Introduction
Seabird populations worldwide have been declining dramatically over the past decades as a result of a range of environmental and anthropogenic stressors. The Atlantic puffin, Fratercula arctica Linnaeus, 1758 (Charadriiformes: Alcidae) has been designated as vulnerable to extinction globally and listed as endangered in Europe (Kersten, Reference Kersten2023). These birds breed in isolated cliff slopes and islands in the North Atlantic Ocean (Greenland, Iceland, Scandinavian Peninsula, British Islands), in late summer they leave their colonies on migration and distribute all around the North Atlantic, having even been seen in the Mediterranean Sea (Clairbaux et al., Reference Clairbaux, Mathewson, Porter, Fort, Strøm, Moe, Fauchald, Descamps, Helgason, Bråthen, Merkel, Anker-Nilssen, Bringsvor, Chastel, Christensen-Dalsgaard, Danielsen, Daunt, Dehnhard, Erikstad, Ezhov, Gavrilo, Krasnov, Langset, Lorentsen, Newell, Olsen, Reiertsen, Systad, Thórarinsson, Baran, Diamond, Fayet, Fitzsimmons, Frederiksen, Gilchrist, Guilford, Huffeldt, Jessopp, Johansen, Kouwenberg, Linnebjerg, Major, Tranquilla, Mallory, Merkel, Montevecchi, Mosbech, Petersen and Grémillet2021). Nevertheless, during the winter of 2022–2023, numerous puffins (hundreds or even thousands as estimated) were found dead on the shores of the Canary Islands (Spain), located near the coast of Morocco, belonging to the Macaronesia region in 13°23′–18°80′ W and 27°37′–29°24′N. The reason for this change in their migration route is not clear, but adverse weather phenomena and changes in food availability due to climate change are the most likely causes (Guilford et al., Reference Guilford, Freeman, Boyle, Dean, Kirk, Phillips and Perrins2011; Dorresteijn et al., Reference Dorresteijn, Kitaysky, Barger, Benowitz-Fredericks, Byrd, Shultz and Young2012).
Because of this rare event, investigation about the parasites present in F. arctica appeared in the Canarian coasts were performed in cooperation with ‘Dirección General de Lucha Contra el Cambio Climático y Medio Ambiente’ as part of the Canarian Network for the Surveillance of the Wildlife Health (Red Vigía, Orden No. 134/2020 of 26 May 2020) and ‘Consejería de Transición Ecológica, Lucha contra el Cambio Climático y Planificación Territorial’ as part of ‘Estudio de patógenos en aves migratorias y en especies exóticas en un escenario de cambio climático’ project (No. 248/2020 of 4 December 2020). The aim of this work was to investigate renal parasites in these birds, following previous findings of renal digeneans in the same species (Hill, Reference Hill1954).
Materials and methods
Animal management
A total of 39 F. arctica (21 adults, 17 juveniles and one age undetermined) found in coastal areas of Tenerife and Gran Canaria islands (Canary Islands, Spain) were dissected for the present study. The kidneys and ureters were examined using stereoscopic microscope and dissection equipment, searching for helminths.
Morphological identification
The helminths found were preserved in 70% ethanol at room temperature. Each one was stained with Semichon acetocarmine, differentiated in 50% acid-alcohol solution, dehydrated in an ethanol series and with 2-propanol, cleared in clove oil and, finally, mounted on slides with Canadian balsam. After that, they were examined under optical microscope. Because of the poor condition of the parasites found, only a single specimen could be measured (see the following section for morphometric data).
DNA extraction
Genomic DNA was obtained from renal tissue portions and the helminths found in bad condition for morphometric analysis, following López et al. (Reference López, Clemente, Almeida, Brito and Hernández2015) protocol. The samples were transferred to 1.5-ml tubes containing 250 μl of lysis solution (10 mM EDTA, 30 mM Tris-HCL pH 8.0 and 0.4% SDS) and 3 μl of proteinase K (20 mg/ml, PanReac AppliChem ITW Reagents, Monza, Italy); after that, they were incubated at 56°C overnight. The following day, 250 μl of 4M ammonia acetate was added, mixed thoroughly and consequently kept for 30 min at room temperature (15–25°C). The mixture was centrifuged at 13,000 rpm for 10 min, and the pellet was discarded. DNA was then precipitated with ethanol, and the sediment was suspended in 20–80 μl of molecular grade water (PanReac AppliChem ITW Reagents). The quantity and quality of genomic DNA was verified using DeNovix DS-11 + Spectrophotometer (DeNovix Inc., Wilmington, DE, USA).
Polymerase chain reaction amplification
The NADH dehydrogenase subunit 1 gene (ND1) was amplified using the primers ND1J and ND1J2A described by Bray et al. (Reference Bray, Littlewood, Herniou, Williams and Henderson1999) and Morgan and Blair (Reference Morgan and Blair1995). The polymerase chain reaction mix contained 1X buffer (VWR International, Haasrode, Belgium), 1.5 mM MgCl2 (VWR International), 0.2 mM of each dNTP (VWR International), 2 μM of each primer (Condalab, Madrid, Spain) and 20–40 ng of total genomic DNA in a total volume of 25 μl filled with molecular grade water. Amplification was performed using a XP Cycler (Bioer Technology, Hangzhou, China) with the following parameters: 95°C for 5 min; 30 cycles at 94°C for 1 min, 50°C for 1 min, 72°C for 1 min; and a final extension step at 72°C for 7 min. The resulting amplicons were visualised on 1.5% agarose gel at 90 V for 1 h.
Sequencing and sequencing data analysis
The polymerase chain reaction products with the expected size (500 bp) were sequenced at Macrogen (Madrid, Spain) along with ND1J and ND1J2A primers, using the Sanger method. The sequences obtained were studied with MEGA X software (Kumar et al., Reference Kumar, Stecher, Li, Knyaz and Tamura2018), applying the multiple alignment program ClustalW and compared with different Renicola Cohn, 1904 species sequences from GenBank database, with minor corrections made by hand. Afterwards, they were analyzed using the Basic Local Alignment Search Tool (BLAST), and their identity was confirmed by homology comparison.
Phylogenetic relationships were stablished based on the maximum likelihood method with the Kimura 2-parameter model (Kimura, Reference Kimura1980) and 1,000 bootstrap replications, exploring the relationships among different Renicola species using MEGA X software. Echinostoma ilocanum Garrison, 1908 sequence (Acc. Number: MN549984.1) was used as outgroup.
Results and discussion
One and three morphologically similar helminth specimens were found in the urinary tract of two (5%) of the 39 birds examined.
The recovered digeneans were identified as belonging to the genus Renicola according to specialised literature (Wright, Reference Wright1954; Gibson, Reference Gibson, Bray, Gibson and Jones2008; Heneberg et al., Reference Heneberg, Sitko, Bizos and Horne2016; Matos et al., Reference Matos, Lavorente, Lorenzetti, Meira-Filho, Nóbrega, Chryssafidis, Oliveira, Domit and Bracarense2019). Body oval to pyriform, round-shaped anteriorly and attenuated posteriorly. Body length 1682 μm, maximum width 671 μm. Body length to width ratio 2.5:1. Oral sucker subterminal, 195 × 193 μm. Ventral sucker postequatorial, 144 × 136 μm. Oral sucker to ventral sucker length ratio 1:0.74 Oral sucker to ventral sucker width ratio 1.42. Pharynx muscular, well-developed 118 × 121 μm. Two testes symmetrical, located at the ventral sucker level. Ovary lobed, preacetabular. Vitellarium consist of two lateral fields, each with numerous follicles. Vitelline fields extend approximately from the pharynx level to the posterior body part, ending well behind the posterior edge of the testes. Eggs 28-31 × 15-18 μm (29.2 × 16.8 μm) (n=5).
These morphological characteristics (Figure 1) show similarities with those of Renicola sloanei Wright, Reference Wright1954. Moreover, our specimens were clearly differentiated from Renicola lari Timon-David, 1933, Renicola sternae Sitko & Heneberg in Heneberg, Sitko, Bizos & Horne, Reference Heneberg, Sitko, Bizos and Horne2016 and Renicola pinguis Mehlis in Creplin, 1846. The main difference between the specimens found in the present study and these three species is related to the vitellarium. Thus, in R. lari, R. pinguis and R. sternae the number of vitelline follicles is low, whereas in our specimens, as occurs in R. sloanei, the number of vitelline follicles is much higher and more extended in the worm’s lateral margins. Comparative measures of R. sloanei found in different hosts are shown in table 1.
Figure 1. Renicola sloanei found in kidneys and ureter tracts of Fratercula arctica arrived at the Canary Islands. a: Whole mount specimen. b: Detail of gonads. c: Eggs. GP, genital pore; LT, left testis; O, ovary; RT, right testis; VS, ventral sucker.
Table 1. Measurements (in μm, except ratios) of Renicola sloanei from different seabirds in diverse localities
OS, oral sucker; VS, ventral sucker.
* Diameter.
A 285-bp sequence from ND1 gene was obtained. The BLAST analysis showed great homology with R. sloanei (Acc. No.: MK463858.1, Query Cover: 88%, Identity: 100.0%, e-value 6e-126). The nucleotide sequence obtained in this study were submitted to the GenBank database under the accession number PP764027.
The results of the maximum likelihood method analysis based on the obtained fragment ND1 gene is shown in Figure 2. The sequence obtained grouped with R. sloanei species and was clearly separated from other Renicola species with high bootstrap value (100%).
Figure 2. Phylogenetic relationships between sequences of the NADH dehydrogenase subunit 1 of Renicola species, including the nucleotide sequence obtained in the present study (showed in bold and highlighted in yellow). The tree was built using the Maximum Likelihood method with p-distance and 1,000 bootstrap replications. Echinostoma ilocanum was used as outgroup.
Renicolidae digeneans are parasites of the urinary system of aquatic birds feeding on infected bivalves and fishes. The intramolluscan stages of this parasite develop in marine and brackish-water gastropods, whereas metacercariae develop in molluscs and fishes (Galaktionov et al., Reference Galaktionov, Solovyeva, Blakeslee and Skírnisson2023). Following the molecular analyses of Galaktionov et al. (Reference Galaktionov, Solovyeva, Blakeslee and Skírnisson2023), R. sloanei belong to the second branch (clade II) of Renicolidae, a group of species that use sea birds as the definitive host, and their cercariae belong to the Rhodometopa group or to ‘transitional morphotype’.
Nevertheless, the status, life cycle and classification of some renicolids are still considered uncertain and several descriptions are incomplete due to the non-observation of some crucial characters, namely ventral sucker, intestine, testes, ovary and vitellarium. Moreover, nothing is known accurately related to the degree of specificity of the different morphological characters used for identification (Heneberg, Reference Heneberg, Sitko, Bizos and Horne2016). According to Galaktionov et al. (Reference Galaktionov, Solovyeva, Blakeslee and Skírnisson2023), molecular biology tools are needed to perform a correct identification of the species, using DNA sequencing methods to upgrade the sequences database, especially on morphologically similar species. A precise description of the life cycles and host ranges of Renicola digeneans, as well as a detailed analysis of their morphological features according to their different life stages is necessary for a better understanding of these parasites.
The genetical analysis based on ND1 gene of the digeneans found in this study confirm the morphological identification of R. sloanei. To date, only a previous single finding of an unidentified species of the genus Renicola in F. arctica has been notified, concretely in Britain in 1953 (Hill, Reference Hill1954), which was picked up in bad condition in London docks and died the day after from renal failure. Thereby, our record represents the second report of this genus of flukes in this bird species. Moreover, present record represents a new host for R. sloanei; the species was previously found parasitizing other species of seabirds, namely Pygoscelis antarcticus, Eudyptes chrysolophus, Uria aalge and Puffinus puffinus (see Table 1).
In conclusion, this work reports the occurrence and first identification, by morphoanatomical analysis and genetic studies, of R. sloanei in the urinary tract of F. arctica. Further investigation is required to better understand the morphological and molecular characteristics of Renicolidae species, together with the determination of their life cycles.
Acknowledgements
The authors thank the ‘Tahonilla’ staff for helping to supply the puffins.
Financial support
This work has been performed with the economic and logistical support from ‘Consejería de Transición Ecológica, Lucha contra el Cambio Climático y Planificación Territorial’ as part of ‘Estudio de patógenos en aves migratorias y en especies exóticas en un escenario de cambio climático’ project (No 248/2020 of 4 December 2020), and "Dirección General de Lucha Contra el Cambio Climático y Medio Ambiente” under the creation of the Canarian Network for the Surveillance of the Wildlife Health (Orden No 134/2020 de 26 May 2020). R.P.-V. was granted a predoctoral scholarship of the training program of the Department of Economy, Knowledge, and Employment of the Canary Government, co-funded by the European Social Fund (ESF) with a co-financing rate of 85% within the framework of the Canary Islands ESF Operational Program 2014–2020, priority axis 3, investment priority 10.2, specific object 10.2.1. (Tesis 2022010038).
Competing interest
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
Ethical standards
Ethical approval was not necessary because all the animals used in this study were already dead before the study. Statements on consent to participate and consent to publish are not applicable.
