Introduction
Members of the genus Andracantha (Schmidt, 1975) are considered to be typical components of helminth fauna of cormorants and shags (Phalacrocoracidae) (see Schmidt, Reference Schmidt1975; Presswell et al., Reference Presswell, García Varela and Smales2018). However, some Andracantha species have been recorded in diverse hosts, such as the brown pelican Pelecanus occidentalis L., the red-breasted merganser Mergus serrator L., the American bald eagle Haliaeetus leucocephalus L, and the little blue penguin Eudyptula novaehollandiae Forster (Schmidt, Reference Schmidt1975; Richardson & Cole, Reference Richardson and Cole1997; Laskowski et al., Reference Laskowski, Jezewski and Zdzitowiecki2008; Presswell et al., Reference Presswell, García Varela and Smales2018). Morphologically, species of Andracantha are identified by the presence of two fields of spines separated by a bare zone in the anterior part of the trunk, a cylindrical proboscis with a slightly swollen region, a cone-shaped neck, six or eight pyriform cement glands, usually arranged in bilateral pairs and eggs with or without polar protrusion in the middle fertilization membrane (Schmidt, Reference Schmidt1975; Presswell et al., Reference Presswell, García Varela and Smales2018). Based on these morphological features, the genus Andracantha currently comprises nine species: five of them were described in the Americas: Andracantha gravida (Alegret, 1941) (type species), Andracantha phalacrocoracis (Yamaguti, 1939), Andracantha mergi (Lundström, 1941), Andracantha baylisi (Zdzitowiecki, 1986) and Andracantha tandemtesticulata (Monteiro et al., 2006); three in Oceania: Andracantha clavata (Goss, 1941), Andracantha sigma (Presswell, García-Varela & Smales, Reference Presswell, García Varela and Smales2018) and Andracantha leucocarboi (Presswell, García-Varela & Smales, Reference Presswell, García Varela and Smales2018); and Andracantha tunitae (Weiss, 1914), which has been recorded in Africa and Europe (Schmidt, Reference Schmidt1975; Zdzitowiecki, Reference Zdzitowiecki1986; Monteiro et al., Reference Monteiro, Amato and Amato2006; Presswell et al., Reference Presswell, García Varela and Smales2018 Reference Presswell, García Varela and Smales2017).
As part of a long-term study on the diversity and distribution of parasites associated with aquatic birds of Mexico, adult specimens of A. gravida were collected from the double-crested cormorant Nannopterum auritus (Lesson) and the brown pelican P. occidentalis from two localities in the Gulf of Mexico: Celestún, Yucatan (south-eastern); and Punta Piedra, Tamaulipas (north-eastern). The aim of the current study was to typify newly collected specimens of A. gravida by combining morphological and molecular methods. In addition, we aimed to explore the genetic structure of the populations sampled using partial sequences of the cytochrome c oxidase subunit 1 (cox1) gene.
Materials and methods
Specimen collection
A total of 14 birds, nine double-crested cormorants and five brown pelicans, were examined. Eight cormorants were collected from Celestún, Yucatán (20°50′53.5″N, 90° 24′22″W), Mexico, in July 2006, while five pelicans and one cormorant were collected from Punta Piedra, Tamaulipas (24° 29′26″N, 97°45′01″W), Mexico, in July 2008. The digestive tract of the birds was dissected and placed in separate Petri dishes containing 0.75% saline and examined using a stereomicroscope. Acanthocephalans were placed in distilled water at 4°C overnight and subsequently preserved in molecular-grade ethanol. Birds were identified following a field guide (Howell & Webb, Reference Howell and Webb1995) and the American Ornithologist Union (1998) guidelines.
Morphological analyses
Selected adult acanthocephalans from brown pelicans and double-crested cormorants were gently punctured with a fine needle in the trunk, stained with Mayer's paracarmine, destained in 70% acid ethanol, dehydrated in a graded ethanol series, cleared in methyl salicylate and mounted in Canada balsam. Specimens were examined using a Leica DM 1000 light emitting diode (LED) compound microscope equipped with a bright field (Leica, Wetzlar, Germany). Measurements were taken using the Leica Application Suite microscope software and are presented in micrometres (μm). Acanthocephalans collected in the present study were identified following Schmidt (Reference Schmidt1975) and Presswell et al. (Reference Presswell, García Varela and Smales2018). For scanning electron microscopy, two adult specimens were dehydrated with an ethanol series, critical point dried, sputter coated with gold and examined with a scanning electron microscope (Hitachi Stereoscan Model S-2469N) operating at 15 kV from the Instituto de Biología, Universidad Nacional Autónoma de México. Voucher specimens were deposited in the Colección Nacional de Helmintos, Instituto de Biología, Universidad Nacional Autónoma de México, Mexico City, Mexico (accession number CNHE: 5997, 6834, 11834, 11835).
DNA isolation, amplification and sequencing
Eight adult acanthocephalans obtained from the double-crested cormorant (one from Punta Piedra and seven from Celestún) and five adult acanthocephalans from the brown pelicans from Punta Piedra were used for the molecular analyses. Acanthocephalans were placed individually in tubes and digested overnight at 56°C in a solution containing 10 mm Tris–hydrochloride (pH = 7.6), 20 mm sodium chloride, 100 mm ethylenediaminetetraacetic acid disodium salt (pH = 8.0), 1% sodium lauroyl sarcosinate and 0.1 mg/ml proteinase K. Following digestion, DNA was extracted from the supernatant using DNAzol reagent. A fragment partial fragment of the cox1 gene was amplified using the forward primer 5′-AGTTCTAATCATAA(R)GATAT(Y)GG-3′ and reverse primer 5′ -TAAACTTCAGGGTGACCAAAAAATCA-3′ (Folmer et al., Reference Folmer, Black, Hoeh, Lutz and Vrijenhoek1994). Polymerase chain reaction (PCR) consisted of 10 μl of each primer, 2.5 μl of 10× buffer, 2 mm MgCl2, 2 μl of the genomic DNA (10–20 ng) and 1 U of Taq DNA polymerase (Platinum Taq, Invitrogen Corporation, São Paulo, Brazil). PCR cycling parameters for the molecular marker consisted of denaturation at 94°C for 1 min, 35 cycles of 94°C for 1 min, 40°C for 1 min and 72°C for 1 min, followed by a postamplification incubation at 72°C for 10 min. Sequencing reactions were performed using ABI Big Dye (Applied Biosystems, Boston, Massachusetts) terminator sequencing chemistry and using an ABI 3730 capillary DNA sequencer reaction products were separated and detected. Contigs were assembled, and base-calling differences were resolved using Codoncode Aligner version 9.0.1 (Codoncode Corporation, Dedham, Massachusetts) and submitted to GenBank (table 1).
Table 1. List of acanthocephalans used in the phylogenetic analyses, with data on the life-cycle stage, host locality and GenBank accession number for mitochondrial cytochrome c oxidase subunit 1 (cox1).
A, adult; C, cystacanth; I, immature specimens. Newly generated sequences are presented in boldface type.
Alignments, phylogenetic analysis and haplotype network
Newly generated sequences were aligned with available sequences for other polymorphid acanthocephalans, including a partial sequence for an isolate of A. gravida from a double-crested cormorant from Mexico of García-Varela et al. (Reference García-Varela, Pérez-Ponce de León, Aznar and Nadler2009) (GenBank EU267822), retrieved from GenBank (see table 1), using Clustal W software (Thompson et al., Reference Thompson, Higgins and Gibson1994). A nucleotide substitution model was selected for the dataset using jModelTest version 2.1.7 (Posada, Reference Posada2008), applying the Akaike criterion. The best nucleotide substitution model was TPM3uf + G + I. Phylogenetic trees were inferred through maximum likelihood (ML) with the program RAxML version 7.0.4 (Stamatakis, Reference Stamatakis2006). A GTRGAMMAI substitution model was used, and 10,000 bootstrap replicates were run to assess nodal support. We also analysed our data in a Bayesian inference (BI) framework using MrBayes 3.2.2 (Ronquist et al., Reference Ronquist, Teslenko and Van Der Mark2012), with two Markov chain Monte Carlo runs for 10 million generations, sampling every 1000 generations, a heating parameter value of 0.2 and a burn-in of 25%. The resulting trees were visualized using FigTree version 1.4.2 (Rambaut & Drummond, Reference Rambaut and Drummond2007). Finally, uncorrected p distances were estimated using MEGA version 11 (Kumar et al., Reference Kumar, Stecher and Tamura2016).
To explore whether piscivorous birds from both localities in Mexico, i.e., Celestún (south-eastern) and Punta Piedra (north-eastern), share the same cox1 haplotypes, an unrooted statistical network was constructed using PopART (Leigh & Bryant, Reference Leigh and Bryant2015), with the minimum spanning network option (Bandelt et al., Reference Bandelt, Forster and Röhl1999). The degree of genetic differentiation between populations was estimated using the fixation index (F st) (see Hudson et al., Reference Hudson, Boos and Kaplan1992) with Arlequin v.3.5 (Excoffier & Lischer, Reference Excoffier and Lischer2010).
Results
Morphological identification (figs 1 and 2).
Acanthocephalans collected from double-crested cormorants and brown pelicans from the Gulf of Mexico were identified as A. gravida, following the key for Andracantha species of Presswell et al. (Reference Presswell, García Varela and Smales2018). In addition, the newly collected specimens show matching morphological characteristics with those assigned as A. gravida by Alegret (Reference Alegret1941) and Schmidt (Reference Schmidt1975), including: (a) pipe-shaped trunk without swellings; (b) testes parallel; (c) absence of small spines between the two field of spines on the foretrunk; (d) six cement glands; (e) hooks arranged in 15–16 rows with 10–12 hooks per row; (f) absence of genital spines in female; and (g) eggs with polar protrusion in the middle fertilization membrane (figs 1 and 2). In addition, our specimens exhibited variability from those previous descriptions in the following characters: trunk; proboscis; hooks; proboscis receptacle testes; and eggs with those previous descriptions (see table 2).
Fig. 1. Andracantha gravida from Nannopterum auritus. (a) Adult male, total view; (b) adult female total view; (c) proboscis; (d) egg.
Fig. 2. Scanning electron micrographs of Andracantha gravida from Nannopterum auritus. (a) Adult male ventral view; (b) adult male anterior trunk; (c) adult male posterior end; (d, e) adult male proboscis. Scale bars: (a, b) 1000 μm; (c) 200 μm; (d) 300 μm; (e) 200 μm.
Table 2. Comparative metrical data for males and females of Andracantha gravida. Measurements in micrometres, unless otherwise indicated.
n =number of specimens analysed.
Phylogenetic analyses and genetic structure
The cox1 dataset included 655 characters and 49 sequences. The phylogenies inferred with the ML and BI methods yielded Andracantha as a monophyletic group, but with weak bootstrap support and Bayesian posterior probabilities (fig. 3). This result was similar and consistent with previous phylogenetic assessments using the same molecular marker or with nuclear molecular markers (e.g. Presswell et al., Reference Presswell, García Varela and Smales2018; Sasaki et al., Reference Sasaki, Katahira, Kobayashi, Kuramochi, Matsubara and Nakao2019; García-Varela et al., Reference García-Varela, Masper, Crespo and Hernández-Orts2021; Santoro et al., Reference Santoro, Palomba, Gili, Marcer, Marchiori and Mattiucci2021; Hernández-Orts et al., Reference Hernández-Orts, Lisitsyna and Kuzmina2022). Our phylogenetic trees showed that Andracantha is divided into five main subclades. The first subclade was formed by two sequences of A. sigma (MF527034 and MF527035) from the little blue penguin E. novaehollandiae and the spotted shag Phalacrocorax punctatus (Sparrman). The second subclade was formed by six sequences of A. phalacrocoracis; one sequence for a cystacanth (LC465356) and one for an adult isolate (LC465403) both from the Japanese cormorant Phalacrocorax capillatus (Temminck & Schlegel) from Japan; three sequences (LC465396–398) from the pelagic cormorant Phalacrocorax pelagicus (Pallas); and one sequence of an immature isolate (MK119254) from a sea lion Zalophus californianus (Lesson) from California, United States. The third subclade was formed by two sequences of A. leucocarboi (MF527023 and MF527024) from an Otago shag Leucocarbo chalconotus (Gray) and a spotted shag P. punctatus, respectively. The fourth subclade includes two sequences of cystacanth isolates from fishes (LC465391 and LC465393) of an unidentified species of Andracantha from Japan. Finally, the fifth subclade was formed by our 13 newly generated sequences for adult isolates for A. gravida and the sequence of an adult isolate identified as A. gravida (EU267822) by García-Varela et al. (Reference García-Varela, Pérez-Ponce de León, Aznar and Nadler2009) from Yucatan, Mexico (fig. 3). The genetic divergence estimated among the isolates A. gravida from Mexico ranged from 0.0% to 2.2%. Finally, the genetic divergence among the four species of Andracantha (A. sigma, A. phalacrocoracis, A. leucocarboi, and A. gravida) plus an unidentified species of Andracantha ranged from 17 to 21%.
Fig. 3. Phylogenetic tree using maximum likelihood and consensus Bayesian inference for the cox1 dataset. Numbers near internal nodes show ML bootstrap percentage (BP) values and Bayesian posterior probabilities (BPP). Scale bars represent the branch length.
The haplotype network, inferred with 14 specimens and 655 characters, represented a total of 9 haplotypes (fig. 4). The most frequent haplotypes, that is, H3 (n = 4) and H9 (n = 2), were shared by the two bird populations analysed (Punta Piedra, Tamaulipas and Celestún, Yucatán). Haplotypes H1, H4, H7 and H8 were found in Punta Piedra (north-eastern Mexico) parasitizing double-crested cormorants and brown pelicans, while haplotypes H2, H5 and H6 were found in Celestún (south-eastern Mexico) parasitizing double-crested cormorants. The level of haplotype diversity (Hd = 0.912) was very high and nucleotide diversity was low (pi = 0.00955) between the two populations. The Fst value was low (0.06949) between isolates of A. gravida from the two sampled localities in Mexico.
Fig. 4. Median-joining network of samples of andracantha gravida built with the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. Each circle represents a haplotype, with size proportional to the haplotype's frequency in the populations.
Discussion
Andracantha gravida was described from the double-crested cormorant in Cuba by Alegret (Reference Alegret1941). Later, this species was redescribed from the same definitive hosts, and it was recorded in brown pelicans and in the neotropical cormorant Nannopterum brasilianus (Gmelin) in Florida, Texas, and Louisiana in the United States (Schmidt, Reference Schmidt1975; Fedynich et al., Reference Fedynich, Pence and Bergan1997; Robinson et al., Reference Robinson, Forbes, Hebert and McLaughlin2008) and in south-eastern Mexico (García-Varela et al., Reference García-Varela, Pérez-Ponce de León, Aznar and Nadler2013). The current records of A. gravida in brown pelicans and double-crested cormorants in north-eastern Mexico represent new locality records of this acanthocephalan species. The morphological characteristics in combination with morphometric data of our specimens fit the descriptions of A. gravida provided by Alegret (Reference Alegret1941) and Schmidt (Reference Schmidt1975). However, we found morphometric variation in the acanthocephalans collected mainly regarding the size of the trunk, proboscis, hooks and proboscis receptacle in males and females (table 2). In addition, all the females recovered in both definitive hosts lacked genital spines (see figs 1a–d and 2a–d), and only two males and two females yielded ventral somatic spines in the hindtrunk, which were also described by Schmidt (Reference Schmidt1975), as genital spines. However, these spines are not genital, because they are displaced to the small field on ventral surface of the hindtrunk. It is well known that the presence or absence of ventral somatic spines in the hindtrunk and genital spines could be due to intraspecific phenotypical variation and represent adaptative features that improve attachment to the host (see Goss, Reference Goss1940; Johnston & Best, Reference Johnston and Best1942; Zdzitowiecki, Reference Zdzitowiecki1991; Aznar et al., Reference Aznar, Crespo, Raga and Hernández-Orts2016). Somatic spines have been traditionally used as a diagnostic morphological character to distinguish species of Andracantha and Corynosoma (Schmidt, Reference Schmidt1975; Aznar et al., Reference Aznar, Pérez Ponce de Leon and Raga2006; Presswell et al., Reference Presswell, García Varela and Smales2018). However, Presswell et al. (Reference Presswell, García Varela and Smales2018) mentioned that somatic spines should be considered with caution to delineate species in Andracantha because it is a variable morphological feature.
Our phylogenetic analyses showed that the genus Andracantha is monophyletic, and it was divided into five main subclades, representing four recognized species (A. sigma, A. phalacrocoracis, A. leucocarboi and A. gravida) and an undescribed species of Andracantha. However, a main subclade was formed with the new 13 sequences of A. gravida plus one isolate previously identified as A. gravida and available in the GenBank dataset (EU267822) (see fig. 3). The intraspecific genetic divergence among all isolates was very low, ranging from 0.0% to 2.2%. The genetic divergence found was consistent with previous studies. For instance, the genetic divergence detected among ten adults of A. sigma recovered from three definitive hosts, the Otago shag L. chalconotus, spotted shag P. punctatus and little blue penguin E. novaehollandiae from New Zealand, ranged from 0.00% to 0.32% (Presswell et al., Reference Presswell, García Varela and Smales2018); that among three adults of A. leucocarboi recovered from two definitive hosts, the Otago shag and spotted shag from New Zealand, ranged from 0.00% to 1.38% (Presswell et al., Reference Presswell, García Varela and Smales2018). Finally, the genetic divergence among cystacanths and adults of A. phalacrocoracis recovered from the Pacific rainbow smelt Osmerus dentex Steindachner & Kner, the Japanese cormorant P. capillatus and the pelagic cormorant P. pelagicus from Japan was 0.013% (Sasaki et al., Reference Sasaki, Katahira, Kobayashi, Kuramochi, Matsubara and Nakao2019).
The haplotype network analysis of cox1 sequences inferred with 14 sequences of A, gravida revealed the presence of nine haplotypes, two of which (H3 and H9) were shared between the two populations sampled, and seven were unique haplotypes (fig. 4). The Fst value was low, suggesting genetic flow between both populations (north-eastern and south-eastern Mexico), which may be explained by the migration of the brown pelican and the double-crested cormorant, which are definitive hosts in the Gulf of Mexico.
Of the nine recognized species of the genus Andracanatha, seven species have been recorded in phalacrocoracids (cormorants and shags), representing 77. 7% of the biodiversity of the genus Andracantha, suggesting that phalacrocoracid could represent an ancestor host with an independent colonization event to other birds, such as pelicans, mergansers, eagles and penguins (Nickol & Kocan, Reference Nickol and Kocan1982; Richardson & Cole, Reference Richardson and Cole1997; Presswell et al., Reference Presswell, García Varela and Smales2018).
Acknowledgements
We thank Laura Márquez and Nelly López Ortiz from LaNabio for their help during the sequencing of the DNA fragments. We also thank Berenit Mendoza Garfias for her help obtaining scanning electron microphotographs.
Financial support
This research was supported by grants from the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT-Universidad Nacional Autónoma de México (UNAM)) No. IN201122 and the Institute of Parasitology, BC CAS (RVO: 60077344). ALJ and MTGG thanks the support of the Programa de Posgrado en Ciencias Biológicas, UNAM and Consejo Nacional de Ciencia y Tecnología, Mexico (ALJ. CVU. No. 706119; MTGG CVU No. 956064), for granting a scholarship to complete her PhD and his Master programme, respectively.
Conflicts of interest
None.
Ethical standards
The sampling in this work complies with the current laws and animal ethics regulations of México. Specimens were collected under the Cartilla Nacional de Colector Científico (FAUT 0202) issued by the Secretaría del Medio Ambiente y Recursos Naturales (SEMARNAT), to MGV.
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