Introduction
Crustaceans of the genus Artemia (Crustacea: Artemiidae), commonly known as brine shrimps, are distributed worldwide and play an essential role as dominant grazers in hypersaline water ecosystems, contributing to nutrient cycling (Sánchez et al., Reference Sánchez, Paredes, Lebouvier and Green2016; Triantaphyllidis et al., Reference Triantaphyllidis, Abatzopoulos and Sorgeloos1998). One of their important ecological functions is serving as intermediate hosts for various species of waterfowl helminths, primarily cestodes. The first report of a metacestode in Artemia salina (Linnaeus, 1758) was from Tunisia in 1926 (Heldt, Reference Heldt1926). The author presented a brief description without identification but, based on the shape and length of the hooks (180 μm), it could be assumed that it was Flamingolepis liguloides (Gervais, 1847). Later on, a metacestode was found in A. salina from the United States (Young, Reference Young1952) and, after infecting a definitive host experimentally, it was identified as Hymenolepis s.l. californicus Young, 1950, a species parasitising gulls. Investigations of various brine shrimp species have been conducted in Kazakhstan (Maksimova, Reference Maksimova1973, Reference Maksimova1976, Reference Maksimova1977, Reference Maksimova1981, Reference Maksimova1986, Reference Maksimova1987, Reference Maksimova1988, Reference Maksimova1989, Reference Maksimova1991, Reference Maksimova1991; Gvozdev & Maksimova, Reference Gvozdev and Maksimova1979, Reference Gvozdev, Maksimova and Gvozdev1985), Romania (Codreanu & Codreanu-Balcescu, Reference Codreanu and Codreanu-Balcescu1978), France (Gabrion & MacDonald, Reference Gabrion and MacDonald1980; Gabrion et al., Reference Gabrion, Boy and MacDonald-Crivelli1982; Thiéry et al., Reference Thiéry, Robert and Gabrion1990; Robert & Gabrion, Reference Robert and Gabrion1991; Vasileva et al., Reference Vasileva, Redón, Amat, Nikolov, Sánchez, Lenormand and Georgiev2009; Sánchez et al., Reference Sánchez, Rode, Flaven, Redón, Amat, Vasileva and Lenormand2012), Spain (Amat et al., Reference Amat, Gozalbo, Navarro, Hontoria and Varó1991a, Reference Amat, Illescas and Fernandezb; Varó et al., Reference Varó, Taylor, Navarro and Amat2000; Georgiev et al., Reference Georgiev, Sánchez, Green, Nikolov, Vasileva and Mavrodieva2005, Reference Georgiev, Sánchez, Vasileva, Nikolov and Green2007, Reference Georgiev, Angelov, Vasileva, Marta, Sánchez, Hortas, Mutafchiev, Pankov and Green2014; Sánchez et al., Reference Sánchez, Georgiev, Nikolov, Vasileva and Green2006, Reference Sánchez, Georgiev and Green2007, 2013; Vasileva et al., Reference Vasileva, Redón, Amat, Nikolov, Sánchez, Lenormand and Georgiev2009; Redón et al., Reference Redón, Green, Georgiev, Vasileva and Amat2015c), Italy (Mura, Reference Mura1995), United Arab Emirates (UAE) (Schuster, Reference Schuster2019; Sivakumar et al., Reference Sivakumar, Hyland and Schuster2020), Algeria (Amarouayache et al., Reference Amarouayache, Derbal and Kara2009), United States (Redón et al., Reference Redón, Berthelemy, Mutafchiev, Amat, Georgiev and Vasileva2015b), and Chile (Redón et al., Reference Redón, Vasileva, Georgiev and Gajardo2019). These studies established that Artemia spp. serve the intermediate host for 16 cestode species and one nematode species. The latter was identified only at the family level, the Acuariidae (Georgiev et al., Reference Georgiev, Angelov, Vasileva, Marta, Sánchez, Hortas, Mutafchiev, Pankov and Green2014; Redón et al., Reference Redón, Berthelemy, Mutafchiev, Amat, Georgiev and Vasileva2015b). Some species, i.e. Flamingolepis caroli (Parona, 1887), Flamingolepis tengizi Gvozdev & Maksimova, 1968 and Hymenolepis s.l. fusa (Krabbe, 1869), were each reported in a single publication (Maksimova, Reference Maksimova1973, Reference Maksimova1987; Gabrion & MacDonald, Reference Gabrion and MacDonald1980; Robert & Gabrion, Reference Robert and Gabrion1991). Flamingolepis megalorchis (Lühe, 1898), found in Artemia franciscana Kellogg, 1906 in the UAE (Schuster, Reference Schuster2019), was previously recorded in chironomids (Gvozdev & Maksimova, Reference Gvozdev and Maksimova1978). These faunistic studies demonstrate a lack of strict metacestode specificity for Artemia species; however, Redón et al. (Reference Redón, Amat, Sánchez and Green2015a) showed that parasite infection may vary depending on the Artemia species. In particular, they demonstrated that the prevalence and species richness of helminth infections in the same ecosystem are higher in the native A. salina compared to the invasive A. franciscana, which is non-native to the European region.
There are few partial sequences of small rRNA gene for metacestodes from Artemia spp. in GenBank: Confluaria podicipina (Szymanski, 1905), F. liguloides H. s.l. californicus, Fimdriarioides sp., and Flamingolepis sp. based on the material from Spain, United States, and Chile (Redón et al., Reference Redón, Quiroz, Lukić, Green and Gajardo2024).
Despite being extensively studied worldwide, brine shrimp helminths have not been investigated in Ukraine. The present research aimed to study the species composition of Artemia spp. helminths in the South and East regions of Ukraine, where several hypersaline water bodies are located.
Materials and methods
Material collection
From 2011 to 2021, 4,347 individuals of brine shrimp (Artemia spp.) were collected for the study. The sample efforts were conducted over different years at three locations in Ukraine: small salt lakes near Sloviansk in Kramatorsk Raion, Donetsk Oblast; an industrial saltern in Skadovsk Raion, Kherson Oblast; and the Adzhibaychik and Sasyk lakes on the western coast of Crimea. The details regarding the brine shrimps collected are provided in Table 1. The brine shrimps were seized using an aquarium net, placed in plastic containers filled with salt water and transported to the laboratory for examination. Because of logistical capabilities, the host individuals obtained from Crimea were treated differently; they were preserved in 70% ethanol before being transported to the laboratory for further examination.
Table 1. Summary data of sampling sites and sample size of Artemia spp. examined
Brine shrimps were examined between the microscope slides under the dissecting microscope. The detected alive helminths were extracted from the brine shrimps using preparatory needles, and the unstained helminth specimens were subsequently studied in 0.9% NaCl under the microscope. Most of the helminth specimens were fixed in 70% ethanol. In addition, several specimens were fixed in 96% ethanol for molecular analysis.
Morphological analysis
For a detailed morphological examination, cestode specimens were stained with iron acetocarmine diluted with 70% ethanol at a 1:1 ratio and subsequently mounted in Berlese’s medium. A cysticercoid of Branchiopodataenia gvozdevi (Maksimova, 1988) was stained with iron acetocarmine, dehydrated through a graded series of ethanol concentrations, cleared in clove oil, and mounted in Canada balsam. Before the examination, nematodes were rinsed in distilled water and cleared in lactophenol, a compound of equal parts water, glycerine, phenol, and lactic acid. The morphology of cestodes and nematodes was examined under a Zeiss Axio Imager M1 microscope equipped with DIC and an AmScope T690B microscope. The photos were made with digital cameras mounted on the microscopes. All measurements in the text are in micrometres and are given as a range followed by the mean and the number of measurements (n) in parentheses.
DNA extraction, polymerase chain reaction, and sequencing
DNA sequences of selected helminth species (10 isolates) were obtained. Total genomic DNA was isolated from cestode and nematode specimens preserved in 96% ethanol using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific) or the Monarch Genomic DNA Purification Kit (New England Biolabs, Inc., Ipswich, MA, USA) according to the manufacturer’s standard protocols. For each polymerase chain reaction (PCR) reaction, the mixture included 2.0 μL of gDNA, 12.5 μL of MyTaq HS Red Mix (Bioline), 1.0 μL of forward primer, 1.0 μL of reverse primer, and 5.5 μL of water.
The amplification of the 18S rDNA (~800 bp) fragment for nematode isolate was conducted using the forward primer 18SU467F (5′-ATC CAA GGA AGG CAG CAG GC-3′) and the reverse primer 18SL1170R (5′-GTG CCC TTC CGT CAA TTC CT-3′) (Indaryanto et al., Reference Indaryanto, Abdullah, Wardiatno, Tiuria and Imai2015). The cycling conditions included denaturation at 94 °C for 2 min, followed by 30 cycles of 30 s at 94 °C, 30 s at 45 °C, and 1 min at 72 °C, with a final extension of 7 min at 72 °C.
The 1,450–1,500 bp long fragments of the 28S rDNA were amplified for cestode isolates using the forward primer ZX-1 (5’-ACC CGC TGA ATT TAA GCA TAT-3’) (Scholz et al., 2013) and the reverse primer 1500R (5’-GCT ATC CTG AGG GAA ACT TCG-3’) (Tkach et al., Reference Tkach, DTJ, Olson, Kinsella and Swiderski2003). The cycling conditions included denaturation at 95 °C for 5 min, followed by 40 cycles of 30 s at 95 °C, 30 s at 55 °C, and 2 min at 72 °C, with a final extension of 7 min at 72 °C.
Fragments of approximately 500 bp and 800 bp of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were amplified for cestode isolates using the forward primer Dig_cox1Fa (5′-ATG ATW TTY TTY TTY YTD ATG CC-3′) and the reverse primer Dig_cox1R (5′-TCN GGR TGH CCR AAR AAY CAA AA-3′) (Wee et al., Reference Wee, Cribb, Bray and Cutmore2017) or the forward primer Dice1F (5′-ATT AAC CCT CAC TAA ATT WCN TTR GAT CAT AAG-3′) and the reverse primer Dice14R (5′-TAA TAC GAC TCA CTA TAC CHA CMR TAA ACA TAT GAT G-3′). The cycling conditions included denaturation at 94 °C for 4 min, followed by 40 cycles of 30 s at 94 °C, 30 s at 51 °C (Dig_cox primers) or 53 °C (Dice primers), and 30 s at 72 °C, with a final extension of 10 min at 72 °C.
For cestodes, partial nad1 + trnN region (~800 bp) was amplified using the forward primer Cyclo_nad1F (5′-GGN TAT TST CAR TNC GTA AGG G-3′) and the reverse primer Cyclo_trnNR (5′-TTC YTG AAG TTA ACA GCA TCA-3′) (Littlewood et al., Reference Littlewood, Waeschenbach and Nikolov2008). The cycling conditions included denaturation at 94 °C for 3 min, followed by 40 cycles of 30 s at 94 °C, 30 s at 55 °C, and 1.5 min at 72 °C, with a final extension of 7 min at 72 °C.
Amplified DNA was purified using ExoSAP-IT PCR Cleanup enzymatic kit from Thermo Fisher Scientific, Inc. (Waltham, MA, USA) and sequenced from both strands using the PCR primers and additional internal sequencing primer 300F (5′-CAA GTA CCG TGA GGG AAA GTT G-3′) and ECD2 (5′-CTT GGT CCG TGT TTC AAG ACG GG-3′) (Littlewood et al., Reference Littlewood, Curini-Galletti and Herniou2000) for 28S rDNA and primer Cyclo_nad1Fb (5′-AGG TTT GAR GCK TGT TTT ATG-3′) for nad1 + trnN region. Sanger sequencing was conducted at the Faculty of Natural Sciences of Comenius University (Bratislava, Slovakia) or a commercial sequencing company, SEQme (Dobříš, Czech Republic). Chromatogram-based contigs were assembled and edited using Geneious Prime 2024.0.5 software (Biomatters, Auckland, New Zealand; https://geneious.com).
Results
Helminth infections were recorded in Artemia spp. at each of the studied locations. Of 4,347 examined individuals of Artemia spp., 953 (21.79%) were infected with helminths. The intensity of infection was low, with one to three specimens per individual. The total values of the mean abundance and the mean intensity were 0.22 and 1.03, respectively. Cysticercoids of six cyclophyllidean species of two families and one nematode species were recorded. The data on helminth prevalence and intensity for each location are provided in Table 2.
Table 2. Summary data of species recorded in Artemia spp. and their infection rates
The information and the description of found helminths are given next.
CESTODA
Family Dilepididae Railliet et Henry, 1909
Anomotaenia tringae (Burt, 1940) (Fig. 1B)
Figure 1. Metacestodes from Artemia spp. (A) Hymenolepis s. l. stellorae Deblock, Biguet et Capron, 1960; (B) Anomotaenia tringae (Burt, 1940); (C) Branchiopodataenia gvozdevi (Maksimova, 1988); (D) Fimbriarioides tadornae (Burt, 1940); (E) Eurycestus avoceti Clark, 1954; (F) Confluaria podicipina (Szymanski, 1905). Scale bars: (A), 200 μm; (B), (E), 50 μm; (C), (D), (F), 100 μm.
Prevalence and intensity of infection: 0.3% and 1 specimen (salt lakes near Sloviansk, Donetsk Oblast).
Description (based on one specimen from Sloviansk): Outer capsule oval, 220 × 230. Cyst rounded, 105 × 125. Cyst-wall consists of three layers: external 1–2 in thickness, median 9–14 in thickness, and internal 3–5 in thickness. Scolex rounded, 85 × 75. Suckers oval muscular, 38–45 × 20–28 (41.5 × 24, n=4). Rostellum 60 with maximum diameter at hooks level 28. Rostellar sheath 70 × 33. Rostellum armed with 18 hooks. Anterior surface of rostellum usually not invaginated and hook blades oriented posteriorly. Total length of hooks 19–20 (19.5, n=4), handle 9 (9, n=4), blade 9–10 (9, n=4). Invagination pore as narrow slit, 11 in depth, at anterior part of cyst. Excretory canal short, 8 in depth, opening at posterior part of cyst. Embryonic hooks 10–13 (12, n=6).
Remarks
Metacestodes of this species were first recorded from Artemia parthenogenetica Bowen et Sterling, 1978 in Spain (Georgiev et al., Reference Georgiev, Sánchez, Green, Nikolov, Vasileva and Mavrodieva2005). In subsequent studies, metacestodes of A. tringae were found in A. franciscana in Portugal (Georgiev et al., Reference Georgiev, Sánchez, Vasileva, Nikolov and Green2007) and in the UAE (Sivakumar et al., Reference Sivakumar, Hyland and Schuster2020), as well as in A. salina in Spain (Sánchez et al., Reference Sánchez, Nikolov, Georgieva, Georgiev, Vasileva, Pankov, Paracuellos, Lafferty and Green2013). The morphology of our specimens corresponds to that of specimens from Spain described by Georgiev et al. (Reference Georgiev, Sánchez, Green, Nikolov, Vasileva and Mavrodieva2005) by the number, shape and size of rostellar hooks, and the shape of cysticercoid. Different charadriiform birds are definitive hosts of this species (Spasskaya & Spassky, Reference Spasskaya and Spassky1978). In Ukraine, A. tringae was reported from Tringa glareola Linnaeus, 1758, Tringa nebularia (Gunnerus, 1767), Tringa stagnatilis (Bechstein, 1803), Tringa totanus (Linnaeus, 1758), Gallinago gallinago (Linnaeus, 1758) and Limosa limosa (Linnaeus, 1758) (Smogorzhevskaya, Reference Smogorzhevskaya1976; Greben & Kornyushin, Reference Greben and Kornyushin2001).
Eurycestus avoceti Clark, 1954 (Fig. 1E)
Prevalence and intensity of infection: 52.5% and one to two specimens (salt lakes near Sloviansk, Donetsk Oblast); 0.1% and one specimen (industrial saltern, Kherson Oblast); 2.5% and one to two specimens (Adzhibaychik and Sasyk lakes, Crimea).
Representative sequence: PQ084087 (28S rDNA), PQ397346 (cox1), PQ156477 (nad1).
Description (based on 10 specimens from Sloviansk): Total length of cysticercoid 115–143 (126, n=10), 75–115 (101, n=10) in diameter. Cysticercoid surrounded by thick external capsule, with thickness of walls 40–70. Wall of cysticercoid delicate, 3–5 thick. Internal covering 10–28 thick. Calcareous corpuscles numerous. Scolex oval or rounded, 60–80 × 55–70 (75 × 62, n=10). Suckers oval, 23–30 × 16–23 (25 × 19, n=34), armed with small spines in anterior part. Rostellum thin and elongate, 33–60 (50, n=10) long; maximum diameter at hooks level, 18–20 (19, n=10). Rostellar sheath 45–71 × 25–30 (62 × 29, n=10). Rostellum armed with 14–16 hooks. Rostellum usually not invaginated; hook blades oriented posteriorly. Total length of hooks 14–16 (16, n=32), handle 10–13 (11, n=32) long, blade 3–4 (4, n=32) long. Invagination pore as narrow slit, 20–35 (26, n=9) in depth, at anterior part of cyst. Excretory canal very short, 1–5 (2, n=9) in depth, at posterior end of cyst. Embryonic hooks not distinct.
Remarks
Initially, metacestodes of E. avoceti were recorded from Artemia sp. in France (Gabrion & MacDonald, Reference Gabrion and MacDonald1980). A detailed description of these metacestodes was presented based on the specimens from A. salina in Kazakhstan (Maksimova, Reference Maksimova1991) and A. parthenogenetica in Spain (Georgiev et al., Reference Georgiev, Sánchez, Green, Nikolov, Vasileva and Mavrodieva2005). This species was registered in A. salina in Spain (Georgiev et al., Reference Georgiev, Sánchez, Vasileva, Nikolov and Green2007; Sánchez et al., Reference Sánchez, Nikolov, Georgieva, Georgiev, Vasileva, Pankov, Paracuellos, Lafferty and Green2013), in A. parthenogenetica in Spain (Georgiev et al., Reference Georgiev, Sánchez, Vasileva, Nikolov and Green2007; Sánchez et al., Reference Sánchez, Georgiev, Nikolov, Vasileva and Green2006, Reference Sánchez, Georgiev and Green2007, 2013), and in France (Sánchez et al., Reference Sánchez, Rode, Flaven, Redón, Amat, Vasileva and Lenormand2012), and in A. franciscana in Spain and Portugal (Georgiev et al., Reference Georgiev, Sánchez, Vasileva, Nikolov and Green2007), in France (Sánchez et al., Reference Sánchez, Rode, Flaven, Redón, Amat, Vasileva and Lenormand2012), and in the UAE (Schuster, Reference Schuster2019; Sivakumar et al., Reference Sivakumar, Hyland and Schuster2020). Our specimens are similar to the specimens described by Gabrion and MacDonald (Reference Gabrion and MacDonald1980) from France, by Maksimova (Reference Maksimova1991) from Kazakhstan, and by Georgiev et al. (Reference Georgiev, Sánchez, Green, Nikolov, Vasileva and Mavrodieva2005) from Spain. They differ only from material from Spain by the smaller size of cysticercoid (126 × 101 vs 182 × 137) and smaller scolex (75 × 62 vs 135 × 98). However, the armament of the rostellum and suckers of specimens from Ukraine correspond to the same material described in the previous publications.
Various charadriiform birds are definitive hosts of E. avoceti (Spasskaya & Spassky, Reference Spasskaya and Spassky1978). In Ukraine, this species was found in Charadrius alexandrinus (Linnaeus, 1758), Himantopus himantopus (Linnaeus, 1758) and Recurvirostra avosetta Linnaeus, 1758 (Smogorzhevskaya, Reference Smogorzhevskaya1976).
Family Hymenolepididae Ariola, 1899
Branchiopodataenia gvozdevi (Maksimova, 1988) (Fig. 1C)
Prevalence and intensity of infection: 0.1% and one specimen (industrial saltern, Kherson Oblast).
Description (based on two specimens from industrial saltern, Kherson Oblast, after Kornyushin and Greben, Reference Kornyushin, Greben and Kharchenko2022, with additional data): Cysticercoid oval, massive, 250–330 in length and 150–200 in maximum width. Cyst-wall 20–30 in thickness. Scolex 150–180 in length and 110–163 in maximum diameter at sucker level. Suckers rounded 55–60 × 50–55. Rostellar sheath 140 × 70. Rostellum 90 in length, with maximum diameter at hooks level 60. Rostellum armed with 10 aploparaksoid hooks. Anterior surface of rostellum usually invaginated and hook blades oriented anteriorly. Total length of hooks 38–40 (39, n=5), handle 13 (13, n=5), blade 20–23 (22, n=5), guard 10–13 (11, n=5), base with guard 20–21 (21, n=5). Invagination pore 60–80 in depth, at anterior part of cyst. Excretory canal 20–30 in depth, at posterior part of cyst. Cercomer short, 630 long, 45–50 in diameter. Embryonic hooks 10–11 (10, n=5), usually localised in cercomer.
Remarks
Metacestodes of B. gvozdevi were first recorded in A. salina in Kazakhstan, where the species was described, and its development in the intermediate host was studied (Maksimova, Reference Maksimova1988). Cysticercoids of this species were also found in A. franciscana, A. salina and A. parthenogenetica in Spain (Vasileva et al., Reference Vasileva, Redón, Amat, Nikolov, Sánchez, Lenormand and Georgiev2009; Redón et al., Reference Redón, Green, Georgiev, Vasileva and Amat2015c).
The specimens in this study were generally similar to the metacestodes described by Maksimova (Reference Maksimova1988) and Vasileva et al. (Reference Vasileva, Redón, Amat, Nikolov, Sánchez, Lenormand and Georgiev2009). They differed from the specimens from Kazakhstan by having a wider scolex (110–163 vs 84–120), a larger rostellum (60 vs 38–50), and a shorter, slightly wider cercomer (630 × 45–50 vs 1.21–1.43 × 40–42). Our specimens differed from the specimens reported from Spain by having a longer scolex (150–180 vs 123–149), a more extended rostellar sheath (90 vs 54–70), a larger rostellum (90 × 60 vs 54–70 × 44–51), a large cercomer (630 × 45–50 vs 400 × 25–39) and a larger maximum scolex width (110–163 vs 84–120).
The gull Chroicocephalus genei (Brème, 1839) is the only known definitive host for B. gvozdevi in Ukraine (Kornyushin & Greben, Reference Kornyushin and Greben2013) and elsewhere (Maksimova, Reference Maksimova1988).
Confluaria podicipina (Szymanski, 1905) (Fig. 1F)
Prevalence and intensity of infection: 3.0% and one to two specimens (Adzhibaychik and Sasyk lakes, Crimea).
Representative sequence: PQ084089 (28S rDNA), PQ096505 (nad1).
Description (based on seven specimens from Crimea): Cysticercoid oval, 160–240 × 130–190 (171 × 141, n=7), with very long think cercomer 8–23 (11, n=14) in diameter. Length of cercomer tens of times greater than length of cysticercoid. Cyst oval, 66–125 × 40–80 (99 × 58, n=7). Cyst wall consists of three layers. External layer thin, 2.5–3 in thickness. Medial layer consists of three parts: first part 4–6 thick, friable part 8–15 thick, and threaded part 2–3 thick. Internal layer consists of three parts: basal part 9–30 thick, fibrous part 2–3 thick, and parenchymal part 2–4 thick. Calcareous corpuscles numerous. Scolex oval, 55–75 × 35–55 (66 × 48, n=7). Suckers slightly oval, 25–32 × 13–25 (30 × 22, n=14). Rostellum 35–65 (47, n=7), with maximum diameter at hooks level 20–40 (29, n=7). Rostellar sheath 55–65 × 35–45 (59 × 40, n=4) with thin wall. Rostellum armed with 10 aploparaksoid hooks. Anterior surface of rostellum usually invaginated and hook blades oriented anteriorly. Total length of hook 18–23 (21, n=18), handle 4–6 (5, n=18), blade 10–13 (11, n=18), guard 5–8 (7, n=18), base with guard 13–15 (13, n=18).
Invagination pore as slit, 13–20 (16, n=6) deep, at anterior part of cyst. Posterior canal short, 5–10 (7, n=6) in depth.
Remarks
Metacestodes of this species were described from A. salina in Kazakhstan (Maksimova, Reference Maksimova1981). Confluaria podicipina was found in A. parthenogenetica and A. salina from Spain (Georgiev et al., Reference Georgiev, Sánchez, Green, Nikolov, Vasileva and Mavrodieva2005, Reference Georgiev, Sánchez, Vasileva, Nikolov and Green2007; Sánchez et al., Reference Sánchez, Georgiev, Nikolov, Vasileva and Green2006, Reference Sánchez, Georgiev and Green2007, Reference Sánchez, Nikolov, Georgieva, Georgiev, Vasileva, Pankov, Paracuellos, Lafferty and Green2013), in A. franciscana from the USA (Redón et al., Reference Redón, Berthelemy, Mutafchiev, Amat, Georgiev and Vasileva2015b), the UAE (Sivakumar et al., Reference Sivakumar, Hyland and Schuster2020), and Chili (Redón et al., Reference Redón, Vasileva, Georgiev and Gajardo2019). Cysticercoids identified C. podicipina from Acanthocyclops viridis in the Czech Republic (Tolkacheva, Reference Tolkacheva1987) likely belong to a different species because this is the only record of this species in freshwater invertebrates; it is unlikely that eggs of the same cestode species can survive in both fresh and saltwater. There are several cestode species with aploparaksoid hooks of the same length as C. podicipina and with an unknown life cycle (Bondarenko & Kontrimavichus, Reference Bondarenko, Kontrimavichus and Movsesyan2006).
The morphology of our specimens corresponds to that of metacestodes from Kazakhstan (Maksimova, Reference Maksimova1981) and Spain (Georgiev et al., Reference Georgiev, Sánchez, Green, Nikolov, Vasileva and Mavrodieva2005). They differ from the specimens described by Maksimova (Reference Maksimova1981) in having a wider cysticercoid (130–190 vs 105) and from the specimens described by Georgiev et al. (Reference Georgiev, Sánchez, Green, Nikolov, Vasileva and Mavrodieva2005) in having a wider range of the scolex length (55–75 vs 72–104).
Grebes are the definitive hosts of C. podicipina (Spasskaya, Reference Spasskaya1966; Vasileva et al., Reference Vasileva, Georgiev and Genov2000). This species was found in Ukraine in various species of grebes (Smogorzhevskaya, Reference Smogorzhevskaya1976).
Fimbriarioides tadornae (Burt, 1940) (Fig. 1D)
Prevalence and intensity of infection: 0.1% and one specimen (industrial saltern, Kherson Oblast); 0.8% and one specimen (Lake Adzhibaychik, Crimea).
Description (based on two specimens from industrial saltern, Kherson Oblast and one specimen from Crimea): Length of rounded cysticercoid 150–215 (185, n=3), diameter 130–150 (142, n=3). One specimen with delicate capsule 10–30 in thickness. Cyst 105–155 × 100–150 (130 × 132, n=3). Cyst-wall thick, consisting of three layers: external 5–6 thick, median 6–10 thick, and internal 10–15 thick. Scolex oval, 73–95 × 80–110 (90 × 86, n=3). Suckers oval, 33–45 × 27–45 (36 × 32, n=7). Rostellum 45–50 (48, n=3) long, with maximum diameter at hooks level 23–30 (28 × 33, n=3). Rostellar sheath 60–73 × 42–45 (68 × 44, n=3). Rostellum armed with 10 hooks. Anterior surface of rostellum usually invaginated and hook blades oriented anteriorly. Total length of hook 25–26 (26, n=7), handle 13–15 (14, n=7), blade 10–11 (10, n=7), guard 4–5 (4, n=7). Invagination pore as narrow slit, 33–43 (37, n=3) deep, at anterior part of cyst. Posterior pore short, 9–18 (13, n=3) deep. Cercomer with maximum length 1.120 mm, 10–20 in diameter. In one specimen cercomer has small tear-shaped widening, 35 × 25, on the end. Embryonic hooks 9–11 (10, n=6), localised in cercomer.
Remarks
Metacestodes of this species were first recorded from A. salina in Kazakhstan with the description of the species (Maksimova, Reference Maksimova1976). Also, F. tadornae has been registered in A. salina in Spain (Sánchez et al., Reference Sánchez, Nikolov, Georgieva, Georgiev, Vasileva, Pankov, Paracuellos, Lafferty and Green2013), in A. parthenogenetica in Spain (Georgiev et al., Reference Georgiev, Sánchez, Vasileva, Nikolov and Green2007; Vasileva et al., Reference Vasileva, Redón, Amat, Nikolov, Sánchez, Lenormand and Georgiev2009), in France (Sánchez et al., Reference Sánchez, Rode, Flaven, Redón, Amat, Vasileva and Lenormand2012), in A. franciscana in Spain (Vasileva et al., Reference Vasileva, Redón, Amat, Nikolov, Sánchez, Lenormand and Georgiev2009) and France (Sánchez et al., Reference Sánchez, Rode, Flaven, Redón, Amat, Vasileva and Lenormand2012; Vasileva et al., Reference Vasileva, Redón, Amat, Nikolov, Sánchez, Lenormand and Georgiev2009).
Our specimens are similar to those reported from Kazakhstan (Maksimova, Reference Maksimova1976), Spain and France (Vasileva et al., Reference Vasileva, Redón, Amat, Nikolov, Sánchez, Lenormand and Georgiev2009). They differ from the specimens described by Maksimova (Reference Maksimova1976) in a shorter scolex (73–95 vs 108), rostellum (45–50 vs 80), rostellar sheath (60–73 vs 96) and a smaller diameter of the suckers (33–45 vs 50). From specimens described by Vasileva et al. (Reference Vasileva, Redón, Amat, Nikolov, Sánchez, Lenormand and Georgiev2009), they differ by a longer cercomer (1.120 vs 720). However, the length and size of rostellar hooks of specimens from Ukraine correspond well to the specimens described in all the previous publications.
Fimbriarioides tadornae is a parasite of Tadorna tadorna (Linnaeus, 1758). In Ukraine, this species was reported as Fimbriarioides intermedia (Fuhrmann, 1918) in this bird (Kornyushin, Reference Kornyushin1969; Smogorzhevskaya, Reference Smogorzhevskaya1976).
Hymenolepis s.l. stellorae Deblock, Biguet et Capron, 1960 (Fig. 1A)
Prevalence and intensity of infection: 8.9% and one to two specimens (salt lakes near Sloviansk, Donetsk Oblast); 0.3% and one to three specimens (industrial saltern, Kherson Oblast); 0.5% and one specimen (Adzhibaychik and Sasyk lakes, Crimea).
Representative sequences: PQ084088 (28S rDNA), PQ397347 (cox1), PQ156478 (nad1).
Description (based on 10 specimens from Sloviansk): Total length of metacestode 1.32–3.48 (2.33, n=5). Cysticercoid oval, 200–280 × 100–175 (249 × 143, n=6). Cyst oval, 165–190 × 120–145 (181 × 127, n=10). Cyst-wall 9–15 in thickness. Calcareous corpuscles numerous, concentrated in anterior and posterior parts of cyst’s cavity. Scolex oval, 85–120 × 75–105 (100 × 92, n=10). Suckers oval, muscular, 35–55 × 30–45 (44 × 35, n=29). Rostellum 52–60 (57, n=9) with maximum diameter at hooks level 35–45 (42, n=9). Rostellar sheath 70–90 × 40–60 (57 × 42, n=9). Rostellum armed with 10 aploparaksoid hooks. Anterior surface of rostellum usually invaginated and hook blades oriented anteriorly. Total length of hook 21–24 (22, n=19), handle 4–6 (5, n=19), blade 13–15 (13, n=19), guard 9–10 (10, n=19), base with guard 14–15 (14, n=19). Invagination pore as narrow slit 45–50 (48, n=10) in depth, at anterior part of cyst. Excretory canal short, 5–10 (7, n=10) in depth, at posterior part of cyst. Cercomer elongate 1.06–3.26 mm (1.94 mm, n=9) and 90–180 (128, n=10) in diameter. It has rare, not deep, protrusions. Embryonic hooks 13–15 (14, n=22), usually localised in cercomer.
Remarks
Cysticercoids of this species were described from A. salina as Aploparaksis parafilum Gasowska, 1932 (Maksimova, Reference Maksimova1973) and later as Wardium stellorae (Maksimova, Reference Maksimova1986) in Kazakhstan. This species was registered in Artemia spp. from France (Robert & Gabrion, Reference Robert and Gabrion1991), A. salina from Spain (Georgiev et al., Reference Georgiev, Sánchez, Vasileva, Nikolov and Green2007; Varó et al., Reference Varó, Taylor, Navarro and Amat2000), A. parthenogenetica from Spain (Georgiev et al., Reference Georgiev, Sánchez, Green, Nikolov, Vasileva and Mavrodieva2005, Reference Georgiev, Sánchez, Vasileva, Nikolov and Green2007; Sánchez et al., Reference Sánchez, Georgiev, Nikolov, Vasileva and Green2006, 2013; Varó et al., Reference Varó, Taylor, Navarro and Amat2000) and France (Sánchez et al., Reference Sánchez, Rode, Flaven, Redón, Amat, Vasileva and Lenormand2012) and A. franciscana from UAE (Sivakumar et al., Reference Sivakumar, Hyland and Schuster2020).
The description of metacestodes of H. s. l. stellorae by Maksimova (Reference Maksimova1973) and Robert and Gabrion (Reference Robert and Gabrion1991) were relatively poor and limited to the size of cysticercoid and hooks. A detailed description of these metacestodes was presented on the material from A. salina in Kazakhstan (Maksimova, Reference Maksimova1986) and A. parthenogenetica in Spain (Georgiev et al., Reference Georgiev, Sánchez, Green, Nikolov, Vasileva and Mavrodieva2005). The morphology of our specimens corresponds to that of cysticercoids described in Kazakhstan and Spain. They differ from metacestodes reported from Spain only by the larger size of the rostellum (52–60 × 35–45 vs 39–45 × 23–30) and from metacestodes from Kazakhstan by a shorter rostellum (52–60 vs 73).
Chroicocephalus genei is the only known definitive host of H. s. l. stellorae. In Ukraine, it was reported in birds on the Black Sea coast (Smogorzhevskaya, Reference Smogorzhevskaya1976).
NEMATODA
Acuariidae gen. sp. (Fig. 2 A–H)
Figure 2. Acuariidae gen. sp. larva from Artemia salina. (A) general view; (B) transverse section of the body at level of muscular oesophagus; (C) transverse section of the body at mid-length; (D) anterior end, lateral view; (E) anterior end, dorso-ventral view; (F) surface of anterior part of the body showing cuticular ridges and deirid (arrow); (G) anterior part of the body showing the shape of the stoma and the nerve ring (arrow), lateral view; (H) posterior part of the body, lateral view. Scale bars: (A), (B), (C), (F), (G), (H), 50 μm; (D), (E), 10 μm.
Prevalence and intensity: 0.1% and one specimen (salt lakes near Sloviansk, Donetsk Oblast); 4.1% and one to three specimens (industrial saltern, Kherson Oblast.); 3.5% and one to two specimens (Adzhibaychik and Sasyk lakes, Crimea).
Representative sequences: PQ084085, PQ084086 (18S rDNA).
Description based on five specimens from Kherson Oblast and five from Crimea (Lake Sasyk).
Third-stage larvae. Total length of body 2.912–3.968 mm (3.428 mm, n=10), maximum width 65–85 (76, n=10). Anterior end with two triangular pseudolabia, each bearing one pair of papillae. Cuticle with longitudinal ridges extending along body. Lateral alae absent. Lateral longitudinal ridges smaller than others. Cuticle forms transverse folds on tail at anus level. Cordons not observed. Deirids spine-like, 5–6 (5, n=16) long, situated at 68–80 (73, n=16) from anterior end, at level of posterior part of buccal cavity. Excretory pore at 175–245 (207, n=8) from anterior end. Tail 120–140 (131, n=8) long; width at anus 40–46 (45, n=8). End of tail with narrowing 13–15 (14, n=10) long, 7–10 (8, n=10) in diameter of proximal part, and 4–6 (5, n=10) in diameter of distal part. Buccal cavity 65–95 (86, n=10) long, 4–6 (4, n=10) wide. Anterior part triangular, posterior part circular. Muscular oesophagus 160–210 (184, n=10) long, 15–27 (21, n=10) wide. Glandular oesophagus 830–1.040 (912, n=10) long, 50–65 (58, n=10) wide. Nerve-ring 20–35 × 25–45 (26 × 33, n=10), at 113–135 (126, n=10) from anterior end. Relative length (ratio) of muscular and glandular oesophagus to body length 0.283–0.376 (0.320, n=10). Relative length of muscular oesophagus to glandular oesophagus (ratio) 0.154–0.205 (0.203, n=10).
Remarks
The presence of muscular and glandular portions of the oesophagus, lateral triangular pseudolabia, and elongated buccal cavity allows for the identification of the studied nematodes as Acuariidae gen. sp. (Chabaud, Reference Chabaud, Anderson and Willmott1975; Anderson, Reference Anderson2000). We identified these nematodes at the family level only because the genera and species of the Acuariidae have been differentiated based on the morphology of the adult stage. Most of these nematodes are parasites of the stomach (under the gizzard lining), proventriculus or oesophagus of birds (Smogorzhevskaya, Reference Smogorzhevskaya and Sharpilo1990). The absence of cordons is characteristic for nematodes of some genera of Acuariidae, in particular for the genus Paracuaria Rao, 1951 (Mutafchiev et al., Reference Mutafchiev, Georgiev and Mariaux2020). There is one species of the genus, Paracuaria adunca (Creplin, 1846), in Ukraine. It is a common parasite of gulls and terns and is found throughout the entire territory. However, freshwater crustaceans are intermediate hosts of this species of nematodes (Smogorzhevskaya, Reference Smogorzhevskaya and Sharpilo1990). It is unlikely that invertebrates living in hypersaline waters can also participate in the circulation of these nematodes.
The first record of Artemia as the intermediate host for the nematodes was published by Georgiev et al. (Reference Georgiev, Angelov, Vasileva, Marta, Sánchez, Hortas, Mutafchiev, Pankov and Green2014). The authors found Acuariinae gen. sp. from A. franciscana in Spain, presented a photo of helminth without description, and noted that the larvae with similar morphology were also recorded in A. parthenogenetica and A. salina. An unidentified nematode of the Acuariidae with detailed description and illustrations was recorded in A. franciscana from the USA (Redón et al., Reference Redón, Berthelemy, Mutafchiev, Amat, Georgiev and Vasileva2015b). The authors admitted similar morphology of their specimens and larvae from Spain and supposed that they belong to the same species.
The morphology of our material differs from the morphology of specimens from Spain and USA by the absence of lateral alae and cordons and the presence of longitudinal ridges extending along the body. Our specimens have a constriction on the distal end of the tail, which is not described in the nematode larvae from Spain and the United States (Georgiev et al., Reference Georgiev, Angelov, Vasileva, Marta, Sánchez, Hortas, Mutafchiev, Pankov and Green2014; Redón et al., Reference Redón, Berthelemy, Mutafchiev, Amat, Georgiev and Vasileva2015b). We found Acuariidae gen. sp. in brine shrimps in all examined localities, which likely suggests that Artemia is the intermediate host for these nematodes.
Discussion
The present study reported seven helminth species (six cestodes and one nematode) in Artemia spp. Two of these species, H. stellorae and E. avoceti, were genetically characterised (cox1 and nad1 mtDNA) for the first time. All identified species of cestodes and the nematode were registered for the first time in intermediate hosts in Ukraine, although they have been recorded elsewhere worldwide. The number of helminth species found in the present study is consistent with those found in other regions where the helminths of Artemia were studied. For instance, nine species were found in both Kazakhstan and Spain (Maksimova, Reference Maksimova1989; Georgiev et al., Reference Georgiev, Sánchez, Green, Nikolov, Vasileva and Mavrodieva2005, Reference Georgiev, Angelov, Vasileva, Marta, Sánchez, Hortas, Mutafchiev, Pankov and Green2014; Sánchez et al., Reference Sánchez, Nikolov, Georgieva, Georgiev, Vasileva, Pankov, Paracuellos, Lafferty and Green2013), seven species in France (Robert & Gabrion, Reference Robert and Gabrion1991; Sánchez et al., Reference Sánchez, Rode, Flaven, Redón, Amat, Vasileva and Lenormand2012) and the UAE (Sivakumar et al., Reference Sivakumar, Hyland and Schuster2020), and five species in the United States (Redón et al., Reference Redón, Berthelemy, Mutafchiev, Amat, Georgiev and Vasileva2015b).
Notably, our study did not detect any species of the genus Flamingolepis Spasskii et Spasskaya, 1952, which are considered typical helminths of brine shrimp and have been found in studies from Italy (Mura, Reference Mura1995), Spain (Amat et al., Reference Amat, Illescas and Fernandez1991b; Varó et al., Reference Varó, Taylor, Navarro and Amat2000; Georgiev et al., Reference Georgiev, Sánchez, Green, Nikolov, Vasileva and Mavrodieva2005, Reference Georgiev, Sánchez, Vasileva, Nikolov and Green2007; Sánchez et al., Reference Sánchez, Nikolov, Georgieva, Georgiev, Vasileva, Pankov, Paracuellos, Lafferty and Green2013; Redón et al., Reference Redón, Green, Georgiev, Vasileva and Amat2015c), France (Thiéry et al., Reference Thiéry, Robert and Gabrion1990; Robert & Gabrion, Reference Robert and Gabrion1991; Sánchez et al., Reference Sánchez, Rode, Flaven, Redón, Amat, Vasileva and Lenormand2012), Algeria (Amarouayache et al., Reference Amarouayache, Derbal and Kara2009), Kazakhstan (Maksimova, Reference Maksimova1973), and the UAE (Sivakumar et al., Reference Sivakumar, Hyland and Schuster2020). This absence is obviously due to the lack of flamingos in our study localities.
The overall prevalence of helminths in Artemia from hypersaline waters in southern and eastern Ukraine was 21.9%. This prevalence was lower than those reported for Artemia in Spain (up to 51.95%) (Sánchez et al., Reference Sánchez, Nikolov, Georgieva, Georgiev, Vasileva, Pankov, Paracuellos, Lafferty and Green2013), France (up to 70.9%) (Sánchez et al., Reference Sánchez, Rode, Flaven, Redón, Amat, Vasileva and Lenormand2012), and the UAE (36.03%) (Sivakumar et al., Reference Sivakumar, Hyland and Schuster2020). Studies in these countries were conducted in protected areas with a high diversity of definitive hosts (birds). In contrast, our research was conducted in water bodies frequently visited by humans – such as resorts in Sloviansk and Crimea – and industrial salt extraction areas in Kherson Oblast, which have lower bird diversity. Anthropogenic habitat alterations and human presence may explain the difference in bird prevalence and diversity between our study and those conducted in protected areas, as human activities can make areas less favourable for bird visits.
Currently, the most accurate method to test whether an adult helminth specimen and a larva belong to the same species is to compare their DNA sequences. This comparison can sometimes be a basis for identifying shared morphological features specific to larval and adult stages. However, this approach can be challenging due to the lack of reliable morphological characters in larval stages, the absence of reference material, or poor species descriptions. Unfortunately, our study did not include adult specimens of cestode species. Nonetheless, we were able to identify the metacestode species based on morphological features that are identical in both adult and larval stages, as is known from studies on the life cycles of specific species (Maksimova, Reference Maksimova1989). These features include the size, shape and number of rostellar hooks (Spasskaya, Reference Spasskaya1966; Spasskaya & Spassky, Reference Spasskaya and Spassky1978). A search in GenBank revealed a very limited number of nucleotide sequences for the cestode species we studied, reflecting a gap in research on cyclophyllidean cestodes (Waeschenbach & Littlewood Reference Waeschenbach, Littlewood, Caira and Jensen2017). This lack of genetic data highlights the need for further molecular studies of the taxa.
Another issue in the molecular identification of helminth species is that some sequences are submitted to GenBank under specific scientific names without accompanying morphological data for the specimens from which they were obtained in related publications. This is particularly problematic when the scientific name is being introduced into the database for the first time because the lack of morphological confirmation leads to inaccuracies, requiring additional efforts to correct. We encountered this problem when analysing sequences of the nematode larvae. The 18S rDNA gene region in the two nematode larvae we sequenced is identical. The GenBank BLAST search service found a nearly identical sequence (EF180064) differing from ours only in a single nucleotide position. The authors of that sequence (Nadler et al., Reference Nadler, Carreno, Mejía-Madrid, Ullberg, Pagan, Houston and Hugot2007) reported that it was obtained from a specimen of Echinuria borealis Mawson, 1956 parasitising Somateria mollissima L. Since the 18S rDNA gene is highly conserved, the minor difference indicates very close phylogenetic relationships between our samples and the sequence from GenBank. According to BLAST results, we should classify the nematodes we examined as E. borealis or at least within the genus Echinuria. However, we have some doubts preventing us from doing so. The reason for doubt is that third-stage larvae of the family Acuariidae typically have well-formed cephalic structures (Anderson, Reference Anderson2000; Smogorzhevskaya, Reference Smogorzhevskaya and Sharpilo1990). We did not observe these structures in our specimens. Considering the sequence similarity and our results of morphological examination, we have chosen to identify our specimens only at the family level for now. To achieve a more accurate identification based on sequencing data, further research is needed to compare the morphology of adult nematodes with the sequences we have obtained.
Overall, studies that provide detailed morphological and molecular information for distinguishing helminth species and contribute verified sequences to public genetic databases are vital for enhancing our understanding of helminth systematics. Such studies are also essential for ecological investigations of parasites as the advancements in next-generation sequencing technologies create new opportunities to study helminths (Thomas et al., Reference Thomas, Milotic, Vaux and Poulin2022), especially for monitoring purposes. Robust genetic databases with comprehensive reference sequences are essential for the broader implementation of these advanced methods.
Acknowledgement
The research was partly supported by the NextGeneration EU through the Recovery and Resilience Plan for Slovakia under project No. 09I03-03-V01-00046.
Competing interest declaration
The authors declare that they have no conflicts of interest.
Ethical standard
Not applicable.
Statement on the use of AI-assisted technologies
During the preparation of this article, the authors used Grammarly (https://www.grammarly.com/) to correct grammatical errors and improve readability. After using this tool, the authors reviewed and edited the content as needed. The authors take full responsibility for the content of the published article.
