Morphological and molecular characterization of Hysterothylacium spp. parasitizing Pomatomus saltatrix and Pagrus pagrus of the State of São Paulo, Brazil

An Acad Bras Cienc (2023) 95(1): e20211046 DOI 10.1590/0001-3765202320211046 Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc | www.fb.com/aabcjournal An Acad Bras Cienc (2023) 95(1) Running title: CHARACTERIZATION OF Hysterothylacium FROM BRAZIL Academy Section: MICROBIOLOGY e20211046 95 (1) 95(1) DOI 10.1590/0001-3765202320211046 MICROBIOLOGY Morphological and molecular characterization of Hysterothylacium spp. parasitizing Pomatomus saltatrix and Pagrus pagrus of the State of São Paulo, Brazil THAISSA D. SERRANO, DIEGO H.M.D. VIEIRA, LARISSA S. PELEGRINI, LÚCIA V. FRAGOSO, BEATRIZ N. AGOSTINHO, MANUEL VERA, FÁBIO PORTO-FORESTI, RODNEY K. DE AZEVEDO & VANESSA D. ABDALLAH Abstract: Raphidascarid nematodes have been the focus of several studies, mainly due to the zoonotic potential of some species, even though the cases are underreported. Due to the difficulty in identifying their larvae, the use of diagnostic techniques involving morphological and molecular analyses has grown in the last 20 years. The present study had as objective the morphological and molecular characterization of the L3 larval types of Hysterothylacium collected in Pomatomus saltatrix and Pagrus pagrus from the Brazilian coast, close to the municipality of Santos, State of São Paulo. Twenty specimens of P. saltatrix were necropsied and Hysterothylacium type V (n = 257) and Hysterothylacium type X (n = 5) larvae were found. Five specimens of P. pagrus were necropsied and all were parasitized by Hysterothylacium type V larvae. The analyses showed a genetic proximity relationship between Hysterothylacium types V with other Hysterothylacium V and with H. deardorffoverstreetorum, although this is a species inquirenda. Haplotypes for Hysterothylacium type X found in the present study formed a monophyletic group with other Hysterothylacium X, H. amoyense, and H. zhoushanense. Through this study, new hosts and localities were registered for Hysterothylacium type V and Hysterothylacium type X. Key words: ITS, Nematoda, phylogeny, Raphidascaridae, taxonomy. INTRODUCTION Raphidascarid nematodes have been extensively studied in recent years since some species have zoonotic potential (Mattiucci et al. 2013). Fish can harbor the larvae and adults of these nematodes, attracting the interest of researchers to the development of tools (classic or modern) that link conspecific individuals in different stages of development (Cannon 1977, Mattiucci et al. 2002, Borges et al. 2012, Shamsi et al. 2015). Additionally, the understanding of epidemiological, biological, and ecological patterns is only possible after the correct identification of a species, regardless of its stage of development (Kijewska et al. 2002, Mattiucci et al. 2005, Nadler et al. 2005, Zhang et al. 2007). Hysterothylacium Ward & Magath, 1917 includes about 72 valid species being the most abundant and diverse group of ascaridoids parasitic in marine fish, with a worldwide distribution (Moravec & Justine 2015). The identification of these larvae is usually problematic, and different tools for this purpose involving morphological and molecular characterization have been frequently used (Ghadam et al. 2018, Jabbar et al. 2012, THAISSA D. SERRANO et al. CHARACTERIZATION OF Hysterothylacium FROM BRAZIL An Acad Bras Cienc (2023) 95(1) e20211046 2 | 14 Khammassi et al. 2020, Pantoja et al. 2016, RocaGeronès et al. 2018, Shamsi et al. 2011, 2013, 2015, 2018, 2020). At least 17 Hysterothylacium larval morphotypes have been described, based mainly on characteristics related to the digestive tract and caudal end, in addition to molecular characterization (Cannon 1977, Shamsi et al. 2011). Hysterothylacium larvae have been widely reported in fishes from Brazilian Atlantic Coast, parasitizing more than 35 species of teleosts (Pantoja et al. 2016). However, the use of integrative taxonomy methods (morphology and molecular analyses) to identify these nematodes is limited to a few studies in Brazil (Borges et al. 2012, Knoff et al. 2012, Pantoja et al. 2015, 2016). The few records of parasitism by Hysterothylacium spp. in humans can also be related to the lack of a specific diagnosis for the identification of their larvae, making morphomolecular studies important for actions to control possible parasitic diseases caused by fish consumption (Shamsi et al. 2018, Rahmati et al. 2020). The anchovy Pomatomus saltatrix (Linnaeus, 1766) and the red porgy Pagrus pagrus (Linnaeus, 1758) are carnivorous fishes with worldwide distribution in tropical and subtropical waters, commonly found on the Brazilian coast, and are widely consumed and marketed (Froese & Pauly 2019). In this study, an integrative taxonomy approach was used to evaluate specimens of Hysterothylacium found in these two hosts and establish their phylogenetic relationships with other congeners. Therefore, morphological data were combined with DNA sequences from the rDNA region comprising ITS-1, gene 5.8S, and ITS-2. MATERIALS AND METHODS Collection, processing, and morphological examination Twenty specimens of bluefish (P. saltatrix) and five specimens of red porgy (P. pagrus) were purchased frozen and not eviscerated at different commercial points in the municipality of Bauru, State of São Paulo, from February to December 2016. Hosts were originally from the coast of the municipality of Santos, State of São Paulo. The fish were eviscerated through an incision close to the opercula until the cloaca and the stomach, intestine, and mesentery were analyzed. The organs were passed through 75 μm sieves and washed with water and the contents and tissue of the organs were analyzed with a stereomicroscope, for nematodes. Subsequently, the macroscopic examination of fish musculature was performed using a stereomicroscope Bel Photonics STM Pro. The technique of filleting with the removal of sections of the host’s musculature and subsequent inspection by transparency using a negatoscope was performed. For the morphological analyses, the larvae were fixed and stored in 70% ethanol. The anterior and posterior extremities of all nematodes were processed for morphological study, cleared in Amann’s Lactophenol, placed on glass slides covered by coverslips, and observed on the Nikon Eclipse E200 microscope equipped with a Moticam 5.0MP image capture system where the photographs were taken and morphometric analyses were performed. The means and ranges measures (in brackets) are given in millimeters. For scanning electron microscopy (SEM), six specimens were taken from the 70% ethanol, passed in phosphate buffer, and stored in 3% glutaraldehyde for three days. Subsequently, the specimens were post-fixed in 1% osmium tetroxide for 6 hours, dehydrated using a series THAISSA D. SERRANO et al. CHARACTERIZATION OF Hysterothylacium FROM BRAZIL An Acad Bras Cienc (2023) 95(1) e20211046 3 | 14 of ethanol, and then dried at the critical point through carbon dioxide. The specimens were coated with gold and examined using a ZEISS EVO-MA10 scanning electron microscope with an acceleration voltage of 15 kV. The SEM images of Hysterothylacium type X were not suitable because the specimens were very dehydrated in this preparation, and we decided to use only the images in light microscopy. To avoid repetition of information, we decided to add only the SEM images of Hysterothylacium type V, as they are more suitable for visualizing the structures. DNA isolation, amplification, and sequencing The median portion of seven specimens of Hysterothylacium from P. saltatrix and five specimens from P. pagrus were fixed in absolute ethanol. Total genomic DNA was extracted according to the information described in the Wizard commercial genomic DNA purification kit (Promega). The primers NC5 (forward: 5’-GTAGGTGA ACCTGCGGAAGGATCATT-3’) and NC2 (reverse: 5’-TTAGTTTCTTTTCCTCCGCT-3’) described by Zhu et al. (1998), were used to amplify the rDNA region comprising ITS-1, 5.8S gene, and ITS-2 (from here and thereafter ITS1-5.8S-ITS2 region). PCR was performed in a 50 µL reaction mixture containing 100-300 ng of template DNA, 1X PCR Gold Buffer, 2.0 mM MgCl2, 0.2 mM of each dNTP, 20 pmol of each primer, and 2.5 U of Ampli Taq Gold™ DNA polymerase (Applied Biosystems, Foster City, CA, USA). PCR cycling conditions were as follows: initial denaturation at 94ºC for 10 min followed by 40 cycles of 94ºC for 45 sec, 55ºC for 45 sec and 72ºC for 45 sec, and a final extension at 72ºC for 7 min. PCR products (10 µL) were enzymatically purified using Exo I (0.6 units) and SAP (0.3 units) enzymes, incubated at 37 ºC for 60 min and 85ºC for 15 min. Finally, the purified product was sequenced following the ABI Prism BigDye™ Terminator v3.1 Cycle Sequencing Kit protocol on an ABI 3730xl DNA sequencer (Applied Biosystems, Foster City, CA, USA). Phylogenetic analysis The obtained sequences were compared to homologous sequences of Hysterothylacium species available in public databases (i.e. NCBI GenBank) (Table I). The software SEQSCAPE 2.5 (Applied Biosystems, Foster City, CA, USA) was used to check variable sites. Sequences from genetically related species that contained the complete ITS region available on GenBank were selected for analysis. Sequences were aligned using Clustal X 2.0 (Thompson et al. 1997) with default parameters. The different haplotypes were detected using the program DnaSP 5.0 (Librado & Rozas 2009) and were compared with sequences available in the NCBI database using the BLAST tool. The software MEGA X (Kumar et al. 2018) was used to estimate the number of nucleotide changes and genetic distances among Hysterothylacium taxa, and to determine the optimal model of nucleotide substitution for the dataset based on the Bayesian Information Criterion (BIC) scores. Phylogenetic analyses were carried out using Bayesian Inference (BI), Neighbor-Joining (NJ), and Maximum Likelihood (ML) methods based on sequences of ITS1-5.8S-ITS2 region. BI analyses were carried out using MrBayes 3.1.2 (Huelsenbeck & Ronquist 2001). For nodal support estimation based on Bayesian posterior probability the Metropolis-Coupled Markov Chain Monte Carlo process was run for 1 cold and 3 hot chains and 1,000,000 generations, with trees being sampled every 100 generations for a total of 10,000 trees in the initial sample and a burn-in of 25% (i.e. 2,500 trees). The Estimation Sample Sizes (ESS) of the model parameters were checked and the chains are converging. THAISSA D. SERRANO et al. CHARACTERIZATION OF Hysterothylacium FROM BRAZIL An Acad Bras Cienc (2023) 95(1) e20211046 4 | 14 Table I. List of nematodes whose sequences were used for analyses and those obtained in the present study. Gene rDNA (ITS1, 5.8S, ITS2). Parasite GenBank accession no. (gene rDNA) Host Locality Source Ascaris lumbricoides AB571298 Homo sapiens Japan Nadler & Hudspeth (2000) Hysterothylacium sp. VI MT635370 Platycephalus richardsoni Australia Unpublished Hysterothylacium sp. MT365529 Eledone sp. Italia Guardone et al. (2020) H. rigidum HF680324 Lophius piscatorius Ireland Unpublished Hysterothylacium sp. MF668813 Rachycentron canadum USA Unpublished H. fortalezae KX098563 Maurolicus weitzmani USA Andres et al. (2016) H. auctum AF115571 Zoarces viviparus Baltic Sea Szostakowska et al. (2001) H. aduncum JX845137 Zoarces viviparus Denmark Haarder et al. (2013) Hysterothylacium sp. HM437225 Gadus macrocephalus South Korea Unpublished H. fabri KX083575 Trigla lyra Italy Costa et al. (2018) Hysterothylacium sp. IV KD203841 - China Shamsi et al. (2013) Hysterothylacium sp. IV-A KP419719 Polydactylus sextarius China Zhao et al. (2016) Hysterothylacium sp. HM545895 Siganus fuscescens China Unpublished H. bidentatum AY603539 - Poland Unpublished H. reliquens MF062509 Boops boops Turkey Şimşek et al. (2018) Hysterothylacium sp. KX083577 Lophius piscatorius Italy Costa et al. (2018) H. thalassini JX982129 Priacanthus macracanthus China Liu et al. (2013) H. liparis KF601900 - China Guo et al. (2014) H. sinense KX084795 Conger myriaster China Unpublished Hysterothylacium sp. AM706344 Astroconger myriaster China Zhu et al. (2007) Hysterothylacium sp. MK039148 Ephippion guttifer Spain Rodríguez et al. (2019) Hysterothylacium sp. MW699927 Epinephelus diacanthus Iraq Unpublished H. zhoushanense KP326557 Lepidotrigla japonica China Zhao et al. (2016) H. amoyense KP252133 Halieutaea stellata China Zhao et al. (2016) H. amoyense KY081889 Otolithes ruber Persian Gulf Unpublished Hysterothylacium sp. MF668871 Scomberomorus maculatus USA Unpublished Hysterothylacium sp. MF668810 Coryphaena hippurus USA Unpublished Hysterothylacium sp. X KU594490 Sarda sarda Brazil Pantoja et al. (2016) Hysterothylacium sp. X KU594489 Priacanthus arenatus Brazil Pantoja et al. (2016) Hysterothylacium sp. X MW817239 Pomatomus saltatrix Brazil Present study Hysterothylacium sp. X MW817240 Pomatomus saltatrix Brazil Present study Hysterothylacium sp. VI MT635373 Platycephalus bassensis Australia Unpublished Hysterothylacium sp. VI MT635372 Platycephalus bassensis Australia Unpublished THAISSA D. SERRANO et al. CHARACTERIZATION OF Hysterothylacium FROM BRAZIL An Acad Bras Cienc (2023) 95(1) e20211046 5 | 14 NJ and ML analyses were performed in MEGA X. For NJ, and applying the Kimura 2-parameter with a gamma value of 0.5529 to estimate the genetic distances of the matrix (see results section), a total of 1,000 nonparametric bootstrap replicates were performed to assess the reliability of the nodes. Indels were not taken into account (complete deletion option activated). The ML method was also based on the Kimura 2-parameter model. Initial trees for the heuristic search were automatically obtained by applying Neighbor-Join and BioNJ algorithms (implemented in MEGA X) to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with a superior log-likelihood value. All positions containing gaps and missing data were eliminated. As for NJ analysis, the reliability of nodes was estimated using 1,000 nonparametric bootstrap replicates. Ascaris lumbricoides Linnaeus, 1758 (accession number AB571298) was used as an outgroup based on previous phylogenetic analyses related to Anisakis (Nadler & Hudspeth 2000, Nadler et al. 2005, Pantoja et al. 2015). RESULTS Morphology Hysterothylacium larval type V sensu Shamsi et al. (2013) Description (based on 10 specimens): thirdstage larvae. Three poorly developed lips, labial papillae not observed; tooth absent (Fig. 2a). Lateral alae present (Fig. 2b). Excretory pore situated immediately posterior nerve ring. Single Hysterothylacium sp. VI MT635371 Platycephalus bassensis Australia Unpublished H. deardorffoverstreetortum JF730204 Paralichthys isosceles Brazil Knoff et al. (2012) Hysterothylacium sp. V KU594488 Zenopsis conchifer Brazil Pantoja et al. (2016) Hysterothylacium sp. V KU594486 Paralichthys isoceles Brazil Pantoja et al. (2016) Hysterothylacium sp. V KU594485 Merluccius hubbsi Brazil Pantoja et al. (2016) Hysterothylacium sp. V KU594484 Menticirrhus americanus Brazil Pantoja et al. (2016) Hysterothylacium sp. V KU594483 Lagocephalus laevigatus Brazil Pantoja et al. (2016) Hysterothylacium sp. V KU594482 Gymnothorax vicinus Brazil Pantoja et al. (2016) Hysterothylacium sp. V KU594481 Caulolatilus chrysops Brazil Pantoja et al. (2016) Hysterothylacium sp. V MW826087 Pomatomus saltatrix Brazil Present study Hysterothylacium sp. V MW826088 Pomatomus saltatrix Brazil Present study Hysterothylacium sp. V MW826089 Pomatomus saltatrix Brazil Present study Hysterothylacium sp. V MW826090 Pomatomus saltatrix Brazil Present study Hysterothylacium sp. V MW826091 Pomatomus saltatrix Brazil Present study Hysterothylacium sp. V MW826092 Pagrus pagrus Brazil Present study Hysterothylacium sp. V MW826093 Pagrus pagrus Brazil Present study Hysterothylacium sp. V MW826094 Pagrus pagrus Brazil Present study Hysterothylacium sp. V MW826095 Pagrus pagrus Brazil Present study Hysterothylacium sp. V MW826096 Pagrus pagrus Brazil Present study Hysterothylacium sp. V KU594487 Prionotus punctatus Brazil Pantoja et al. (2016) Table I. Continuation. THAISSA D. SERRANO et al. CHARACTERIZATION OF Hysterothylacium FROM BRAZIL An Acad Bras Cienc (2023) 95(1) e20211046 6 | 14 small cuticular projection (mucron) present on terminal region of tail (Fig. 2c). Measurements from specimens infecting P. saltatrix were: 8.00 (1.00-9.63) length, 0.18 (0.15-0.32) width, 0.48 (0.160.65) nerve ring to anterior end, 0.55 (0.20-0.69) excretory pore to anterior end, 0.54 (0.18-1.17) esophagus length, 0.06 (0.02-0.08) ventricule length, 0.58 (0.25-0.69) ventricular appendix length, 0.07 (0.03-0.10) intestinal caecum length and 0.26 (0.16-0.32) tail lenght. The average measures presented in P. pagrus were: 9.67 (1.7811.64) length, 0.26 (0.12-0.40) width, 0.52 (0.340.66) nerve ring to anterior ring, 0.58 (0.22-0.75) excretory pore to anterior end, 0.63 (0.22-1.32) esophagus length, 0.09 (0.04-0.13) ventricule length, 0.78 (0.29-1.02) ventricular appendix length, 0.09 (0.05-0.20) intestinal caecum length, 0.33 (0.21-0.45) tail length (Table III). Host: Pomatomus saltatrix and Pagrus pagrus. Location: Santos, State of São Paulo, Brazil. Prevalence: 75% in P. saltatrix and 100% in P. pagrus. Mean abundance: 10.95± 0.56 in P. saltatrix and 3.57±0.8 in P. pagrus. Mean intensity: 14.6±0.75 in P. saltatrix and 3.57±0.8 in P. pagrus. Site of infection: intestine lumen Hysterothylacium larval type X sensu Shamsi et al. (2013) Description (based on 5 specimens): thirdstage larvae. Three poorly developed lips, unseen labial papillae; tooth absent (Fig. 1a). Excretory pore at level of nerve ring. The average measures presented were: 6.71 (4.01-8.92) length, 0.19 (0.12-0.5) width, 0.35 (0.11-0.46) nerve ring to anterior end, 0.38 (0.16–0.50) excretory pore to anterior end, 0.19 (0.12-0.61) esophagus length, 0.94 (0.53-0.99) ventricule length, 0.70 (0.43-0.81) ventricular appendix length, 0.54 (0.12-0.65) intestinal caecum length and 0.11 (0.5 -0.22) tail length (Table II). Tail presented four delicate spines at the rounded tip (Fig. 1b). Host: Pomatomus saltatrix. Location: Santos, State of São Paulo, Brazil. Prevalence: 15%. Mean abundance: 0.25± 0.05. Mean intensity: 1.67±0.33. Site of infection: intestine lumen. Molecular and phylogenetic analysis The BLAST search-related our haplotypes with Hysterothylacium sequences available from GenBank. The sequences of ten specimens (PH11, HT06, HT10, HT12, HT13, HT15, PH01, PH06, PH08, PH10) were identical to the Hysterothylacium type V species when edited and aligned sequences retrieved from GenBank, while two specimens (H301, H303) had identical sequences to those available for Hysterothylacium type X (Supplementary Material - Table SI). The nucleotide sequences obtained in the present study were deposited in GenBank (Accession numbers for 10 specimens identical to Hysterothylacium type V: MW826087-MW826096; accession numbers for Figure 1. Hysterothylacium type X (third-stage larva). a) Anterior end. b) Posterior end with mucrons. Bars: = 30 μm. THAISSA D. SERRANO et al. CHARACTERIZATION OF Hysterothylacium FROM BRAZIL An Acad Bras Cienc (2023) 95(1) e20211046 7 | 14 sequence alignment used had 979 bp using only positions that were unequivocally aligned in all taxa. All samples of Hysterothylacium type V were identical. The two samples of Hysterothylacium type X were also identical. Among the haplotypes of Hysterothylacium. type V and Hysterothylacium type X, the difference found was 0.092% about the sequences obtained in this study. The Kimura 2-parameter with a gamma value of 0.5529 was selected as the optimal model of nucleotide substitution rate using BIC scores. Phylogenetic analyses were based on the alignment of the ITS1-5.8S-ITS2 region sequences together with those of the Hysterothylacium genus and other Anisakidae and Raphidascarididae species of the GenBank database (see Accession Numbers on Table I). Many of the analyzed specimens (10) were assigned to Hysterothylacium Type V. All of them presented the haplotype which was identical to those described by Knoff et al. (2012) from the host Paralichthys isosceles Jordan, 1891, and the Hysterothylacium type V haplotypes described by Pantoja et al. (2016) in several species of fish of the Brazilian coast. The other specimens (2) analyzed were genetically related to H. amoyense (Hsü, 1933) and H. zhoushanense Li, Liu & Zang, 2012. The haplotypes of these two specimens were identical to the sequences KU594489 and KU594490 of Hysterothylacium type X larvae from Priacanthus arenatus Cuvier, 1829 and Sarda sarda (Bloch, 1793), respectively, captured in Brazil (Pantoja et al. 2016). However, this haplotype showed slight nucleotide differences from the previously described haplotypes of H. amoyense and H. zhoushanense. The three phylogenetic reconstructions (Fig. 3) methods used in the present study (i.e. BI, NJ, and ML) showed that haplotypes of Hysterothylacium sp. were related to Hysterothylacium larvae X, H. amoyense, and H. zhoushanense formed a two specimens identical to Hysterothylacium type X species: MW817239-MW817240). A 900 bp fragment of the ITS1-5.8S-ITS2 region was amplified for Hysterothylacium type V and Hysterothylacium type X it was 933 bp. The multiple Figure 2. Hysterothylaciumtype Vfound in the present study inPomatomus saltatrix. a) cephalic extremity of the body showing three poorly developed lips; b) lateral view showing the lateral ala (arrowed); c) posterior end of the body showing a single prominent terminal spine (mucron) (arrowed). THAISSA D. SERRANO et al. CHARACTERIZATION OF Hysterothylacium FROM BRAZIL An Acad Bras Cienc (2023) 95(1) e20211046 8 | 14 monophyletic group including the haplotype detected in samples of Hysterothylacium type X (01) and Hysterothylacium type X (03). DISCUSSION Hysterothylacium species are distributed worldwide and often found in fish (Mattiucci & Nascetti 2008, Mattiucci et al. 2014, Shamsi et al. 2016). The fish will be intermediate or final hosts for these parasitic nematodes. The effect of Hysterothylacium species on the fish will depend on the site of infection and the parasite abundance, causing tissue erosions and necrosis (Felizardo et al. 2009). Some authors also attribute zoonotic potential to Hysterothylacium spp. (Yagi et al. 1996, Moravec 1998). However, such speculations are controversial, mainly due to the few records of human infection by these parasites (Yagi et al. 1996, FernándezCaldas et al. 1998, Valero et al. 2003, GonzálezAmores et al. 2015). This panorama is also due to the lack of specific diagnostic techniques, and the knowledge regarding the zoonotic larval types responsible for causing human infection remains incomplete (Shamsi et al. 2018). Since Hysterothylacium larvae have problematic identification, morphological and molecular analyses are important for these purposes (Shamsi et al. 2011, 2013, 2015, 2018). In this sense, a single morphotype may exhibit wide morphometric variations (Shamsi et al. 2015). Such variations will depend on the characteristics of the hosts, the intensity of infection, and the parasite ontogeny since its development between the second and fourth larval stages directly influences the morphometric features (Pantoja et al. 2016). Therefore, it is not possible to determine the consistency of morphological characters is consistent for different stages of larval development (Shamsi et al. 2013). The type V third-stage larvae of Hysterothylacium found in this study showed morphological and morphometric similarities with the larvae described by Bicudo et al. (2005), Borges et al. (2012), Knoff et al. (2012), Saad et al. (2012), Jabbar et al. (2012), Shamsi et al. (2013), Pantoja et al. (2016) and Khammassi et al. (2020), collected in several species of marine fish from Brazil and other continents (Shamsi et al. 2013, Khammassi et al. 2020). The morphotype of the present study, even with a shorter body length, presented morphological structures proportionally similar to the specimens found by the cited authors. Table II. Morphometric comparison of larvae L3 of Hysterothylacium type X collected in Pomatomus saltatrix with larvae L3 of Hysterothylacium type X previously reported. All measurements are in mm. Present study Pantoja et al. (2016) Shamsi et al. (2013) Jabbar et al. (2012) Host Pomatomus saltatrix Priacanthus arenatus Lutjanus carponotatus Atherinomorus endrachtensis Length 6.71 (4.01-8.92) 6.4 (3.7–8.5) 2.78 - Body width 0.91 (0.50.12) 0.16 (0.12–0.21) 0.13 - Nerve ring 0.35 (0.11-0.46) 0.29 (0.21–0.40) - 0.23 Esophagus 0.19 (0.12-0.61) 0.63 (0.48–0.86) 0.40 0.40–0.60 (0.48) Ventricle 0.94 (0.53-0.99) 0.72 (0.40–0.10) - - Ventricular appendix 0.70 (0.43-0.81) 0.53-0.38 (0.66) 0.38 - Intestinal cecum 0.54 (0.12-0.65) 0.17 (0.12–0.24) 0.12 - Tail 0.11 (0.5 -0.22) - 0.10 0.12–0.20 (0.16) THAISSA D. SERRANO et al. CHARACTERIZATION OF Hysterothylacium FROM BRAZIL An Acad Bras Cienc (2023) 95(1) e20211046 9 | 14 The larvae identified as Hysterothylacium type V in the present study resembled the first description of Hysterothylacium type V described by Shamsi et al. (2013). The phylogenetic tree of the ITS1-5.8S-ITS2 region obtained in this study is well-founded and grouped with Hysterothylacium type V described by Pantoja et al. (2016) and with H. deardorffoverstreetorum described by Knoff et al. (2012). Hysterothylacium type VI described by Shamsi et al. (2013) showed some morphological characteristics similar to the Hysterothylacium type V specimens of the present study (undeveloped lips, absent flat tooth, blunt tail with a single terminal spine or mucron). In the studies by Shamsi et al. (2013) and Shamsi et al. (2015), Hysterothylacium type VI larvae have a surface with tiny spines and intestines with a sinusoidal pattern. The original morphological description of Hysterothylacium type V (Shamsi et al. 2013), as well as the Figure 3. Phylogenetic tree of the ITS1-5.8SITS2 region, evidencing the clustering of Hysterothylacium type V with other Hysterothylacium species available in Genbank and Hysterothylacium type X with Hysterothylacium type X species available from Genbank (MEGA 7). Samples from the present study are in bold.