Morphological and molecular characterization of Contracaecum australe (Nematoda: Anisakidae) parasitizing Phalacrocorax brasilianus (Aves: Phalacrocoracidae) on the north coast of Brazil

Abstract For the first time in Brazil, Contracaecum australe is recorded parasitizing Phalacrocorax brasilianus (Aves, Suliformes, Phalacrocoracidae) from the Marine Extractive Reserve of Soure on Marajó Island, Brazilian Amazon. Its morphology revealed a body with a transversally striated cuticle, smooth or slightly cleft interlabia, lips with auricles, labial papillae, and conspicuous amphids. In males, the presence of the median papilla on the upper lip of the cloaca and spicules that reach almost half of the body of the parasite. These morphological characters, added to the number and distribution of the pre- and postcloacal papillae of the male specimens, and supported by the molecular phylogeny from the analysis of the ITS-1, 5.8S and ITS-2 genes, allowed the identification of these parasites.


Introduction
Nematodes of the Anisakidae family infect a variety of aquatic organisms at various developmental stages of their life cycle (Anderson, 2000). Among the anisakids, the genus Contracaecum Railliet & Henry, 1912, stands out, having been recorded in several locations on the planet (Biolé et al., 2012). They are parasites described in freshwater, brackish, and marine ecosystems that use fish as intermediate and/or paratenic hosts and aquatic mammals and piscivorous birds as definitive hosts (Anderson, 2000;Mattiucci & Nascetti, 2008;Shamsi et al., 2009a;Garbin et al., 2011;Shamsi, 2014).
The species C. australe was described for the first time in lagoon Santa Elena in Chile, as a parasite of P. brasilianus, using morphological and molecular analyses (Garbin et al., 2011). Biolé et al. (2012), recorded the species on the same host in central Argentina and later P. gaimardi Lesson & Garnot, 1828 was added as a new host for C. australe, the southernmost record of the species in Argentina, thus expanding its geographical distribution and definitive host range (Garbin et al., 2014).
Given the above, this study aims to investigate the nematode parasites of Phalacrocorax brasilianus from the Marine Extractive Reserve of Soure, Marajó Island, Pará, employing the perspective of integrative taxonomy.

Material and Methods
From 2020 to 2022, twenty specimens of P. brasilianus were obtained from the coastal zone of the municipality of Soure (Marine Extractive Reserve of Soure) on the Marajó Island, Pará, Brazil (Figure 1) (Latitude: -0.742862°, Longitude: -48.507732°). The birds are used as an alternative source of food by fishers in the region, who kindly provided the dead birds that were used in this study. The animals were transported individually in bags and kept refrigerated in isothermal boxes with ice for transport to the Laboratory. In the laboratory, each organ was individualised in a Petri dish containing 0.9% NaCl saline solution and analysed under a stereomicroscope (Leica ES2), cleaned, quantified, and stored in AFA solution (93 parts of 70% ethyl alcohol, 5 parts of formaldehyde, and 2 parts of glacial acetic acid) for morphological studies, and representative specimens were fixed in 70% ethyl alcohol for molecular analyses.

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Contracaecum australe in Brazil Light microscopy For morphology, the nematodes were clarified in Aman Lactophenol 70%, observed under a microscope (LEICA DM 2500) with a digital capture system (LEICA ICC50 HD) and using the Leica Application Suite software version 4.4.0, being drawn under a microscope (LEICA DM 2500) with attached camera lucida, from which photomicrographs and morphological drawings were respectively obtained. For morphometric analyses, fifteen adult males and fifteen gravid females were used, measuring twenty eggs in each female. Measurements are given in millimetres, unless otherwise indicated, and are presented as mean values followed by minimum and maximum values in parentheses. The taxonomic classification of nematodes was performed according to Baruš et al. (1978), Fagerholm (1991), and Garbin et al. (2011). The ecological indices of parasitism were calculated according to Bush et al. (1997) and Bautista-Hernández et al. (2015).

Scanning Electron Microscopy
Twelve nematodes (eight males and four females) were washed in distilled water for 1 hour, post-fixed in 1% Osmium Tetroxide (OSO 4 ) for 2 hours, and then submitted to dehydration in an increasing series of ethanol from 70% ethanol until 100% for 1 hour in each battery of alcohol, subsequently subjected to the critical point of CO 2 , mounted on metallic aluminium supports (stubs), metallized with gold+palladium, and analysed in a scanning electron microscope (VEGA 3 LMU/TESCAN).

Molecular and Phylogenetic Analyses
Four adult nematodes (two males and two females) fixed in 70% ethyl alcohol had a fragment of approximately 5 mm from the central region removed after measuring the total length of the specimens for allocation to molecular analyses by sequencing the regions of the first and second internal spacers transcribed from ribosomal DNA (ITS-1, 5.8S, and ITS-2). The rDNA extraction was performed at the Biomolecular Technology Laboratory of the Federal University of Pará, using a DNA extraction kit (Spin Tissue Mini Kit, Stratec®), following the protocol indicated by the manufacturer.
The final reaction volume was 25 μL, with 2.5 μL of reaction buffer (BUFF), 1 μL of MgCl2, 2 μL of dNTP's, 0.5 μL of each primer, 0.2 μL of Taq-DNA polymerase unit, 17.3 μL of H2O, and 1 μL of extracted DNA. The samples were processed in a thermal cycler (Applied Biosystems™ ProFlex™ PCR System, 3 x 32-well) and subjected to the following conditions: 95ºC for 5 minutes followed by 35 cycles at 95ºC for 1 minute (denaturation), 56ºC for 1 minute (annealing), 72ºC for 1 minute (extension), and a final extension at 72ºC for 7 minutes. The amplicons were submitted to 1.5% agarose gel electrophoresis. The PCR product was purified with ExoSAP-ITTM, quantified in Nanodrop equipment, and sequenced using NC5 and NC2 primers in AB 3500 Genetic Analyzer equipment, generating approximately 700 nucleotides each.
For phylogenetic reconstruction, alignment was performed with sequences of ribosomal genes available in GenBank having the ITS-1, 5.8S, and ITS-2 regions or the ITS-1 and ITS-2 interval regions using the BioEdit programme (7.2.5). Bayesian inference (BI) analysis were used based on Markov Chain Monte Carlo (MCMC) tree searches performed with MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). Two parallel runs of four simultaneous MCMC searches, each with ten million generations, were performed, with a tree being sampled every 500 generations. Results from the first 1000 trees were discarded as burn-in. The remaining trees were analysed in MrBayes to estimate the posterior probability of each node in the phylogenetic reconstruction. The evolutionary model of nucleotide substitution was determined by the Bayesian Information Criterion (BIC) with the JModelTest programme (Posada, 2008), and the most appropriate model chosen was TPM2uf+G. Genetic distances were determined for sequences from the ITS-1, 5.8S, and ITS-2 regions of Contracaecum species using PAUP 4.0b (Swofford, 1998).

Morphological data
Examination of the specimens revealed morphological characters that resemble descriptions in the literature (Garbin et al., 2011(Garbin et al., , 2014Biolé et al., 2012). Below is the morphological characterization. Morphological and morphometric data for C. australe are presented in Table 1.
General Morphology (based on 42 specimens): Body totally striated transversely. Very evident cephalic collar with a V-shaped lateral region without striations (Figures 2A, 3A, 3B, 4C, 5A, 5B). Three smooth or slightly cleft interlabia reach 4/5 of the length of the lips. Excretory pore is located immediately below the ventral interlabia. Lips longer than wide, each lip bearing three notable medial apical notches ( Figures 3B, 4C), with two conspicuous lobed auricles directed laterally. Dorsal lip with two large laterally directed papillae ( Figures 4C, 5B). Ventrolateral lips with one large papilla and a very evident amphid, displaced to the lateral line of the body ( Figure 4C). Buttonshaped deirids located at the level of the nerve ring or immediately posterior (   Mean intensity and range: 43.7 (7-360).

Molecular data
In our study, the tree topology derived from the phylogenetic analyses inferred from the ITS-1, 5.8S, and ITS-2 intergenic regions of the rDNA of the molecularly analysed specimens (GenBank accession number: OQ397677), demonstrated a 100% correspondence with C. australe (Figure 2), grouping it in the same clade and showing it to be distinct from the other species previously genetically characterized and considered for comparison purposes. Parasitic specimens of P. brasilianus from Brazil matched previously reported sequences for the ITS-1 and ITS-2 genes of C. australe characterised in Chile by Garbin et al. (2011) and deposited in GenBank under accession numbers (ITS-1: HQ389545; ITS-2: HQ389547).  A matrix of genetic distances based on the ITS-1, 5.8S, and ITS-2 sequences (2-parameter index from Kimura, 1980) between members grouped according to tree topology is presented in Table 2. The genetic distances between the taxa studied ranged from 0.012 to 0.064. The values between C. rudolphii C and C. ogmorhini (0.012) were the lowest observed in this study.

Discussion
In this study, nematodes recovered from the proventriculus and ventriculus of P. brasilianus on the north coast of the State of Pará, presented morphological characters compatible with C. australe (Garbin et al., 2011;2014;Biolé et al., 2012), making it possible to assign them to this specific taxon, which was found parasitizing P. brasilianus from the Marine Extractive Reserve of Soure, Pará-Brazil.
Among the Contracaecum species that occur in Brazil, this is the first record of C. australe in the national territory. This species was described by Garbin et al. (2011) in the Santa Elena lagoon in Chile as a parasite of P. brasilianus using morphological and molecular techniques. Biolé et al. (2012) recorded the species on the same host in the central Argentina region, and later P. gaimardi was added as a new host for C. australe in the southernmost record of the species in Argentina, thus expanding its geographical distribution and definitive host range (Garbin et al., 2014).         Garbin et al. (2011), when describing C. australe based on morphological characters considered diagnostic for the species of the genus (sensu Hartwich, 1964), such as the length of the spicules, morphology of the distal end of the spicule, and the presence of a slit in the interlabial tip, reported that, a priori, this parasite species of P. brasilianus from Chile could be easily attributed to C. rudolphii lato sensu. However, after the morphological comparison of the new specimens and due to the presence of characters such as a well-marked constriction in the tail just after the pairs of paracloacal papillae, presence of a median plaque (median papilla), parasites being apparently smaller, more robust and presenting longer spicules than C. rudolphii s.l., and supported by phylogenetic analyses of sequences from multiple loci, it was confirmed as a highly supported clade distinct from the rest of the Contracaecum taxa considered, thus validating its specific status.

Phalacrocorax brasilianus
Observing the morphological characteristics of C. australe and comparing them to their parasitic congeners of birds, we can see that this species can be easily distinguished from several of them using morphological characters with high diagnostic value (Fagerholm 1988(Fagerholm , 1991Moravec & Scholz, 2016), such as C. multipapillatum s.l.; C. pyripapillatum; C. overstreeti; C. gibsoni; C. bancrofti; C. spasskii; C. tricuspis; C. mexicanum; C. ovale; C. heardi; C. variegatum, C. travassosi that have one or more pairs of double postcloacal caudal papillae. However, sometimes this morphological differentiation is more difficult and requires the use of distinctive diagnostic characters together, such as body length/body width ratio, body length/spike length ratio, and oesophagus length/ventricular appendage length ratio, among others. Garbin et al. (2011Garbin et al. ( , 2014, to differentiate between species, such as between C. australe and C. rudolphii complex, as these species have a large similarity in the number and pattern of distribution of caudal papillae. According to Moravec & Scholz (2016), the shape and length of cephalic structures, such as lips and interlabia, the number and arrangement of pre-and postcloacal papillae, as well as the shape of the distal end of the spicules, are taxonomic features that distinguish between C. rudolphii and three other congeners parasites of birds, C. microcephalum, C. micropapillatum, and C. variegatum. Currently, the C. rudolphii complex has six described cryptic species (C. rudolphii A, B, C, D, E, and F) (D'Amelio et al., 2007(D'Amelio et al., , 2012Shamsi et al., 2009b).
Contracaecum australe can be differentiated from C. fagerholmi and C. rudolphii F described by D' Amelio et al. (2012), by the length of the spicules that vary from (4.15-4.85 and 5.96-7.30 mm), respectively, as opposed to (9.6-15.88 mm) in C. australe described by Garbin et al. (2011), values like those found in the present study (9.54-13.91 mm). However, Biolé et al. (2012), when noting the occurrence of C. australe in P. brasilianus in Argentina, saw morphometric variations that were considered intraspecific variations, until molecular studies can prove otherwise or corroborate their results. The authors observed a more anterior position of the nerve ring and deirids, a smaller ventriculus and ventricular appendix, a greater number of precloacal papillae, pre-equatorial location of the vulva, and smaller size of the eggs. Garbin et al. (2014), when adding a new host parasitized by C. australe also in Argentina (P. gaimardi), pointed out morphometric variations that partially corroborated the findings of Biolé et al. (2012), such as greater amplitude in the number of precloacal papillae, smaller size of the ventriculus and ventricular appendix in male specimens, more pre-equatorial vulva and smaller size of eggs in females. However, the most important morphometric difference occurred in the length of the spicules (7.2-10.44 mm), which were almost a third shorter than those described in specimens found in P. brasilianus from Chile (9.6-15.88 mm) and in the present study (9.54-13.91 mm). And as we can observe within the congener species of Contracaecum parasites of piscivorous birds, there is a wide range of variation in several metric characters that allow the fitting of multiple species and make it difficult to clearly differentiate.
Contracaecum australe can be differentiated from C. jorgei, also recorded in P. brasilianus (Syn. Nannopterum brasilianus), by the greater length of the spicules (9.54−13.91 vs 2.03-3.63) and the greater number of precloacal papillae (27-38 vs 26), respectively. Furthermore, when describing the species, Sardella et al. (2020) reported the presence of two papillae on the ventrolateral lips, as well as the dorsal lip being longer than the ventrolateral lips, whereas in C. australe the lips are the same size and the ventrolateral lips have only one labial papilla and a phasmid in each (Garbin et al., 2011). These results are similar to those found in the present study for the species.
According to Garbin et al. (2011), morphological analyses and differential diagnosis of male specimens of C. australe allowed the detection of differences in several characters, including the length of the spicule, the peculiar shape of the male tail, the disposition of the paracloacal papillae, and the depth and shape of the cleft in the interlabium. As for the characters mentioned by the authors, such as caudal constriction after the paracloacal papillae and presence of the median plaque (median papilla), they have not proven to be strong characters for differentiating, for example, between C. australe and C. rudolphii s.l., since, despite not having been described by some authors, they seem to be clearly present in their illustrations or even photomicrographs. (See, for example, Abollo et al., 2001;Amato et al., 2006;D'Amelio et al., 2012;Moravec & Scholz, 2016), as previously stated by the authors (Garbin et al., 2011). Garbin et al. (2014) suggested that the specimens described by Amato et al. (2006) as C. rudolphii parasites of P. brasilianus in Brazil may be C. australe, because they share certain morphological characteristics (such as lips, interlips, arrangement, and number of caudal papillae) and the same host. However, when taking into account the length of the spicules and the BL/SL ratio between (C. rudolphii 4.5-8.2 mm and 3.8-4 Amato et al., 2006) vs (9.6-15.88 mm and 1.45-1.79 C. australe Garbin et al., 2011) and (9.54−13.91 mm and 2.07-2.08 C. australe present study), we can see that these species clearly differ and show that these morphological characters seem to be the most consistent ones for differentiating between species, given that, so far, the species of the C. rudolphii complex described molecularly and morphologically have smaller spicules than C. australe. C. rudolphii D 3.90-6.99 mm; C. rudolphii E 5.53-6.13 mm Shamsi et al. (2009b); C. rudolphii F 5.96-7.3 mm D' Amelio et al. (2012), and higher BL/SL ratios in the species of the C. rudolphii complex (C. rudolphii D 3.7-3.8; C. rudolphii E 4.3-4.4; C. rudolphii F 2.5-2.7) than in C. australe (1.45-1.79 Garbin et al. 2011; 2.07-2.08 present study). The sister species C. rudolphii A, B, and C, despite having been characterised molecularly, have not been morphologically described, and only the length of the spicules is available for these species (D'Amelio et al., 2007;. We agree with Garbin et al. (2014) when they state the need to review the specimens described by Amato et al. (2006) and, if possible, evaluate them molecularly to complement the morphological diagnosis of these parasites. As previously seen, P. brasilianus is a bird capable of harbouring multiple species of Contracaecum at the same time (Lent & Freitas, 1948), raising the possibility that this bird presents co-infection with C. australe and C. rudolphii s.l., and/or more species of the genus in the same individual (Ricardo et al., unpublished data).
For Sardella et al. (2020), the use of molecular techniques is fundamental not only for defining the taxonomic status of these species but also for enabling their recognition. Result observed in the topology of the tree, derived from the inferred phylogenetic analysis of the ITS-1, 5.8S and ITS-2 genic regions of the rDNA of four specimens analysed molecularly in our study, being observed 100% of correspondence with C. australe described in Chile (Garbin et al., 2011), if grouping in the same corresponding clade the previously reported sequences for the ITS-1 and ITS-2 genes deposited in GenBank.
In our study, the clade formed in the phylogenetic tree by C. australe specimens was distinct from all Contracaecum species previously genetically characterized and considered for comparison purposes. In the phylogenetic analyses, it was possible to observe that C. chubutensis was the species that was genetically closest to C. australe, with a distance of 0.021, but forming two distinct clades. This genetic proximity can be justified by both species being parasites of birds (Phalacrocoracidae) and having the same biogeographical distribution (See Figure 6). However, the smallest genetic distance seen in our study occurred between the species C. rudolphii F and C. ogmorhini (0.004). Small genetic distances were also observed between C. ogmorhini and C. rudolphii E (0.007) and between C. rudolphii E and C. rudolphii F (0.009). Furthermore, the analyses of the data from the ITS-1, 5.8S, and ITS-2 sequences of C. australe from Brazil supported its distinction from the cryptic species of the C. rudolphii complex, corroborating the results found by Garbin et al. (2011).

Conclusion
Phalacrocorax brasilianus from the north coast of Brazil is the definitive host of C. australe; this is the first record of the species in the national territory. In this study, we have expanded the biogeographical distribution of this parasite, in addition to highlighting the need for the application of integrative taxonomy for the characterization of species of Contracaecum.