Phylogenetic relationship between Contracaecum spp. (Nematoda, Anisakidae) parasitizing cormorants (Aves, Phalacrocoracidae) from Argentina

doi:10.21203/rs.3.rs-3209950/v1

Download PDF

Research Article

Phylogenetic relationship between Contracaecum spp. (Nematoda, Anisakidae) parasitizing cormorants (Aves, Phalacrocoracidae) from Argentina

https://doi.org/10.21203/rs.3.rs-3209950/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 19 Dec, 2023

Read the published version in Parasitology Research →

You are reading this latest preprint version

Anisakidae nematodes have a wide host and geographic distribution. Species of the genus Contracaecum parasitize aquatic organisms such as piscivorous birds and mammals as their definitive hosts. Several Contracaecum species have been reported parasitizing Phalacrocoracidae in South America. The aim of this work is to highlight phylogenetic relationships between Contracaecum species parasitizing shags based on both molecular markers and the papillae pattern arrangement on the male tail. Some Contracaecum specimens parasitizing Red-legged cormorants from the Ría Deseado (RD), and other nematodes parasitizing eight dead Neotropic cormorants from San Miguel del Monte lagoon (SMML), Argentina, were collected and analyzed. An integrative analysis allowed us to recognize Contracaecum chubutensis parasitizing Phalacrocorax gaimardi, and Contracaecum australe parasitic in Phalacrocorax brasilianus. According to obtained mtDNAcox2, ITS1, ITS2, and SSrRNA isolates, Contracaecum sp. parasitizing P. gaimardi matched with the previously reported C. chubutensis parasitizing P. atriceps from Bahía Bustamante, Chubut province. Likewise, Contracaecum sp. isolates parasitizing P. brasilianus clustered with C. australe from Chile. Besides, the papillae pattern arrangement of the male tail allowed us to understand the interspecific and genetic relationships between the Contracaecum species. The analysis confirm that C. chubutensis specimens parasitizing P. gaimardi from RD represent a new host and the southernmost record for the species. Whereas, those C. australe specimens parasitizing P. brasilianus from SMML provide a new geographical record for the species and the extension of its distribution range. Present results also confirm the inland and marine distribution of C. australe and C. chubutensis, respectively.

Contracaecum

Phalacrocoracidae

ITS regions

Cox2 gene

SSrRN

Anisakidae nematodes have a wide host and geographic distribution. In particular, the species of the genus Contracaecum Railliet and Henry 1912, parasitize aquatic organisms in different parts of the world (Rhode 2005). Their life cycles generally include aquatic invertebrates and fish as intermediate and/or paratenic hosts, and piscivorous birds and mammals as definitive hosts (Anderson 2000). Several species of Contracaecum spp. have been reported parasitizing Phalacrocoracidae species in South America so far (Garbin et al. 2023). Among them, five species were reported from shags in Argentina: Contracaecum pelagicum Johnston and Mawson 1942, Contracecum travassosi Guitiérrez 1943, and Contracecum chubutensis Garbin, Diaz, Cremonte, and Navone 2008, parasitizing the Imperial shag Phalacrocorax atriceps (King 1828) from the north Patagonian marine coasts (Gutiérrez 1943; Garbin et al. 2008, 2013), Contracaecum australe Garbin, Mattiucci, Paoletti, González-Acuña, and Nascetti 2011, and Contracaecum jorgei Sardella, Mancini, Salinas, Simões and Luque 2020, parasitizing the Neotropic cormorant Phalacrocorax brasilianus (Gmelin 1789) in inland wetlands from Córdoba, Argentina (Garbin et al. 2011; Biolé et al. 2012; Sardella et al. 2020). In addition, specimens described as C. australe was reported parasitizing the Red-legged cormorant Phalacrocorax gaimardi (Lesson and Gamot 1828) from the Ría Deseado, Santa Cruz province, Argentina (Garbin et al. 2014). Four of the mentioned species were characterized by molecular markers to date, i.e. C. pelagicum, C. chubutensis, C. australe, and C. jorgei (Garbin et al. 2011, 2013; Sardella et al. 2020).

Morphological characterization of the Contracaecum species is sometimes difficult due to the overlap of diagnostic characters and often the poor material fixation which has an impact on the nematode cuticle. Therefore, misdiagnosis of the species can occur.

The Red-legged cormorant is distributed from Northern Peru along the Pacific Ocean coast to southern Chile (Frere et al. 2004), and on the Atlantic Ocean coasts, restricted to the Santa Cruz province, Argentina (Gandini and Frere 1995; Zavalaga et al. 2002, Frere et al. 2005). The Neotropic cormorant lives in both freshwater and marine environments (Harrison, 1985), and is widely distributed in southern South America, i.e., Argentina and Chile to north of Texas, USA (Araya and Millie 1991; Telfair and Morrison 1995). The imperial cormorant (both color morphs ‘‘atriceps’’ and ‘‘albiventer’’ sensu Rasmussen 1994) is one of several that breed along the southern coast of South America (Malacalza and Bertellotti 2001; Punta et al. 2003).

The aim of the present paper is to highlight the phylogenetic relationships between species of Contracaecum parasitizing shags from two different localities of Argentina based on both molecular markers and the papillae pattern arrangement on the male tail, also considering their inland or marine distribution.

Sampling

Three dead Red-legged cormorants were collected on the Ría Deseado shore (47°45'S, 65°56'W) near Puerto Deseado town, Santa Cruz province, Argentina, from September 2009 and March 2011. Some extracted Contracaecum specimens from P. gaimardi -identified previously as C. australe- were re-examined (see Garbin et al. 2014). In addition, eight dead Neotropic cormorants P. brasilianus from San Miguel del Monte lagoon (35°27'30" S; 58°48'15" W), Buenos Aires province, were collected from March 2011 to August 2016, and necropsied. Nematodes found in the oesophagus and stomach from both shags were collected, 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

Some nematode specimens from each host species were cleared with lactophenol and studied with an optical microscope (OM) Olympus BX51. Some specimens were dehydrated in ethanolic graduations, treated with the critical point technique, mounted, and metalized for observation under a scanning electron microscope (SEM) (JEOL JSV 6063 LV®, Tokyo, Japan). Parasite measurements are expressed in mm (unless otherwise indicated) with mean values followed by the range in parentheses. For taxonomical determination, nematodes were analyzed according to the diagnostic features of the family Anisakidae, the classification criteria of Fagerholm (1990) regarding to the papillae pattern arrangement on the male tail, and specific bibliography (Hartwich 1964; Garbin et al. 2019). Prevalence and mean intensity were calculated according to Bush et al. (1997). Voucher specimens were deposited in the Helminthological Collection of the Museo de La Plata (MLP He ………), Buenos Aires, Argentina.

DNA extraction, amplification, and sequencing

DNA of 4 males and 4 females specimens parasitizing the Red-legged cormorant from the Ría Deseado, and 4 specimens parasitizing one Neotropic Cormorant individual from the San Miguel del Monte lagoon was extracted using Wizard® Genomic DNA Purification Kit (Promega, USA) according to the manufacturer’s instructions. The mtDNA cox2 gene from each Contracaecum specimen 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 (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. Sequences were edited and aligned using Chromas v2.6.6® and Gap v4.11.2® (Bonfield et al., 1995) and then compared to the NCBI database using BLAST version 2.2.26 to identify sequences with high similarity to DNA sequences obtained. The nucleotide sequences of both Contracaecum species reported in the present study are available from the DDBJ/EMBL/GenBank databases under the accession numbers (MZ605037-9, MZ603426-8, MZ579647, and MZ6342121-2).

Phylogenetic analysis

GenBank sequences of nuclear (ITS-1 and ITS-2), and mitochondrial (SSrRNA and cox2) markers of Anisakidae species were aligned with the sequences newly obtained (Table 2). Since there were different taxa available for these markers, they were analyzed separately. Only the ITS-1 and ITS-2 regions could be concatenated. Alignment of the sequence data was done using Clustal W implemented in MEGAX and the resulting output was adjusted manually to check and edit inconsistencies (Kumar et al. 2018). Aligned sequence matrices of ITS-1 and ITS-2 regions were concatenated with Mesquite software v.3.04. The best-fit partitioning scheme and substitution models were evaluated using PartitionFinder v. 1.1.1 (Lanfear et al. 2012).

Phylogenetic analyses were conducted using the Maximum Likelihood (ML) algorithm, implemented in MEGAX (Kumar et al. 2018), and the Bayesian Inference (BI), implemented in MrBayes Ver. 3.2.6 software (Ronquist and Huelsenbeck 2003). Nodal support of ML analysis was estimated using nonparametric bootstrapping of 500 pseudoreplicates. MrBayes analyses consisted of two sets of four simultaneous chains of 1,000,000 generations with sample frequency set at 100. Log-likelihood scores were plotted and only the final 75% of trees were used to produce the consensus trees by setting the “burn-in” parameter at 2500. This number of generations was considered sufficient because the SD dropped below 0.01.

Nodal supports were considered as strong if they reached more than 70% for ML bootstrap values and 0.95 for posterior probability (pp) support values (Huelsenbeck and Rannala 2004).

A pairwise distance matrix among mtDNAcox2 Contracaecum sequences was calculated using the nucleotide p-distance algorithm implemented in MegaX (Kumar et al. 2018).

The integrative analysis of the studied specimens allowed us to recognize two different species:

Contracaecum chubutensis Garbin, Diaz, Cremonte, & Navone 2008

General morphology: (based on 10 males and 10 females) (Fig. 1, Table 1): Smooth or slightly bifurcated interlabia (Figs. 1a-c). Well notorious auricles tips (Figs. 1a-c).

Table 1

Morphometric data of *Contracaecum chubutensis* parasitizing *Phalacocorax gaimardi* from Ría Deseado, Santa Cruz province, and *C. australe* parasitic in *P. brasilianus* from San Miguel del Monte lagoon, Buenos Aires, Argentina, and other authors’ works. All measurements are expressed in millimetres (mm). dae: distance to anterior end.
References	Garbin et al., 2011	Biolé at al., 2012	Present work	Garbin et al., 2008	Present work
Species	Contracaecum australe	Contracaecum australe	Contracaecum australe	Contracaecum chubutensis	Contracaecum chubutensis
Hosts	Phalacrocorax brasilianus	Phalacrocorax brasilianus	Phalacrocorax brasilianus	Phalacrocorax atriceps	Phalacrocorax gaimardi
Locality	Santa Helena lagoon (Chile)	Córdoba Province lagoons (Argentina)	San Miguel del Monte lagoon, Buenos Aires province (Argentina)	Bahía Bustamante, Chubut, Argentina	Ría Deseado, Santa Cruz province (Argentina)
Males (n)	10	10	10	10	10
Total length (TL)	23.24 (13.90–28.40)	24.37 (19.25–27.37)	19.35 (17-65-21.60)	25.06 (14.32–38.58)	24.87 (15.24–32.23)
Maximum body width (MBW)	0.75 (0.64–0.93)	0.90 (0.65-1)	0.725 (0.58–0.80)	0.77 (0.43–0.98)	0.69 (0.49–0.81)
Nerve ring (dae)	0.63 (0.58–0.68)	0.35–0.39	0.57 (0.45–0.67)	0.52 (0.36–0.60)	0.50 (0.43–0.60)
Deiridos (dae)	0.65 (0.58–0.79)	0.35–0.38	0.61 (0.55–0.73)	0.64 (0.46–0.84)	0.60 (0.44–0.77)
Esophagous length	3.62 (2.62–4.60)	4.12–4.40	2.97 (2.51–3.45)	3.39 (2.32–4.50)	2.86 (2.23–3.45)
Intestinal cecum length (ICL)	2.41 (1.56–3.24)	3.57-4.00	2.15 (1.65–3.04)	2.25 (1.50–2.76)	2.04 (1.60–2.60)
Ventricle length	0.28 (0.20–0.38)	0.10–0.15	0.18 (0.16–0.21)	0.23 (0.13–0.30)	0.20 (0.14–0.25)
Ventricular appendix length (VAL)	1.17 (0.87–1.41)	0.75–0.85	0.69 (0.53–0.88)	0.67 (0.46–0.80)	0.88 (0.73–1.36)
Spicule lenght (SL) (+)	11.97 (9.60-15.88)	9.20-10.45	9.70 (8.90-10.56)	9.95 (6.40–12.60)	9.11 (7.20-10.44)
(-)			10.31 (9.52–11.10)
Tail length (TL)	0.22 (0.18–0.24)	0.16 (0.12–0.35)	0.20 (0.18–0.22)	0.20 (0.17–0.26)	0.20 (0.17–0.25)
Precloacal papillae pairs	27–32	32–40	30–38	35–43	27–43
BL/MBW	34.12 (28.31–39.12)	29.61	27	33.19 (27.68–39.32)	38,40 (34,50 − 43,82)
BL/EL	7.14 (6.03–8.87)	4.67	6.56	7.33 (6.15–9.27)	9,09 (7,75 − 10,37)
BL/TL	117.42 (97.92-138.89)	152.31	94.35	124 (83.74–214.32)	126,82 (106,54–143,58)
EL/ICL	1.52 (1.37–1.68)	1.15	1.66	1.52 (1.44–1.63)	1,52 (1,39 − 1,96)
EL/VAL	3.13 (2.25–3.99)	5.49	3.97	4.90 (3.49–6.02)	3,28 (2,10 − 4,61)
BL/SL	1.9 (1.41–2.77)	2	1.9	2.58 (2.18–3.14)	2.88 (2.69–3.32)
Females (n)	10	10	10	10	10
Total length	31.6 (25.44–41.23)	31.6 (27–37)	29.56 (24.62–38.25)	29.60 (21.98–35.33)	27.28 (15.64–36.20)
Maximum body width	0.94 (0.66–1.16)	0.82 (0.7–0.9)	0.88 (0.63–0.99)	0.90 (0.61–1.27)	0.85 (0.65–1.05)
Nerve ring (dae)	0.58 (0.50–0.68)	0.40-0.475	0.61 (0.49–0.75)	0.56 (0.48–0.62)	0.52 (0.46–0.60)
Deirids (dae)	0.65 (0.58–0.79)	0.46–0.55	0.63 (0.59–0.81)	0.69 (0.58–0.80)	0.58 (0.49–0.65)
Esophagous length	3.24 (1.52–3.95)	4.50-3.625	3.05 (2.75–3.68)	3.05 (1.19–4.28)	2.91 (2.56–3.50)
Intestinal caecum length	2.13 (1.30–2.86)	3.70–4.25	2.28 (1.79–3.15)	1.91 (1.08–2.93)	2.07 (1.66–2.57)
Ventricle length	0.25 (0.14–0.28)	0.19–0.23	0.21 (0.18–0.32)	0.24 (0.16–0.26)	0.25 (0.20–0.33)
Ventricular appendix length	0.70 (0.57–0.91)	0.62–0.92	0.73 (0.58–0.90)	0.74 (0.66–0.94)	0.90 (0.69–1.33)
Vulva (dae)	9.26 (8.25–10.87)	8.32–8.45	8.85 (8.23–10.15)	9.57 (8.32–11.56)	8.58 (4.70-15.36)
Tail length	0.39 (0.28–0.58)	0.25 (0.12–0.30)	0.35 (0.28–0.45)	0.41 (0.30–0.65)	0.31 (0.22–0.40)
Embryonated egg length	0.068 (0.063–0.071)	0.050 (0.047–0.057)	0.063 (0.052–0.070)	0.070 (0.06–0.07)	0.060 (0.05–0.07)

Male

Tail distal constriction present immediately behind the pair of paracloacal papillae (Fig. 1d, arrow). Spicule free-distal end variable in length 47 (32–58) µm (Figs. 1e, f).

Female

(Table 1, see description in Garbin et al. 2008). No significant morphometric differences were observed between the specimens found in the present work and those ones described by Garbin et al. (2008).

Taxonomic summary

Type host: Phalacrocorax atriceps (King 1828) (Pelecaniformes: Phalacrocoracidae).

Present study host: Phalacrocorax gaimardi (Lesson and Garnot 1828) (Pelecaniformes: Phalacrocoracidae).

Type locality: Bahía Bustamante (45°11’S, 66°30’W), Chubut Province, Argentina.

New locality: Ría Deseado shore (47°45'S, 65°56'W), Santa Cruz Province, Argentina.

Infection site: stomach.

Prevalence: 100% (3/3)

Mean intensity: adults = 18; L4 larvae (fourth-stage larvae) = 88.7

Contracaecum australe Garbin, Mattiucci, Paoletti, González-Acuña & Nascetti 2011

General morphology: (based on 10 males and 10 females) (Fig. 2, Table 1): Smooth interlabia.

Male

Tail distal constriction present immediately behind the pair of paracloacal papillae (Fig. 2d). Spicule free-distal end low variable 18 (25 − 11) µm.

Female

(Table 1, see Garbin et al. 2011). No significant morphometric differences were observed between the specimens found in the present work and those ones described by Garbin et al. (2011)

Taxonomic summary

Type host: Phalacrocorax brasilianus (Gmelin 1789) (Phalacrocoracidae).

Present study host: Phalacrocorax brasilianus

Type locality: Santa Elena lagoon, VIII Región, Chile.

New locality: San Miguel del Monte lagoon (35°27'30" S; 58°48'15" W), Buenos Aires province, Argentina.

Infection site: Stomach.

Prevalence: 87.5% (7/8)

Mean intensity: 31.37 (6–111).

Molecular characterization and phylogenetic analysis

Six isolates belonging to Contracaecum specimens parasitizing P. gaimardi from the Ría Deseado (RD), Santa Cruz province were sequenced: one for mtDNA cox2, two for ITS1, two for ITS2, and one for SSrRNA genes. On the other hand, three isolates from Contracaecum specimens parasitizing P. brasilianus from San Miguel del Monte lagoon (SMML) were obtained: one for ITS1, one for ITS2, and one for SSrRNA genes. Unfortunately, mitochondrial DNA Cox2 sequences could not be obtained from these nematodes for unknown reasons. GenBank accession numbers, hosts and sampling site, and DNA markers are provided in Table 2.

The only sequenced mtDNAcox2 isolate for Contracaecum parasitizing P. gaimardi from RD matched 99% with the previously reported gene sequences of C. chubutensis parasitizing P. atriceps from Bahía Bustamante, Chubut (HQ328504). A very well-supported clade was obtained for both ML and BI trees (97 / 1.0) inferred from the mtDNA cox-2 sequences analysis (Fig. 3). Besides, Fig. 3 shows the papillae pattern arrangement of the male tail in order to understand the interspecific and genetic relationships between Contracaecum species. Two large groups of Contracaecum congeners can be appreciated: a large clade (I) that groups only species that own a simple papillae pattern arrangement on the male tail (A), and another group (II) that clusters species with the three morphotypes of papillae distribution (A, B, and C). A monophyletic clade was observed for Contracaecum species that own the intermediate morphotype (B) within the large clade II. Also, another monophyletic clade for species with morphotype C is observed into clade II. Following this phylogenetic hypothesis, morphotype A would turn out to be paraphyletic.

Similar topologies were observed according to both ITS1 and ITS2 isolates sequences for Contracaecum specimens parasitizing P. gaimardi from RD. Latter isolates (MZ605037, MZ605037, MZ303426, MZ603428) clustered with C. chubutensis parasitizing P. atriceps from Bahía Bustamante, Chubut (HQ389546, HQ389548), very well-supported (98–100 / 0.70–0.99) in the concatenated BI and ML tree sequences analysis (Fig. 4). Likewise, isolates from Contracaecum specimens parasitizing P. brasilianus from SMML (MZ 605039, MZ603428) clustered with C. australe from Chile (HQ389545, HQ389547) (Fig. 4). According to the papillae pattern arrangement of the male tail, a large monophyletic clade was observed for those Contracaecum species that own the simple morphotype (A). And, a small clade of those species with intermediate morphotype (B) can be observed.

Finally, SSrRNA isolates of Contracaecum specimens parasitizing P. gaimardi from RD (MZ634121) clustered with C. chubutensis from Bahia Bustamante (HQ333521), very well supported (96/ 0.99) in both ML and BI trees (Fig. 5). In the same way, SSrRNA isolates of Contracaecum from P. brasilianus at SMML (MZ634122) matched (83/ 0.97) with C. australe isolates from Chile (HQ333520) (Fig. 5). All congeners show simple mophotype (A) according to the papillae pattern arrangement of the male tail.

Pairwise DNA analyses revealed that the divergence between the newly obtained sequence of mtDNA cox2 for C. chubutensis and C. australe, and other sequences included in this study ranged from 1.1–16.0% (5 to 72 nucleotides) (Fig. 6).

Table 2

List of *Contracaecum* sequences included in the phylogenetic analysis with the information on DNA markers, GenBank accession numbers, definitive host, sampling site, and references when data were available.
Species	Cox2	ITS1	ITS2	SSrRNA (18s)	Host	Country	Reference
Contracaecum ogmorhini				NC031647	Arctocephalus pusillus doriferu	Australia	Liu et al. 2016 a
				KU558725	Arctocephalus pusillus doriferus	Australia	Liu et al. 2016 a
		AJ291470	AJ291473		Zalophus californianus	Canada:pacific Canada	Zhu et al., 2001
	MN624186				Zalophus californianus	USA: California	Unpublished
	MN624197				Zalophus californianus	USA: California	Unpublished
Contracaecum rudolphii				JF423899	Pelecanus occidentalis	USA: Florida	D’amelio et al., 2012
		AJ634783	AJ634911		Phalacrocorax carbosinensis	Italy: Venice lagoon	Li et al., 2005
Contracaecum rudolphii	EF122201				Phalacrocorax carbo sinensis	Italy: Oristano	Li et al., 2005
Contracaecum rudolphii	EF122202				Phalacrocorax carbo sinensis	Italy: Venezia	Li et al., 2005
Contracaecum chubutensis	MZ579647	MZ605037-8	MZ603426-7	MZ634121	Phalacrocorax gaimardii	Argentina	This study
	HQ328504	HQ389546	HQ389548	HQ333521	Phalacrocorax atriceps	Argentina: Chubut	Shamsi et al., 2009
Contracaecum australe	GQ847535	HQ389545	HQ389547	HQ333520	Phalacrocorax brasilianus	Chile	Shamsi et al., 2009
Contracaecum australe		MZ605039	MZ603428	MZ634122	Phalacrocorax brasilianus	Argentina	This study
	GQ847543-4				Phalacrocorax brasilianus	Chile	Unpublished
Contracaecum pelagicum				JN580990	Phalacrocorax atriceps	Argentina	Garbin et al., 2013
Contracaecum pelagicum	KC435448				Spheniscus magellanicus	Brazil	Unpublished
Contracaecum pelagicum	KC435449				Spheniscus magellanicus	Brazil	Unpublished
Contracaecum pelagicum	EF122210				Spheniscus magellanicus	Argentina: Chubut	Unpublished
Contracaecum pelagicum	EF535568				Spheniscus magellanicus	Argentina: Chubut	Unpublished
Contracaecum microcephalum	EF513519			EF014282	Phalacrocorax pygmeus	Yugoslavia: Montenegro	D’amelio et al., 2012
Contracaecum microcephalum	EF513518				Phalacrocorax pygmeus	Yugoslavia: Montenegro	Unpublished
Contracaecum septentrionale	EF513512				Phalacrocorax aristotelis	Norway	Unpublished
Contracaecum septentrionale	EF558898				Phalacrocorax carbo carbo	Iceland	Unpublished
Contracaecum margolisi	EU477212				Zalophus californianus	USA	Unpublished
Contracaecum bioccai	EF513500				Pelecanus occidentalis	Colombia	Unpublished
Contracaecum bioccai	EF513499				Pelecanus occidentalis	Colombia	Unpublished
Contracaecum multipapillatum	MH044685				Rhamdia sp.	Guatemala: Rio Dulce	Unpublished
Contracaecum micropapillatum	EF513516				Pelecanus onocrotalus	Egypt	Unpublished
Contracaecum micropapillatum	EF513515				Pelecanus onocrotalus	Egypt	Unpublished
Contracaecum mirounga	EU477213				Mirounga leonina	Antarctica	Unpublished
Contracaecum radiatum	EU477210				Leptonychotes weddellii	Antarctica	Unpublished
Contracaecum osculatum	EF122211				Halichoerus grypus	Colombia	Unpublished
		AJ250414	AJ250417		Leptonychotes weddelli	Antarctica	Zhu et al., 2000
Contracaecum bancrofti		FM177888	EU839572		Pelecanus conspicillatus	Unknow	Shamsi et al., 2009
Contracaecum eudyptulae		FM177561	FM177578		Eudyptula minor	Unknow	Shamsi et al., 2009
Contracaecum variegatum		FM177535	FM177541		Anhinga melanogaster	Unknow	Shamsi et al., 2009
Contracaecum septentrionale		AJ634784	AJ634787		Alca torda	Spain:Northern coasts	Li et al., 2005
Contracaecum microcephalum		FM177526	FM177530		Pycnonotus melanoleucos	Unknow	Shamsi et al., 2009
Contracaecum pyripapillatum		AM940065	AM940069		Pelecanus conspicillatus	Australia: Victoria	Shamsi et al., 2008
Contracaecum multipapillatum		FM210414	FM210416		Mugil cephlaus	Australia	Shamsi et al., 2011
Contracaecum ovestreeti	EU852348				Pelecanus crispus	Greece	Mattiucci et al., 2010
Anisakis pegreffii				LC222461	Scomber japonicus	Japan:Nagasaki	Yamada et al., 2017
Anisakis simplex				JX500049	Globicephala melas	Pacific Ocean: New Zealand coast	Mattiucci et al., 2014

Despite Contracaecum specimens parasitizing P. gaimardi from the RD have a great similarity with C. australe, the present integrative analysis allow us to conclude that they actually belong to C. chubutensis, not C. australe as previously identified by Garbin et al. (2014). However, slight morphometric differences were observed with those C. chubutensis specimens found in P. atriceps from Bahía Bustamante, Chubut (Garbin et al. 2008). Thus, the length of the spicules is shorter, which is reflected in the body length/spicule length ratio (Table 1). On the other hand, the present specimens have entire or barely bifid interlabia, and lips have either a single shallow notch or three barely noticeable notches (Figs. 1a, b, c). The tail distal constriction between the paracloacal and subventral/sublateral papillae from present specimens seems to be less notorious than those from the original description (Fig. 1d).

The main dietary items of P. gaimardi in the study area are sardines Sprattus fuegensis (Jenyns 1842), and Ramnogaster arcuata (Jenyns 1842) (Pisciformes: Clupeidae) (Frere et al. 2005; Morgenthaler et al. 2016). Garbin et al. (unpub. data) found Contracaecum L3 larvae on both sardine species. Hence, both fish might likely act as intermediate/paratenic hosts of C. chubutensis in the foraging area of shags P. gaimardii and P. atriceps. Other shared prey items on both cormorants in the area are Patagonotothen spp., Odontesthes spp., and Pinguipes brasilianus (Garbin et al. 2019). All these fish are recorded to host Contracaecum L3 larvae (Carballo et al. 2007; Timi et al 2008; Garbin et al. 2019). The specific correspondence between Contracaecum L3 larvae found in those fish species, and adults of C. chubutensis found in P. gaimardi should be corroborated using molecular tools.

On the other hand, integrative studies on nematodes recovered from P. brasilianus from SMML allowed us to confirm that we are dealing with C. australe. Present specimens look very similar to those of C. australe from Chile (Garbin et al. 2011), sharing almost all morphological features like lips, interlabia, tail distal constriction, arrangement and number of caudal papillae. Also, it resembles those specimens of C. australe described by Biolé et al. (2014) parasitizing P. brasilianus from Cordoba province lagoons, although its spicules showed smaller dimensions. These three records on C. australe (i.e. Chile, Cordoba, and Buenos Aires province in Argentina) note the wide geographic range of the species on South America. Presumably, Silversides Odontesthes spp. or Sabalito Cyphocharax voga (Hensel 1870) (Characiformes: Curimatidae) could play the parathenic/intermediate host role of C. australe L3 larvae (Carballo et al. 2007; Garbin pers. obs.). Besides, according to the phylogenetic analyses these findings would appear to be different populations of C. australe parasitizing P. brasilianus, or haplotypes of the same species (Tang and Hyman 2007). Further molecular analyses are needed to test this hypothesis. The results would indicate that C. australe carries its cycle out in continental environments whereas C. chubutensis does it in marine environments.

Amato et al. (2006) reported C. rudolphii parasitizing P. brasilianus in the Guaiba Lake, Rio Grande do Sul, Brazil. Taking into account the great intraspecific variability observed for C. australe, it is likely those specimens from Brazil actually belong to C. australe even more considering that both findings were carried out on P. brasilianus. Similar is the case of Contracaecum jorgei parasitizing the Neotropic cormorant and Tararira Hoplias argentinensis Rosso et al. 2018 (Characiformes: Erythrinidae) from a shallow Pampean lake in central Argentina. However, the molecular characterization of larval and adult stages separated this species from C. australe within another lineage (Sardella et al. 2020).

Regarding to the ML – BI tree based on the mtDNA cox2 marker, it seems that the different analyzed Contracaecum spp. clusterize according to the papillae pattern arrangement (PPA) on the male tail (Fig. 3). Eight species which hold a simple PPA -morphotype A- grouped in a big paraphyletic clade. Whereas, the other nine Contracaecum spp. grouped in three different clusters and morphotypes. It can be inferred that the Contracaecum species of morphotype B would probably derive from a simple morphotype ancestor. The same would occur for the species that hold morphotype C (Fig. 2). A similar topology is shown by the BI and ML tree based on concatenated ITS1- ITS2 markers in which Contracaecum species of morphotype A are grouped into a large superior clade, whereas few species of morphotypes B and C are separated into other lower clades (Fig. 3). The available SSrRNA sequences only show clustering of morphotype A species, since there are no gene available sequences for Contracaecum species of morphotypes B and C so far (Fig. 4). These findings highlight the potential of combining molecular and morphological data in understanding the evolutionary relationships and diversification of these parasitic nematodes. However, more molecular marker sequences from different Contracaecum species in different hosts and localities would be required to better explain this inferred order on the ML and BI trees. In addition, new molecular markers which are related to the gene expression of the male tail papillae could be synthesized to solve this uncertainty.

The integrative analysis confirm that Contracaecum specimens parasitizing P. gaimardi from RD, Argentina, belongs to C. chubutensis representing a new host and the southernmost record for the species. Whereas, those specimens parasitizing P. brasilianus from SMML belong to C. australe providing a new geographical record for the species and the extension of its distribution range. In addition, present results confirm the inland and marine distribution of C. australe and C. chubutensis, respectively.

Acknowledgements

The authors would like to thank the staff of the Scanning Electron Microscopy Services from the La Plata Museum, UNLP. The support personnel Walter Ferrari for its contribution on the PCR assays. Dr. Diego Montalti for his help in collecting the Neotropic cormorant samples.

Authors' contributions

Lucas E. Garbin: Wrote the main manuscript text, bird digestive tract prospection, nematode specimen collection, morphometric and taxonomical nematode studies, photographs to scanning electron microscopy, phylogenetic analysis, preparation of figures 1 and 2.

Andrea Servián: Prepared figures 3-6, DNA extractions and PCR assays, phylogenetic analysis, secondary writer of the manuscript.

Lautaro Fuentes: Bird digestive tract prospection, morphometric nematodes studies.

Annick Morgenthaler: Collected the Red-legged cormorant samples.

Ana Millones: Collected the Red-legged cormorant samples.

Daniela Fuchs: Collected the Neotropic cormorant samples.

Julia I. Diaz: Prepared figures 1 and 2, parasite imaging and figure preparation, morphometric and taxonomical nematode studies, obtaining funding.

Graciela T. Navone: Morphometric and taxonomical nematode studies, obtaining funding.

All authors reviewed the manuscript.

Funding information

This research was funded by the grant Universidad Nacional de La Plata (N859 to JID).

Data availability statement

The SEM-photographed Contracaecum specimens (3 males and 3 females) from Phalacrocorax gaimardi are stored at the Electron Microscopy Service of La Plata Museum. All paratypes of Contracaecum spp. parasites of Phalacrocorax gaimardi and P. brasilianus 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 (MZ605037-9, MZ603426-8, MZ579647, and MZ6342121-2).

Ethical approval

Cormorant individuals were captured according to permissions, legal procedure rules, and the animal protection law from the Dirección de Flora y Fauna, Ministerio de Asuntos Agrarios, Buenos Aires province, Argentina. Expediente Nº22500-31095/15.

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.

Amato JFR, Monteiro CM, Amato SB (2006) Contracaecum rudolphii Hartwich (Nematoda, Anisakidae) from the Neotropical Cormorant Phalacrocorax brasilianus (Gmelin) (Aves, Phalacrocoracidae) in Southern Brazil. Rev Bras Zool 23(4):1284–1289. https://doi.org/10.1590/S0101-81752006000400046
Anderson RC (2000) Nematode parasites of vertebrates. Their development and transmission. CABI Publishing, Farnham Royal, UK. https://doi.org/10.1079/9780851994215.0001
Araya B, Millie G (1991) Guía de campo de las aves de Chile. Editorial Universitaria, Santiago, Chile
Biolé FG, Guagliardo SE, Mancini MA, Tanzola RD, Salinas V, Morra G (2012) Primer registro de Contracaecum australe (Nematoda: Anisakidae) en Phalacrocorax brasilianus (Aves: Phalacrocoracidae) de la región central de Argentina. BioScriba 5:1–11.
Bonfield JK, Smith KF, Staden R (1995) A new DNA sequence assembly program. Nuc Ac Res 23:4992–4999. https://doi.org/10.1093/nar/23.24.4992
Bush A O, Lafferty K D, Lotz JM, Shostak AW (1997) Parasitology meets ecology on its own terms: Margolis et al. revisited. J. Parasitol 83:575–583. https://doi.org/10.2307/3284227
Carballo MC, Diaz JI, Garbin L, Cremonte F, Navone G (2007) Parasite fauna of two sympatric silversides species (Odontesthes smitti and O. nigricans, Atherinopsidae) from the Patagonian coast. VII Int Symp Fish Parasitol. Parassitologia 49:298.
D'Amelio S, Barros NB, Ingrosso S, Fauquier DA, Russo R, Paggi L (2007) Genetic characterization of members of the genus Contracaecum (Nematoda: Anisakidae) from fish-eating birds from west-central Florida, USA, with evidence of new species. Parasitology 134(7):1041–1051. https://doi.org/10.1017/S003118200700251X
Fagerholm HP (1990) Systematic position and delimitation of ascaroid nematode parasites of the genus Contracaecum with a note on the superfamily Ascaridoidea. Dissertation. Department of Biology, Abo and National Veterinary Institute, Helsinky, Finland
Frere E, Gandini P, Ruiz J, Vilina YA (2004) Current status and breeding distribution of Red-legged Cormorant Phalacrocorax gaimardi along the Chilean coast. Bird Conserv Int Jour 14:115–123. https://doi.org/10.1017/S0959270904000139
Frere E, Quintana F, Gandini P (2005) Cormoranes de la costa patagónica: estado poblacional, ecología y conservación. Hornero 20:35–52. https://doi.org/10.56178/eh.v20i1.818
Gandini P, and Frere E. 1995. Distribución, abundancia y ciclo reproductivo del Cormorán gris (Phalacrocorax gaimardi) en la costa patagónica, Argentina. Hornero 14:57–60. https://doi.org/10.56178/eh.v14i1-2.1029
Garbin LE, Diaz JI, Cremonte F, Navone GT (2008) New anisakid species parasitizing the Imperial Cormorant Phalacrocorax atriceps from the North Patagonian coast, Argentina. J Parasitol 94(4):852–859. https://doi.org/10.1645/GE-1369.1
Garbin LE, Mattiucci S., Paoletti M., González-Acuña D, Nascetti G (2011) Genetic and morphological evidences for the existence of a new species of Contracaecum (Nematoda: Anisakidae) parasite of Phalacrocorax brasilianus (Gmelin) from Chile and its genetic relationships with congeners from fish-eating birds. J Parasitol 97:476–492. https://doi.org/10.1645/GE-2450.1
Garbin LE, Mattiucci S, Paoletti M, Diaz JI, Nascetti G, Navone GT (2013) Molecular identification of Contracaecum pelagicum (Nematoda: Anisakidae) from the anchovy Engraulis anchoita (Engraulidae) and fish-eating birds from Argentinian North Patagonian Sea, with larval morphological description. Parasitol Int 62:309–319. https://doi.org/10.1016/j.parint.2013.03.001
Garbin LE, Capasso S, Diaz JI, Morgenthaler A, Millones A, Navone G (2014) Nuevo hospedador y registro geográfico de Contracaecum australe (Nematoda, Anisakidae) parasitando a Phalacrocorax gaimardi (Aves, Phalacrocoracidae) en costas del Atlántico Sudoccidental. Rev Arg Parasitol 2:6–14.
Garbin LE, Diaz JI, Navone GT (2019) Species of Contracaecum parasitizing the Magellanic Penguin Spheniscus magellanicus (Spheniscidae) from the Argentinean coast. J Parasitol 105 (2):222–231. https://doi.org/10.1645/17-91
Garbin LE, Diaz JI, Servián A, Fusaro B, Navone GT (2023) The genus Contracaecum Raillet & Henry (Nematoda: Anisakidae): host and geographical distribution on Neotropical and Antarctic species. Zootaxa 5256(1): 043–062. https://doi.org/10.11646/ZOOTAXA.5256.1.3
Gutiérrez RO (1943) Sobre la morfología de una nueva especie de Contracaecum (Nematoda: Ascariroidea). Rev Bras Biol 3:159–172.
Harrison P (1985) Seabirds, an identification guide. Houghton Mifflin, Boston, Massachusetts, USA
Hartwich G (1964) Die Typen Parasitischer Nematoden in der Helminthen- Sammlung des Zoologischen Museums in Berlin. I. Ascaridoidea. Mitteil Zool Mus 40:1–53. https://doi.org/10.1002/mmnz.4830400105
Huelsenbeck JP, Rannala B. (2004) Frequentist properties of Bayesian posterior probabilities of phylogenetic trees under simple and complex substitution models. Syst Biol 53(6):904–913. https://doi.org/10.1080/10635150490522629
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) Mega X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy096
Lanfear R, Calcott B, Ho SY, Guindon S (2012) PartitionFinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Mol Biol Evol 29: 1695–1701. https://doi.org/10.1093/molbev/mss020
Madison WP, Madison DR (2011) Mesquite: A modular system for evolutionary analysis, version 2.75. http://mesquiteproject.org. Accessed April 2023
Malacalza V, Bertellotti M (2001) Cambios poblacionales de los Cormoranes (Phalacrocorax) en Punta Lobería, Patagonia Argentina. Ornitol Neotrop 11(1):83–86.
Mattiucci S, Paoletti M, Consuegra-Solorzano A, Nascetti G (2010) Contracaecum gibsoni n. sp. and C. overstreeti n. sp. (Nematoda: Anisakidae) from the Dalmatian pelican Pelecanus crispus (L.) in Greek waters: genetic and morphological evidence. Syst Parasitol 75:207–24. https://doi.org/10.1007/s11230-009-9220-8.
Morgenthaler A, Millones A, Gandini PA, Frere E (2016): Pelagic or benthic prey? Combining trophic analyses to infer the diet of a breeding South American seabird, the Red-legged Cormorant Phalacrocorax gaimardi. Emu 116:360–369. http://dx.doi.org/10.1071/MU15101
Nadler SA, Hudspeth DS (2000) Phylogeny of the Ascaridoidea (Nematoda: Ascaridida) based on three genes and morphology: Hypotheses of structural and sequence evolution. J Parasitol 86:380–393. https://doi.org/10.1645/0022-3395(2000)086[0380:POTANA]2.0.CO;2
Punta G, Yorio P, Saravia J, Borboroglu PG (2003) Breeding Habitat Requirements of the Imperial Cormorant and Rock Shag in Central Patagonia, Argentina. Waterbirds 26(2): 176–183. https://doi.org/10.1675/1524-4695(2003)026[0176:BHROTI]2.0.CO;2
Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574. https://doi.org/10.1093/bioinformatics/btg180
Sardella CJ, Mancini M, Salinas V, Simões RO, Luque JL (2020) A new species of Contracaecum (Nematoda: Anisakidae) found parasitizing Nannopterum brasilianus (Suliformes: Phalacrocoracidae) and Hoplias argentinensis (Characiformes: Erythrinidae) in South America: morphological and molecular characterization of larval and adult stages. J Helminthol 94(184):1–11. https://doi.org/10.1017/S0022149X20000644
Shamsi S, Gasser RB, Beveridge I (2008) Contracaecum pyripapillatum n. sp. (Nematoda: Anisakidae) and a description of C. multipapillatum (von Drasche, 1882) from the Australian pelican Pelecanus conspicillatus. Parasitol Res 103:1031–1039. https://doi.org/10.1007/s00436-008-1088-z
Shamsi S, Norman R, Gasser R, Beveridge (2009). Redescription and genetic characterization of selected Contracaecum spp. (Nematoda: Anisakidae) from various hosts in Australia. Parasitol Res 104(6):1507–1525. https://doi.org/10.1007/s00436-009-1357-5
Rohde K (2005) Marine Parasitology. CSIRO Publishing, Collingwood, Autralia. https://doi.org/10.1079/9780643090255.0000
Telfair RC, Morrison ML (1995) Neotropic cormorant (Phalacrocorax brasilianus). In: Poole A, Gill F (ed) The birds of North America. The Academy of Natural Sciences, Washington D.C., pp 1–22.
Tang S, Hyman BC (2007) Mitochondrial Genome Haplotype Hypervariation Within the Isopod Parasitic Nematode Thaumamermis cosgrovei. Genetics 176:1139–1150. https://doi.org/10.1534/genetics.106.069518
Timi JT, Lanfranchi AL, Etchegoin JA, Cremonte F (2008) Parasites of the Brazilian sandperch Pinguipes brasilianus Cuvier: A tool for stock discrimination in the Argentine Sea. J Fish Biol 72(6):1332–1342. https://doi.org/10.1111/j.1095-8649.2008.01800.x

No competing interests reported.

Download PDF

Journal Publication

published 19 Dec, 2023

Read the published version in Parasitology Research →

Editorial decision: Major revision
05 Sep, 2023
Reviews received at journal
13 Aug, 2023
Reviewers agreed at journal
07 Aug, 2023
Reviewers invited by journal
07 Aug, 2023
Editor assigned by journal
07 Aug, 2023
Submission checks completed at journal
07 Aug, 2023
First submitted to journal
27 Jul, 2023

You are reading this latest preprint version

Phylogenetic relationship between Contracaecum spp. (Nematoda, Anisakidae) parasitizing cormorants (Aves, Phalacrocoracidae) from Argentina

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Material and methods

Sampling

Morphological study

DNA extraction, amplification, and sequencing

Phylogenetic analysis

Results

Taxonomic summary

Taxonomic summary

Molecular characterization and phylogenetic analysis

Discussion

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1