC. multipapillatum\u003c/em\u003e. In addition, the papillae arrangement on the male tail is simple in PES, whereas in \u003cem\u003eC. multipapillatum\u003c/em\u003e it is intermediate (Table 2, Fig. 1). Present study specimens show simple papillae arrangement on the male tail with respect to \u003cem\u003eC. pyripapillatum\u0026nbsp;\u003c/em\u003eparasitizing \u003cem\u003eP. conspicillatus\u003c/em\u003e from Australia (Shamsi et al. 2008), and the spicules are twice in length (Table 2).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eContracaecum jorgei\u003c/em\u003e was first described parasitizing \u003cem\u003eN. brasilianum\u003c/em\u003e and \u003cem\u003eHoplias argentinensis\u003c/em\u003e (Sardella et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), a frequent item prey for \u003cem\u003eA. cocoi\u003c/em\u003e (Ducommun and Beltzer 2010). Therefore, this fish species might facilitate the entry of \u003cem\u003eC. jorgei\u003c/em\u003e third-stage larvae (L3) acting as an intermediate/paratenic host. Present report suggests an intermediate host-specificity of this nematode species parasitizing two different Pelecaniformes families. Montes (pers. obs.) found otoliths of several fish species in the Cocoi Heron from the coast of Magdalena, R\u0026iacute;o de la Plata, Buenos Aires Province. Therefore, this could indicate that there are more than one paratenic/intermediate host for \u003cem\u003eC. jorgei\u003c/em\u003e L3 larvae.\u003c/p\u003e \u003cp\u003eAccording to the molecular analysis conducted in this study, PES clustered with the sister species \u003cem\u003eC. microcephalum\u003c/em\u003e on the SSrRNA BI tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). As mentioned above, both species are morphologically similar and parasitize Ardeidae not only in South America but also in other parts from the world. However, the phylogenetic analysis results are restricted due the scarcity of sequences available in Genbank resulting in limited conclusions.\u003c/p\u003e \u003cp\u003eConsidering the BI analysis of ITS1-ITS2 genes, we can observe the topologies differ slightly each other (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Present study specimens appear as a sister species to the node consisting of \u003cem\u003eC. multipapillatum\u003c/em\u003e, \u003cem\u003eC. pyripapillatum, C. quadripapillatum\u003c/em\u003e, and \u003cem\u003eC. gibsoni\u003c/em\u003e. As mentioned before, \u003cem\u003eC. jorgei\u003c/em\u003e and \u003cem\u003eC. multipapillatum\u003c/em\u003e would parasitize Ardeidae in Argentina (Navone et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Sardella et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)d \u003cem\u003epyripapillatum\u003c/em\u003e in Australia (Shamsi et al. 2019). But, \u003cem\u003eC. quadripapillatum\u003c/em\u003e, and \u003cem\u003eC. gibsoni\u003c/em\u003e parasitize Pelecanidae in Israel and Greece, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Therefore, this similarity should be explained from a parasite-host physiology point of view taking into account that both families belong to the Pelecaniformes order.\u003c/p\u003e \u003cp\u003eFinally, the topology of the mtDNA \u003cem\u003ecox2\u003c/em\u003e BI tree and the genetic distances between \u003cem\u003eContracaecum\u003c/em\u003e taxa supports the identification of the PES as \u003cem\u003eC. jorgei\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). \u003cem\u003eContracaeum jorgei\u003c/em\u003e is closely related to \u003cem\u003eC. multipapillatum\u003c/em\u003e, as shown in the BI analysis. However, despite their genetic similarity, the two species can be easily distinguished by their morphology. For instance, the arrangement of tail papillae in males varies significantly between the two species. While \u003cem\u003eC. jorgei\u003c/em\u003e exhibits a simple pattern of postcloacal papillae (simple morphotype), \u003cem\u003eC. multipapillatum\u003c/em\u003e is characterized by having a greater number of pre- and postcloacal papillae (intermediate morphotype), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (Navone et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Garbin et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe sequence MH044685 (unpublished data) identified as \u003cem\u003eC. multipapillatum\u003c/em\u003e in GenBank groups with \u003cem\u003eC. jorgei\u003c/em\u003e in the same branch. According to the information, this sequence belongs to a \u003cem\u003eContracaecum\u003c/em\u003e larvae specimen found parasitizing a catfish (\u003cem\u003eRhamdia\u003c/em\u003e sp.) in Costa Rica and Guatemala (locality not specified). It is not correct to determine species with larval specimens as these do not develop secondary characters such as spicules and pre- and postcloacal papillae. Therefore, we believe that this sequence misidentified as \u003cem\u003eC. multipapillatum\u003c/em\u003e belongs to \u003cem\u003eC. jorgei\u003c/em\u003e. The genetic proximity between \u003cem\u003eC. jorgei\u003c/em\u003e and \u003cem\u003eC. multipapillatum\u003c/em\u003e can be explained from a parasite-host physiological point of view. This is because both \u003cem\u003eContracaecum\u003c/em\u003e species parasitize members of the Ardeidae family, specifically \u003cem\u003eA. cocoi\u003c/em\u003e and \u003cem\u003eA. alba\u003c/em\u003e. These birds have similar feeding habits and share a geographical distribution. The same can be observed for \u003cem\u003eC. microcephalum\u003c/em\u003e, which is also a parasite of Ardeidae in Argentina (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The tree reflects these similarities, as the species that closely group with \u003cem\u003eC. jorgei\u003c/em\u003e are those that use Ardeidae as hosts.\u003c/p\u003e \u003cp\u003eThe record of \u003cem\u003eC. jorgei\u003c/em\u003e parasitizing \u003cem\u003eA. coccoi\u003c/em\u003e is the second report of an Anisakidae for this host species in Argentina, and also for any Ardeidae. Integrative molecular studies including morphological and molecular tools are important to know the real host and geographical distribution of parasite diversity and establish specific correspondences to determine phylogenetic relationships on the \u003cem\u003eContracaecum\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003eIn conclusion, our work represents the second report of \u003cem\u003eC. jorgei\u003c/em\u003e from Argentina. Based on morphological analysis conducted using optical and scanning electron microscopes, we identified the species approximately 600 km away from its type collection site. Additionally, we correct the identification of the sequence deposited in Genbank MH044685 as \u003cem\u003eC. jorgei\u003c/em\u003e. And therefore, the range of the species is extended to Central America.\u003c/p\u003e \u003cp\u003eWe have included new sequences of mtDNA \u003cem\u003ecox2\u003c/em\u003e and provided the firsts sequences of SSrRNA, ITS1 and ITS2 on \u003cem\u003eC. jorgei\u003c/em\u003e. This study shed light on the limited information available regarding this conspicuous nematode and sets the stage for further investigations into its life cycles. We hope that new records, particularly those involving intermediate and definitive hosts, will contribute to elucidating the distribution of these parasites in the Americas, and potentially lead to the discovery of new species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments.\u003c/strong\u003e We are grateful to the CEPAVE for providing laboratory space for processing and studying the specimens, and the CONICET and FONCYT for the grants assigned to researchers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLucas E. Garbin: Conceived and designed research. Wrote the main manuscript text, nematode specimen collection, morphometric and taxonomical nematode studies, phylogenetic analysis, photographs to scanning electron microscopy, preparation of figures 1, 2, and tables 1 and 2.\u003c/p\u003e\n\u003cp\u003eMart\u0026iacute;n M. Montes: Conceived and designed research. Wrote the main manuscript text, bird digestive tract prospection, nematode specimen collection, phylogenetic analysis, preparation of figures 3, 4, 5 and 6, tables 3a-d.\u003c/p\u003e\n\u003cp\u003eN. Arredondo: Made morphometric and taxonomical nematode studies, photographs to scanning electron microscopy, phylogenetic analysis.\u003c/p\u003e\n\u003cp\u003eJ. Barneche: Collected the Red-legged cormorant samples, bird digestive tract prospection.\u003c/p\u003e\n\u003cp\u003eM. Ib\u0026aacute;\u0026ntilde;ez: DNA extractions and PCR assays.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eM. Moncada: DNA extractions and PCR assays.\u003c/p\u003e\n\u003cp\u003eJulia I. Diaz: Morphometric and taxonomical nematode studies, images and figure preparation.\u0026nbsp;Contributed to conceptualization and provide financial support.\u003c/p\u003e\n\u003cp\u003eAll authors reed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Consejo Nacional de Investigaciones Cient\u0026iacute;ficas y T\u0026eacute;cnicas (CCT-CONICET-La Plata) (MMM., Grant number: 11220200101713CO); and Fondo para la Investigaci\u0026oacute;n Cient\u0026iacute;fica y Tecnol\u0026oacute;gica (FONCyT) (M.M.M., Grant number: PICT-2020-SERIEA-01531; N.J.A. Grant number: PICT-2020- SERIEA-00660).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SEM-photographed \u003cem\u003eContracaecum jorgei\u003c/em\u003e specimens (3 males and 3 females) from \u003cem\u003eArdea cocoi\u003c/em\u003e are stored at the Servicio Nacional de Microscop\u0026iacute;a (SNM), Facultad de Ciencias M\u0026eacute;dicas, Universidad de Buenos Aires. All paratypes of \u003cem\u003eContracaecum\u003c/em\u003e \u003cem\u003ejorgei\u003c/em\u003e mounted on lactophenol are preserved in 70% alcohol at the Helminthological Collection of CEPAVE and will be deposited in the Helminthological Collection of the La Plata Museum once the manuscript is accepted.\u003c/p\u003e\n\u003cp\u003eThe nucleotide sequences of both \u003cem\u003eContracaecum\u003c/em\u003e species reported in the present study are available from the DDBJ/EMBL/GenBank databases under the accession numbers (xxxxx).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors give their consent to participate in this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors give their consent for publication of this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eAmato JFR, Monteiro CM, Amato SB (2006) \u003cem\u003eContracaecum rudolphii\u003c/em\u003e Hartwich (Nematoda, Anisakidae) from the Neotropical Cormorant \u003cem\u003ePhalacrocorax brasilianus\u003c/em\u003e (Gmelin) (Aves, Phalacrocoracidae) in Southern Brazil. Rev Brasil Zool 23(4):1284\u0026ndash;1289. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/S0101-81752006000400046\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAnderson RC (2000) Nematode parasites of vertebrates. Their development and transmission. CABI Publishing, Farnham Royal, UK. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1079/9780851994215.0001\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBiol\u0026eacute; FG, Guagliardo SE, Mancini MA, Tanzola RD, Salinas V, Morra G (2012) Primer registro de \u003cem\u003eContracaecum australe\u003c/em\u003e (Nematoda: Anisakidae) en \u003cem\u003ePhalacrocorax brasilianus\u003c/em\u003e (Aves: Phalacrocoracidae) de la regi\u0026oacute;n central de Argentina. 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J Helminthol 1:35\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/s0022149x00002698\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eVicente JJ, Rodrigues HO, Gomes DC, Pinto RM (1995) Nemat\u0026oacute;ides do Brasil. Parte IV: Nemat\u0026oacute;ides de aves. Rev Brasil Zool 12(1):1\u0026ndash;273.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eVidal-Mart\u0026iacute;nez VM, Osorio-Sarabia D, Overstreet RM (1994) Experimental infection of \u003cem\u003eContracaecum multipapillatum\u003c/em\u003e (Nematoda: Anisakinae) from Mexico in the domestic cat. J Parasitol 80:576\u0026ndash;579. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2307/3283194\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eVon Drasche R (1882) Helmlinthologische Notizen. Abhandlungen der K. K. Zool.-Botan. Gesellschaft Wien 32:139\u0026ndash;142.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Contracaecum, Ardeidae, SSrRNA gene, ITS regions, mtDNA cox2 gene","lastPublishedDoi":"10.21203/rs.3.rs-4450708/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4450708/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnisakidae nematodes of the \u003cem\u003eContracaecum\u003c/em\u003e genus are known for their wide host and geographic distribution. These parasites commonly infest aquatic organisms worldwide. The life cycles of \u003cem\u003eContracaecum\u003c/em\u003e species typically involve aquatic invertebrates and fish as intermediate and/or paratenic hosts, while piscivorous birds and mammals serve as definitive hosts.\u003cstrong\u003e \u003c/strong\u003eThe aim of the present work is to identify the \u003cem\u003eContracaecum\u003c/em\u003especimens parasitizing \u003cem\u003eArdea cocoi\u003c/em\u003efrom the Magdalena city coast, Argentina, and to determine their phylogenetic relationships with its congeners based on both morphometric and phylogenetic analyses. One Cocoi Heron specimen was found dead at the Magdalena coast, Buenos Aires Province, and 53 nematodes were recovered from its esophagus and stomach. Some nematode specimens were cleared with lactophenol and studied using an optical microscope. Three males and females were examined in a scanning electron microscope. DNA extraction, PCR and sequencing of mtDNA \u003cem\u003ecox2\u003c/em\u003e, ITS1, ITS2, and SSrRNA genes were performed from three \u003cem\u003eContracaecum\u003c/em\u003e specimens. Both morphometric and phylogenetic analyses of the present studied specimens allowed us to recognize the species \u003cem\u003eContracaecum jorgei. \u003c/em\u003eIn the constructed tree using SSrRNA sequences, a node aligns with \u003cem\u003eC. microcephalum\u003c/em\u003e sequences. Conversely, ITS1 and ITS2 sequences respectively, establish a robust node positioning our sequence as the sister species to a node consisting of \u003cem\u003eContracaecum pyripapillatum\u003c/em\u003e, \u003cem\u003eC. multipapillatum, C. gibsoni\u003c/em\u003e, and\u003cem\u003e C. quadripapillatum\u003c/em\u003e. The mtDNA \u003cem\u003ecox2\u003c/em\u003egene shows our sequences exhibiting concordance with \u003cem\u003eC. jorgei\u003c/em\u003e. The p-distances calculated for the SSrRNA gene matrix displayed a distance of 14% from \u003cem\u003eC. microcephalum \u003c/em\u003eand the present study specimens (PES)\u003cem\u003e.\u003c/em\u003e For ITS1, the calculated distance is 16% from both \u003cem\u003eC. pyripapillatum\u003c/em\u003e and \u003cem\u003eC. multipapillatum\u003c/em\u003e. In the case of ITS2, the distances are 35% from \u003cem\u003eC. multipapillatum,\u003c/em\u003e and 38% from \u003cem\u003eC. pyripapillatum\u003c/em\u003e. Finally, the mtDNA \u003cem\u003ecox2\u003c/em\u003egene displayed a distance of 1% observed for among our sequences and \u003cem\u003eC. jorgei. \u003c/em\u003eAccording to the molecular analysis, PES clustered with the sister species \u003cem\u003eC. microcephalum \u003c/em\u003eon the SSrRNA BI tree. Considering the BI analysis of ITS1-ITS2 genes, PES appear as a sister species to the node consisting of \u003cem\u003eC. multipapillatum\u003c/em\u003e, \u003cem\u003eC. pyripapillatum,\u003c/em\u003e \u003cem\u003eC. quadripapillatum\u003c/em\u003e, and \u003cem\u003eC. gibsoni\u003c/em\u003e. Finally, the topology of the mtDNA \u003cem\u003ecox2\u003c/em\u003e BI tree and the genetic distances between \u003cem\u003eContracaecum\u003c/em\u003etaxa supports the identification of the PES as \u003cem\u003eC. jorgei\u003c/em\u003e. \u003cem\u003eContracaeum jorgei\u003c/em\u003e is closely related to \u003cem\u003eC. multipapillatum\u003c/em\u003e. The record of \u003cem\u003eC. jorgei\u003c/em\u003e parasitizing \u003cem\u003eA. coccoi\u003c/em\u003e is the second report of an Anisakidae for this host species in Argentina, and also for any Ardeidae. Integrative molecular studies including morphological and molecular tools are important to know the real host and geographical distribution of parasite diversity and establish specific correspondences to determine phylogenetic relationships on the \u003cem\u003eContracaecum\u003c/em\u003e species. This work represents the second report of \u003cem\u003eC. jorgei\u003c/em\u003e from Argentina based on morphological analysis conducted using optical and scanning electron microscopy. This study shed light on the limited information available regarding this conspicuous nematode and sets the stage for further investigations into its life cycles.\u003c/p\u003e","manuscriptTitle":"Unveiling the phylogeny of Contracaecum jorgei (Nematoda, Anisakidae) parasitizing Ardea cocoi (Aves, Ardeidae) in Argentina based on an integrative analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-07 19:01:39","doi":"10.21203/rs.3.rs-4450708/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ecac5f2c-8fe0-4aeb-8718-45f51dbb5d27","owner":[],"postedDate":"June 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-24T07:16:53+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-07 19:01:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4450708","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4450708","identity":"rs-4450708","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}