Introduction
The genus Contracaecum Railliet and Henry 1912 comprises heteroxenous parasites with life cycles dependent on aquatic intermediate hosts (crustaceans, fish) and definitive hosts that are predominantly piscivorous birds and, less frequently, marine mammals (e.g. pinnipeds) (Abollo et al., Reference Abollo, Gestal and Pascual2001; Mattiucci et al., Reference Mattiucci, Sbaraglia, Palomba, Filippi, Paoletti, Cipriani and Nascetti2020). Humans (accidental hosts) can become infected by ingesting raw or undercooked fish carrying third-stage larvae (Shamsi and Butcher, Reference Shamsi and Butcher2011).
In freshwater systems, herons (Ardeidae) contribute substantially to the transmission and maintenance of Contracaecum spp. (Barson and Marshall, Reference Barson and Marshall2004; Deng et al., Reference Deng, Li, Wang, Liu and Tu2022). For example, 6 nematode species, including Contracaecum, were found in 6 ardeids in Spain (Navarro et al., Reference Navarro, Lluch and Font2005). In a breeding colony in central Poland (Dziekońska-rynko et al., Reference Dziekońska-rynko, Mierzejewska and Hliwa2015), larval and adult stages of C. rudolphii Hartwich 1964 were found in all grey heron (Ardea cinerea L.) chicks examined, demonstrating that infections with this parasite occur very early in life, with possible adverse impacts on host health, and indicating a transfer of parasites from adult birds to chicks during feeding.
The black-crowned night heron (Nycticorax nycticorax L.), an ardeid host of Contracaecum spp. with a wide geographical range, has a broad breeding range across Eurasia, extending from southern and central Europe eastward through western and central Asia into China and Japan. In Europe, breeding populations exist from Iberia through Greece and Turkey, reaching northward into France, the Netherlands and Russia; northern European populations are largely migratory, wintering in sub-Saharan Africa. In Asia, N. nycticorax breeds from the Caspian region eastward into China and Japan; many northern populations migrate southward, while southern Asian populations tend to be resident year-round (IUCN, 2025).
Colonies at Israel’s Huleh Nature Reserve include both resident and migratory individuals, highlighting partial migration behaviours in the Middle East (Ashkenazi and Yom-tov, Reference Ashkenazi and Yom-tov1997). In eastern Spain, characterized by mild temperatures and high food availability, winter breeding occurs as late as February (Dies et al., Reference Dies, Ramón and Prosper2003).
During the breeding season in Eurasia, N. nycticorax exhibits an opportunistic feeding behaviour, with fish as the dominant prey across populations. In northern Honshū (Japan), black-crowned night herons primarily consumed Japanese loach (Misgurnus anguillicaudatus Cantor 1842), shifting foraging sites between rivers during daylight and irrigated paddy fields at night to match prey activity rhythms (Endo and Sawara, Reference Endo and Sawara2000). In western Spain, nestling diets comprised over 93% fish, predominantly Barbus comiza Steindachner 1864, while amphibians were almost absent, likely due to local river fauna dynamics (Pérez et al., Reference Pérez, De Lope, Turégano and De la Cruz1991). In the floodplain wetlands of the Paraná River (Argentina), fish were also the most important dietary item of black-crowned night herons, followed by insects and crustaceans (Quiroga et al., Reference Quiroga, León, Beltzer and Olguín2013). Together, these studies show a strong piscivorous preference among widely separated populations of this species.
Nycticorax nycticorax harbours a diverse community of helminths, including nematodes of the genus Contracaecum, which are commonly reported across different geographic regions. European studies have shown a notable prevalence of Contracaecum species (e.g. C. microcephalum (Rudolphi 1819 (Baylis 1920)), suggesting that this heron plays an important role in the parasite’s life cycle, likely through trophic transmission associated with the consumption of infected fish (Sitko and Heneberg, Reference Sitko and Heneberg2015; Syrota et al., Reference Syrota, Lisitsyna, Greben, Kornyushin and Du Preez2021).
Adult stages of Contracaecum have been recovered from N. nycticorax in southern Italy (Santoro et al., Reference Santoro, D’alessio, Di Prisco, Veneziano, Galiero, Cerrone, Barca, Kinsella and Aznar2026), Japan (Yoshida et al., Reference Yoshida, Tsuji and Iwata2012), the USA (Groves et al., Reference Groves, Achatz, Kinsella, Calhoun, Wilcox, Greiman and Johnson2025), China (Zhang et al., Reference Zhang, Meng, Huang, Feng, Liu, Tang, Fan, Yuan, Han, Luo, Zhao and He2021) and Iraq (Mhaisen and Abdul-ameer, Reference Mhaisen and Abdul-ameer2021). Infection levels vary geographically and are influenced by local ecological factors such as diet and intermediate host availability. With respect to Israel and the Middle East, Ashkenazi and Yom-tov (Reference Ashkenazi and Yom-tov1996) reported that black-crowned night herons in the Hula Valley, Israel, were found to harbour C. microcephalum larvae in their stomachs.
Although N. nycticorax represents an important host for Contracaecum in aquatic ecosystems, many reports in this host species do not identify the parasite to species level. A species-level understanding of Contracaecum diversity, distribution and pathogenic potential in ardeids is essential for elucidating host–parasite dynamics and assessing ecological and potential public health implications. In the present work, which is part of a larger study of parasites of piscivorous birds in Israel, morphological and molecular methods are employed to describe a new species of Contracaecum from N. nycticorax in Israel.
Materials and methods
Sampling and morphological study
Fourteen Contracaecum specimens were collected from the gastric mucosa of 3 black-crowned night herons (N. nycticorax) from a fish farm belong to Gesher, situated along the Jordan Rift Valley, Israel, collected under permits 2020/42659 and 2021/42855 from the Israel Nature and Parks Authority. The nematodes were taken from freshly processed hosts, washed in saline and preserved in 70% ethanol for morphological and molecular analysis.
A total of 7 male and 3 female adult worms were examined under a dissection microscope to assess gross morphological characteristics and record total length. Detailed morphological observations were subsequently conducted using a light microscope (Nikon Ci-L Plus, Nikon Europe B. V. Amstelveen, The Netherlands) equipped with a Nikon DS-Fi1 digital camera and image acquisition software (Nikon NIS-Elements U D-SUA). Except where noted, measurements are given in micrometres. The central region of each specimen, which lacks taxonomically diagnostic features, was excised for genomic DNA extraction. The anterior and posterior regions of the body were cleared in Amman’s lactophenol to facilitate visualization and measurement of internal structures. Morphometric analysis was carried out following Hartwich (Reference Hartwich1964) and Baruš et al. (Reference Baruš, Sergeeva, Sonin, Ryzhikov, Ryšavý, Ryšavý and Ryzhikov1978), while the nomenclature of the caudal structures of males follows Fagerholm (Reference Fagerholm1991).
For scanning electron microscopy (SEM), anterior and posterior portions of male and female specimens were dehydrated through a graded ethanol series, dried in hexamethyldisilazane, sputter-coated with gold palladium and observed using a Phenom XL G2 Desktop SEM (Thermo Fisher Scientific, Eindhoven, The Netherlands) operating at 5 kV.
The morphology of the specimens was quantitatively compared to those of other species using multivariate analysis implemented in Primer-E (Auckland, New Zealand). These analyses employed morphometrics of 10 specimens (7 male, 3 female) from the present study and up to 3 data points from other species, consisting of the minimum, maximum and mean reported in other studies. For each variable, data were normalized (the mean subtracted from each observed value, and the result divided by the standard deviation). Analysis of similarity (ANOSIM) with 9999 permutations was used to test for differences among species (crossed by worm sex) in the matrix of Euclidean distances among specimens. The resemblance matrix was visualized using a metric multidimensional scaling (MDS) ordination, after removing variables and species with >50% missing data.
Molecular study
For molecular analysis, genomic DNA was extracted using a PureLink® Genomic DNA Kit (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. The ITS rDNA was amplified with primers NC5_f (5′-GTAGGTGAACCTGCGGAAGGATCATT-3′) and NC2_r (5′-TTAGTTTCTTCCTCCGCT-3′) (Zhu et al., Reference Zhu, Gasser, Podolska and Chilton1998), while a fragment of cox2 mtDNA was amplified from with primers 211_f (5′-TTTTCTAGTTATATAGATTGRTTTYAT-3′) and 210_r (5′-CACCAACTCTTAAAATTATC-3′) of Mattiucci et al. (Reference Mattiucci, Paoletti, Webb, Sardella, Timi, Berland and Nascetti2008) following the same protocol. The PCR products were electrophoresed on a 1% agarose gel stained with SYBR Safe DNA Gel Stain (Thermo Fisher Scientific, Carlsbad, CA, USA) in 0.5× TBE. For sequencing, the amplicons were excised and purified by Nucleo-Spin Gel and PCR Clean-up (Mackerey-Nagel, Düren, Germany), and sequenced with an ABI 3730 DNA analyser (StarSEQ, Mainz, Germany). The DNA trace files were assembled with Contig Express (VectorNTI Advance 11 software, Invitrogen, Carlsbad, CA, USA), and the consensus sequences of the ITS rDNA after separating the 2 regions (ITS1 and ITS2) and cox2 mtDNA were compared with published data by BLAST tools (Altschul et al., Reference Altschul, Gish, Miller, Myers and Lipman1990). Multiple sequence alignments were performed using BioEdit 7.2.5 (Hall, Reference Hall1999). Genetic distances (uncorrected p) and maximum-likelihood (ML) trees (using GTR + G + I substitution model for both ITS and cox2 and bootstrap of 1000 replicates) were obtained using MEGA12 (Kumar et al., Reference Kumar, Stecher, Suleski, Sanderford, Sharma and Tamura2024). The ITS1 and ITS2 rDNA sequences were concatenated and used to build an ML tree together with the sequences of Contracaecum spp. reported by Mattiucci et al. (Reference Mattiucci, Sbaraglia, Palomba, Filippi, Paoletti, Cipriani and Nascetti2020) together with other Contracaecum sequences retrieved from GenBank. The cox2 gene was also aligned with the sequences reported by Mattiucci et al. (Reference Mattiucci, Sbaraglia, Palomba, Filippi, Paoletti, Cipriani and Nascetti2020), with Pseudoterranova ceticola (DQ116435) and Anisakis pegreffii (MT912471) as outgroups. The sequences generated in this study have 
$^\sim$been deposited in GenBank under accession numbers PZ240692-69, PZ240700-01 (ITS rDNA) and PZ244216-19 (cox2 mtDNA).
Results
Morphological description
Contracaecum fioravantii n. sp. (n = 10) (Figures 1–4)
Line drawing of adult male and female specimens of C. fioravantii from N. nycticorax: (A) and (B) male specimen, anterior end; (C) and (D) male specimen, posterior end; (E) female specimen, anterior end; and (F) female specimen, posterior end.
Host: N. nycticorax
Site in host: stomach
Locality: Gesher, fish farm along the Jordan Rift Valley, Israel
Type-material: Holotype (1 male) and allotype (1 female) are deposited at the Helminthological Collection of the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic (IPCAS N-1295).
Representative DNA sequences: PZ240692-69, PZ240700-01 (ITS rDNA) and PZ244216-19 (cox2 mtDNA).
Etymology: Contracaecum fioravantii n. sp. is named after Professor Marialetizia Fioravanti, whose scientific contributions and passion for parasitology have inspired our work and fostered our appreciation of parasites.
Adult stage: Body stout. Cuticle transversally striated (Figures 1–4). Lips longer than wide (Figure 2A), 1 dorsal and 2 subventral (Figure 4B). Two oval cephalic papillae on dorsal lip and a single papilla on each subventral lip (Figures 2A, 4B). Interlabium reaching approximately 4/5 of lip length, wider at base, with pointed tip. Excretory pore at base of lips. Collar area short, with fine cuticular annulations interrupted laterally (Figure 2A). Amphids approximately at level of nerve ring (Figures 1B, 2B). Oesophagus muscular, ending in round ventriculus with short ventricular appendix (Figures 1A, 3B). Intestinal caecum 3 times longer than ventricular appendix (Figures 1A, 3A).
Adult male of C. fioravantii from N. nycticorax, SEM: (A) anterior end with detail of dorsal lip and interlabium (scale bar 50 µm); (B) detail of amphid (scale bar 100 µm); (C) posterior end with everted spicules (scale bar 300 µm); (D) detail of distal papillae (scale bar 50 µm); (E) detail of caudal tip (scale bar 20 µm); and (F) detail of spicule tip (scale bar 50 µm).
Adult male of C. fioravantii from N. nycticorax, light microscopy: (A) anterior end with terminal part of intestinal caecum (ic); (B) detail of ventricular appendix (va); (C) posterior end with spicules (sp); and (D) detail of posterior end with caudal papillae. All scale bars 100 µm.
Males (n = 7). Body length 11–35 (18 ± 8.36) mm. Labia 55–98 (69 ± 17.45), interlabia 49–89 (60 ± 16.63). Oesophagus 2584–3905 (3099 ± 552.20). Nerve ring 325–638 (470 ± 102.61) from anterior extremity. Amphids 319–569 (431 ± 90.04) from anterior extremity. Intestinal caecum 1968–3306 (2479 ± 481.78) long. Ventricular appendix 845–1441 (1067 ± 215.70) long. Spicules subequal, robust, folded, with longitudinal alae (Figures 1C, D, 2F, 3C). Right spicule 2520–3849 (3247 ± 539.82), left spicule 2538–4043 (3309 ± 566.66). 21–23 pairs of simple proximal caudal papillae forming 2 subventral rows, 2 pairs of paracloacal papillae, 2 pairs of distal subventral papillae and 2 pairs of distal sublateral papillae, 1 pair of phasmids between distal subventral and distal sublateral papillae (Figures 1D, 2C, D). Tail 205–364 (270 ± 53.51), with sagittate tip (Figure 2E).
Females (n = 3). Body length 16–27 (22 ± 5.56) mm. Labia 89–143 (111 ± 28.54), interlabia 79–105 (91 ± 13.29). Oesophagus 4341–5368 (4948 ± 538.27) long. Nerve ring 497–676 (583 ± 90.08) from anterior extremity. Intestinal caecum 3455–4655 (4176 ± 636.08) long. Ventricular appendix 1194–1522 (1371 ± 165.42) long. Tail conical, 494–632 (544 ± 76.93) long, with pointed tip (Figures 4E, F). Vulva 8798–15274 (11623 ± 3316.48) from anterior extremity (Figure 4C). Eggs smooth, ovoid (Figure 4D), 125–171 (146 ± 171.1) long, 111–147 (128 ± 146.85) wide.
Adult female of C. fioravantii from N. nycticorax, light and SEM: (A) anterior end, lateral view (scale bar 50 µm); (B) anterior end, apical view (scale bar 100 µm); (C) detail of vulva (v) (scale bar 100 µm); (D) eggs (scale bar 50 µm); (E) posterior end, lateral view (scale bar 100 µm); and (F) posterior end, ventral view (scale bar 300 µm).
Remarks
Contracaecum fioravantii n. sp. presents a few similarities but also marked morphometric differences with respect to other Contracaecum species previously reported in N. nycticorax. Among other, C. microcephalum shows a similar pattern of distal papillae and a similar appearance of the spicule tip, however the spicules in C. microcephalum are on average smaller than in C. fioravantii n. sp. (2600–3120 vs 3247–3309 right-left spicule, respectively); furthermore, the 2 species differ with respect to the appearance of the caudal tip, which is pointed in C. microcephalum (Cram, Reference Cram1927; Baruš et al., Reference Baruš, Sergeeva, Sonin, Ryzhikov, Ryšavý, Ryšavý and Ryzhikov1978) and sagittate in C. fioravantii n. sp. The arrangement of distal papillae in C. fioravantii n. sp. is similar to that of members of the C. rudolphii complex. However, in C. fioravantii n. sp., phasmids are located between distal subventral and distal sublateral papillae, while in C. rudolphii they are dorsal to distal sublateral papillae. More importantly, spicule morphometry is markedly distinct, with considerably longer, narrower spicules in C. rudolphii (8054 right/7756 left) and shorter, more robust spicules in C. fioravantii n. sp. Furthermore, the appearance of spicule tip is markedly distinct (Figure 2F, as compared to SEM micrographs of C. rudolphii in Caffara et al., Reference Caffara, Tedesco, Davidovich, Rubini, Luci, Cantori, Glogowski, Fioravanti and Gustinelli2023b).
Multivariate analysis revealed varying degrees of morphometric distinction between C. fioravantii n. sp. and other species reported or described for the first time in China (Figure 5). In the matrix of Euclidean distances based on 5 morphometric variables, worms differed in terms of species (R = 0.345, P = 0.0004) and sex (R = 0.361, P = 0.018). The variables included in the analysis were the lengths of the body, oesophagus, intestinal cecum, ventricular appendix, nerve ring and tail. In pairwise comparisons, C. fioravantii n. sp. differed significantly from C. multipapillatum Drasche 1882 of Shen (Reference Shen1982), C. pingii Xu 1957, C. accipiterum Shen 1981, C. rudolphii B and C. milvi Yamaguti 1935, and with marginal significance from C. cantonense Xu 1957, C. pelecanii Hu, Lin and Qiu 1997, and C. multipapillatum of Navone et al. (Reference Navone, Etchegoin and Cremonte2000).
Metric MDS of morphometric resemblance among specimens of C. fioravantii n. sp. and data from other species obtained from other studies (1 or more of minimum, maximum, mean). The ordination and ANOSIM tests are based on Euclidean distances calculated from 5 normalized variables (lengths of the whole body, oesophagus, intestinal cecum, ventricular appendix, nerve ring and tail). The 2-dimensional stress of the MDS is 0.13. The results of ANOSIM pairwise tests between C. fioravantii n. sp. (2-way crossed with worm sex) are to the right of the ordination, together with MDS symbol legends.
Figure 5 Long description
Two-dimensional metric multidimensional scaling (MDS) ordination illustrating morphometric similarity among specimens of Contracaecum fioravantii n. sp. and comparative data for other Contracaecum species obtained from published studies. The analysis is based on Euclidean distances calculated from six normalized morphometric variables: body length, oesophagus length, intestinal caecum length, ventricular appendix length, nerve ring position, and tail length. Each point represents one specimen or one published morphometric dataset (minimum, maximum, or mean values), with symbols distinguishing species and, for C. fioravantii, male and female specimens. Pairwise ANOSIM test results comparing C. fioravantii with the other taxa are presented alongside the ordination. The two-dimensional MDS solution has a stress value of 0.13, indicating an acceptable representation of the multivariate relationships in two dimensions.
Molecular results
The ITS rDNA of all 10 specimens yielded a product of approximately 850 bp. BLAST searches showed 100% identity with an unidentified Contracaecum species collected from N. nycticorax in China (MN089475–76, Zhang et al., Reference Zhang, Meng, Huang, Feng, Liu, Tang, Fan, Yuan, Han, Luo, Zhao and He2021; PX498555 and PX459832, Yin, 2025, unpublished; PP837473, Long, 2024 unpublished and unknown host), with ON158309 (Deng et al., Reference Deng, Li, Wang, Liu and Tu2022) from Egretta ardesiaca Wagler 1827, and with Contracaecum sp. from Butorides striata L. in China (MW538933–36, Cheng, 2021, unpublished). The genetic distance (uncorrected p) between the new species and the above sequences retrieved from GenBank was 0–0.3%. In phylogenetic analysis, the concatenated ITS1–2 regions of C. fioravantii n. sp. formed a well-supported clade (99% bootstrap support) separated from other Contracaecum species (Figure 6). This separation clearly supported the morphological differences between our new species.
Phylogenetic tree inferred from ITS rDNA sequences showing the position of C. fioravantii n. sp. (in bold), reconstructed using the Maximum Likelihood method under the General Time Reversible model of nucleotide substitution. Bootstrap support values (1000 replicates) are shown at the nodes as the percentage of replicate trees in which the associated taxa clustered together. The final dataset comprised 37 nucleotide sequences. Evolutionary analyses were performed in MEGA12 using up to 5 parallel computing threads.
Figure 6 Long description
Maximum Likelihood phylogenetic tree inferred from ITS rDNA sequences illustrating the evolutionary relationships of Contracaecum fioravantii n. sp. among other representatives of the genus Contracaecum. The new species is highlighted in bold. Branch labels indicate bootstrap support values based on 1,000 replicates and represent the percentage of replicate trees recovering each clade. The phylogeny was reconstructed under the General Time Reversible model of nucleotide substitution using a dataset of 37 ITS rDNA sequences. Evolutionary analyses were performed in MEGA12 using up to five parallel computing threads.
Four sequences of cox2 mtDNA were obtained; BLAST analysis revealed a similarity ranging from 98% to 99.1% with Contracaecum sp. (ON149889, Deng et al., Reference Deng, Li, Wang, Liu and Tu2022) from E. ardesiaca in China and an intraspecific p-distance of 0.1–0.2%. The ML tree topology clearly places the new species in a well-supported (100% bootstrap) and distinct clade that includes the Contracaecum sp. collected in China (Figure 7).
Phylogenetic tree inferred from cox2 mtDNA sequences showing the position of C. fioravantii n. sp. (in bold), reconstructed using the Maximum Likelihood method under the Kimura 2-parameter model of nucleotide substitution. Bootstrap support values (1000 replicates) are shown at the nodes as the percentage of replicate trees in which the associated taxa clustered together. The final dataset comprised 23 nucleotide sequences. Evolutionary analyses were performed in MEGA12 using up to 5 parallel computing threads.
Figure 7 Long description
Maximum Likelihood phylogenetic tree inferred from cox2 mtDNA sequences illustrating the evolutionary relationships of Contracaecum fioravantii n. sp. among other representatives of the genus Contracaecum. The new species is highlighted in bold. Branch labels indicate bootstrap support values based on 1,000 replicates and represent the percentage of replicate trees recovering each clade. The phylogeny was reconstructed under the Kimura 2-parameter model of nucleotide substitution using a dataset of 23 cox2 mtDNA sequences. Evolutionary analyses were performed in MEGA12 using up to five parallel computing threads.
Discussion
In Israel, N. nycticorax is a widespread wetland-associated species occurring in marshes, lakes, rivers and coastal lagoons, where it behaves as both a resident and partial migrant. Its distribution is largely determined by the availability of suitable aquatic habitats, including key areas such as the Hula Reserve and coastal wetlands, despite increasing anthropogenic pressures (Ashkenazi and Yom-tov, Reference Ashkenazi and Yom-tov1997; Ashkenazi and Dimentman, Reference Ashkenazi and Dimentman1998; Yom-tov, Reference Yom-tov2012). The species exhibits an opportunistic diet, feeding on fish, amphibians, crustaceans and aquatic insects, thereby acting as an important host for trophically transmitted parasites, including Contracaecum spp.
Interestingly, C. fioravantii n. sp. has never been detected in previous molecular surveys of Contracaecum in Israel, including extensive sampling of larvae in fish (Davidovich et al., Reference Davidovich, Tedesco, Caffara, Yasur-landau, Gustinelli, Drabkin, Minkova, Aflalo, Morick and Fioravanti2022, Reference Davidovich, Tedesco, Caffara, Luci, Cantori, Morick, Fioravanti and Gustinelli2023) and adult Contracaecum from other piscivorous birds from the same area as the present study (Caffara et al., Reference Caffara, Tedesco, Davidovich, Locke, Gustinelli, King, Nuytten, Nuzzo and Fioravanti2023a, b). This may indicate that infections in N. nycticorax were acquired at other sites along migratory routes, or that the parasite occurs in intermediate hosts not previously investigated.
The identical ITS rDNA sequences and high similarity (99%) in cox2 mtDNA with unidentified Contracaecum specimens from the oesophagus of N. nycticorax collected near Beijing, China (Zhang et al., Reference Zhang, Meng, Huang, Feng, Liu, Tang, Fan, Yuan, Han, Luo, Zhao and He2021; Deng et al., Reference Deng, Li, Wang, Liu and Tu2022) strongly suggest that this record corresponds to C. fioravantii n. sp. This finding is particularly significant, as it links the new species to the only available complete mitochondrial genome sequences of an unidentified Contracaecum species (MN892395, ON149889).
Within the Chinese avifauna, the genus Contracaecum is represented by species infecting a wide range of birds, particularly raptors, herons and cormorants (Supplementary Table 1; Zhang et al., Reference Zhang, Bu, Huang, Wen and Zhang2012; Li et al., Reference Li, Gibson and Zhang2016). These nematodes have been recorded from various Chinese provinces and have been isolated from a diverse range of hosts (Supplementary Table 1). Some taxa, such as C. microcephalum, C. multipapillatum and C. rudolphii, exhibit relatively low host specificity, although species boundaries within the C. multipapillatum and C. rudolphii complexes require further investigation, whereas others appear restricted to a limited number of hosts.
Genetic sequencing (ITS and mitochondrial data) of C. rudolphii from cormorants (Phalacrocorax carbo sinensis) sampled along the southern coasts of China confirmed their affinity with the species C. rudolphii B (Li et al., Reference Li, Xu and Zhang2013). However, genetic data are available only for a subset of the Contracaecum species from China (e.g. C. ovale); nevertheless, many species are readily distinguishable from C. fioravantii n. sp. based on morphology. Particularly, C. ovale (Linstow 1907) Baylis 1920 (synonym of C. nehli and C. spasskii; Presswell and Bennett, Reference Presswell and Bennett2022) is found mainly in grebes and shows a distinct appearance of the anterior end, with markedly rounded upper margins of labia and greatly reduced non-bifurcated interlabia. Moreover, the comparison of our sequences with the ITS1 of C. ovale available in GenBank (OL470520, from Australasian crested grebe, Podiceps cristatus australis L., Presswell and Bennet, 2022) showed a 1.8% distance with C. fioravantii n. sp.
Contracaecum micropapillatum and C. pelecani show a characteristic pattern of distal papillae with 3 pairs of double papillae. Contracaecum pingii has been reported only from the Eurasian goshawk Astur (formerly Accipiter) gentilis L. (Li et al., Reference Li, Gibson and Zhang2016); however, this species has never been reported after its original description, and has not been recorded in the same host from other parts of the Eurasian continent (Yakovleva and Ieshko, Reference Yakovleva and Ieshko2025). In China, A. gentilis is a reported host for C. accipiterum, firstly described by Shen (Reference Shen1981) from Jiangsu province: male individuals of this species are characterized by having 42 pairs of proximal caudal papillae and by 2 pairs of single and 2 pairs of double distal papillae, a pattern distinct from the species under study; also, this species has never been reported after its first description.
Contracaecum fioravantii n. sp. shows morphological similarities, with respect to the pattern of distal papillae, with C. microcephalum and species within the C. rudolphii complex, both already reported from N. nycticorax. Nevertheless, the latter 2 species/species complexes markedly differ from C. fioravantii n. sp. in the spicule morphometry and morphology of distal posterior end; C. rudolphii B also differs in other aspects of overall morphometry from C. fioravantii n. sp. (Figure 5). Molecular analyses based on amplification and sequencing of nuclear and mitochondrial markers support this distinction by placing C. fioravantii n. sp. in a distinct clade far from C. multipapillatum and C. rudolphii complexes.
The high similarity with sequences from China suggests that C. fioravantii n. sp. may have a broader geographical distribution than currently documented, potentially linked to the migratory behaviour of N. nycticorax and other ardeid hosts. This finding also indicates that some previously unidentified Contracaecum sequences available in GenBank likely correspond to this species. The recognition of C. fioravantii n. sp. underscores the underestimated diversity within Contracaecum and the key role of migratory hosts in parasite dispersal. Integrative taxonomy remains essential for resolving species boundaries in this complex genus.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0031182026102558.
Data availability statement
All the data that support the findings of this study are available from the corresponding author. Molecular sequences are available in GenBank under accession numbers PZ240692-69, PZ240700-01 (ITS rDNA) and PZ244216-19 (cox2 mtDNA).
Acknowledgements
The authors thank Valentina Luci and Aurora Lattanzi for their technical support and assistance with molecular and SEM analyses during the experimental work.
Author contributions
P.T.: methodology, data curation, SEM analyses, writing original draft; N.D.: sampling, review and editing; S.L.: methodology, data curation, review and editing; A.G.: review and editing; M.C.: conceptualization, data curation, methodology, writing original draft. All authors reviewed the manuscript and approved the final version.
Financial support
This study was supported by the Israeli Veterinary Services and by the University of Bologna, Italy. Scanning Electron Microscopy acquired thanks to the Department of Excellence Project 2018–2022 funded by the Italian Ministry of Education, Universities and Research.
Competing interests
The author(s) declare none.
Ethical standards
The permission to collect the night great heron was obtained from Israel Nature and Parks Authority ‘Permits 2020/42659 and 2021/42855’.
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