Redescription and resurrection of the status of Joyeuxiella gervaisi (Setti, 1895) (Eucestoda, Dipylidiidae)⁵

Joyeuxiella Fuhrmann, 1935 is one of the three genera of the family Dipylidiidae and according to a revision of the genus by Jones (1983) includes three different species J. echinorhynchoides (Sonsino, 1889), J. pasqualei (Diamare, 1893) and J. fuhrmanni (Baer, 1924). Starting from the description of its first species onwards, the current genus Joyeuxiella underwent several changes. Joyeuxiella echinorhynchoides was originally described from a fennec, Vulpes zedra (Zimmermann) in Egypt under the name Taenia echinorhynchoides

– Diamare (1892) transferred this species into the genus Dipylidium.

Joyeuxiella gervaisi fell into oblivion and was declared a species inquirenda by Jones (1983). However, the allegedly lost syntype material collected by Setti (1895) and used for the description of J. gervaisi was found in the zoological museum of the University of Naples.

The presence of J. fuhrmanni in feral cats in Dubai was reported in two papers (Schuster et al., 2009, 2016). Beside other helminths collected at the Central Veterinary Research Laboratory in Dubai, Poon et al. (2017) sequenced the cytochrome oxidase subunit 1 (COI) gene of J. pasqualei and J. fuhrmanni from feral cats in Dubai and registered the sequence in the GenBank Database (KY310707 for J. fuhrmanni and KY310708 for J. pasqualei).

The allocation of small cestodes of the genus Joyeuxiella from cats in Dubai to J. fuhrmanni was based on the revision of the genus Joyeuxiella by Jones (1983) but the eventual availability of the original description of J. gervaisi by Setti (1895) caused doubts about the correctness of the determination and was the reason for this paper.

Twenty-five specimens of J. gervaisi initially identified as J. fuhrmanni from the parasitological collection of the Central Veterinary Research Laboratory in Dubai were used. All samples were originally preserved in 70 % alcohol. For the creation of temporary mounts, samples were rehydrated and stained with lactic acid carmine before being dehydrated in rising alcohol concentrations and transferred into clove oil. Temporary preparations were examined under the microscope. For the preparations of permanent mounts, stained and dehydrated cestodes were cleared in clove oil and mounted in DPX medium. Since the structural integrity of rostellar hooks was partly lost through the use of this medium, another 15 cestodes collected from a freshly deceased cat sent to the CVRL for necropsy were fixed in hot 10 % formalin for examination. For comparison and identification, detail pictures of J. pasqualei tapeworms were utilized. Measurements are given in μm unless stated otherwise.

The molecular analysis involved genomic DNA isolation from the individual worms of Joyeuxiella pasqualei (n = 8) and Joyeuxiella gervaisi (n = 3) which were washed thrice with phosphate buffered saline (PBS) to remove residual ethanol and the DNA was extracted following manufacturer's protocol (G-spin total DNA extraction kit, Intron Biotechnology, South Korea). A partial segment of mitochondrial COI gene (400 bp) was amplified using previously described JB3/JB4.5 primer pair (Bowles et al., 1992). The PCR conditions were: initial denaturation: 95 °C for 5 min, denaturation: 95 °C for 1 min, annealing: 45 °C for 1 min, extension: 72 °C for 1 min and final extension: 72 °C for 5 min. The PCR products were sent for bidirectional sequencing using the same primer pair as previously mentioned.

The obtained sequence chromatograms were viewed through FinchTV viewer (Geospiza, Seattle, WA, USA) and checked individually for base-calling errors after ascertaining the peak qualities. Comparison for generated DNA sequences was carried out in NCBI BLAST algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to search for the reference sequences. The sequences were submitted in the GenBank database under following accession numbers: OR081741-OR081751. Additionally, a maximum likelihood tree was generated through MEGA-X software through Kimura-2-parameter substitution model with gamma distributed sites and 1000 bootstrap replicates to understand phylogenetic patterns and species alignment with the reference sequences (Joyeuxiella pasqualei: KY310707 (Poon et al., 2017) and ON981095 (Bezerra-Santos et al., 2022); Joyeuxiella fuhrmanni: KY310708 (Poon et al., 2017)) and an outgroup (Taenia hydatigena NC_012896; Jia et al., 2010).

Not applicable. For this study, formal consent is not required.

Redescription of Joyeuxiella gervaisi (Setti, 1895) Fuhrmann, 1935 (Figs. 1 – 4)

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Cyclophyllidea, Dipylidiidae. Strobila 30 to 60 mm long, consisting of 52 to 85 segments. Mature segments with maximum width of 1.2 mm. Scolex (Fig. 1) dorso-ventrally flatted 282 – 436 by 360 – 470; slightly oval, unarmed suckers 115 – 135; rostellum 205 – 225 long and 205 – 225 wide stinkhorn-shaped, divided into lower unarmed cylindrical, followed by conical upper part (Fig. 2), armed with up to 10 – 12 (13) alternating rows of rose-thorn shaped hooks, base of hooks slightly longer than blades, upper rows 10 – 12, rows at the base 6 – 7; neck 388 – 760 × 245 – 425. First proglottids with primordia of reproductive organs (ovaries and vasa deferens) in segments 11 to 25, first signs of testes 2 – 5 segments later. Three to four fully developed rectangle-shaped mature proglottids (Fig. 3), broader (825 – 1230) than long (330 – 690), slightly convex in anterior third at genital pores; genital pores funnel-shaped, 30 – 78 wide and 31 – 69 deep. Gravid segments (Fig. 4) longer than wide, 2100 – 4050 × 840 – 1170, with genital pores in first quarter. Round testes 50 – 70 in diameter, numbering between 28 and 38, positioned anteriorly, medially and posteriorly to female reproductive glands, not reaching anterior part of the segment; vasa deferentia in anterior part of segments, heavily coiled; cirrus sac oval-shaped 150 – 240 × 75 – 120, oblique, with basis directed anteriorly and crossing excretory vessels; unarmed cirrus everted in some segments, 360 – 520 long, diameter 15 – 21. Fan shaped ovaries posterior and median to cirrus pouch, 130 – 250, seminal receptacle fusiform, 75 – 130 × 25 – 60 between ovary lobes; vitelline glands round, oval or kidney-shaped postovarian, 60 – 96. Vagina with diameter comparable to that of cirrus, opening posterior to cirrus sac.; egg capsules in gravid segments 50 – 80 in diameter, located medially to longitudinal excretory vessels; eggs 30 – 42, oncospheres: 28 – 38; embryonal hooks: 15 – 18.

Taxonomic summary

Type host: Felis catus Linneus, 1758 (Carnivora: Felidae)

The molecular analysis backed the morphological assessment of cestodes revealing two species namely J. pasqualei and J. gervaisi. The BLAST results yielded 99.11 % homology and 100 % query coverage for the J. pasqualei partial COI sequences with the reference sequence (GenBank accession number: ON981095). For the J. gervaisi sequences, the homology was low as the available sequence of J. fuhrmanni (GenBank accession number: KY310708) is only a stretch of 100 base pairs, however the multiple alignment showed high similarity for the partial COI segment. To further discern the genealogical relationships between the species, a maximum likelihood tree was generated in which COI sequences for both cestode species, J. pasqualei and J. gervaisi, positioned along the respective reference sequences having a strong tree support.

Cestodes found by E. Setti in the small intestine of a genet from Eritrea and described as Dipylidium gervaisi were part of the helminthological collection created by Corrado Parona, director of the Natural History Museum of the University of Genova. After Paronas's death in 1922, this collection housing more than 700 different worm species was donated to the Zoological Institute of the University of Naples and was united with the helminthological collections of Monticelli and Stossich to form the ‘Collezione Elmintologica Centrale Italiana’ (Salefi, 1956). A jar labeled ‘Cest 254’ in the aforementioned collection contained the type material (syntype) still classified under the old name of Dipylidium gervasi from Genetta abyssinica from Eritrea. The species name ‘gervasi’ on the label is obviously a typo (Fig. 5, 6).

Fig. 5.

Fig. 6.

According to Setti (1895), the main features of this small, up to four-centimeter long dipylidiid cestode were the striking long cirri (0.5 mm) and the positioning of testes posterior to the heavily coiled vasa deferentia. Setti's illustrations showed the number of testes to be between 30 – 46 and the positioning of egg capsules in gravid segments to lay between the longitudinal excretory vessels. However, taking into consideration that solely 10 tapeworms and many gravid segments were in Setti's disposition, it is possible that J. gervaisi might have had a longer total length.

As far as molecular analysis is concerned, only a single sequence of J. furhmanni is available in the GenBank (accession no. KY310708; 100 bp) which differs at four nucleotide positions from the currently sequenced specimens (partial COI; 372 bp) of J. gervaisi. However, these sequences shared the same clade with a strong nodal support and that the haplotypic variations exist within the genome manifesting such differences (Fig.7).

Fig. 7.

The morphological examination of small Joyeuxiella cestodes found in feral cats in Dubai matched the typical characteristics described for J. gervaisi as well as for J. fuhrmanni. Since J. gervaisi was described 29 years earlier, J. fuhrmanni has to be considered a junior synonym.

Although the verbal characterization of J. gervaisi by Setti (1895) is limited, drawings of the scolex, the rose-thorn-shaped rostellar hooks and mature and gravid proglottids completed the description of the species. The author drew attention to the dichotomous structure of the rostellum consisting of a club-like armed upper part and a bulb-like base. In reality, the whole rostellum resembles a stinkhorn with a slender and longer stalk and a conical shaped armed cap.

Unfortunately, rostellar hooks easily fall off and can only be perceived in freshly isolated cestodes. In such specimens, 10 to 12 rows of hooks can be seen. In a few specimens however, up to 13 alternating rows with a bare outmost apex of the rostellum were observed. In the original description, Setti (1895) counted eight to 12 rows of rostellar hooks.

Mature segments were wider than long and gravid segments resembled a miniature cucumber seed. Within the material observed in this research, in each strobila, only three to four fully developed mature segments were seen. Ovaries, yolk glands and testes were replaced rapidly by the developing uterus in the following proglottids. Baer (1926) acknowledged J. fuhrmanni to show certain affinities with J. gervaisi but to differ in terms of ovarian and yolk gland structure

– In the description of J. gervaisi, Setti did not mention yolk glands. The pictured hermaphrodite segments had not reached the full maturity since yolk glands could not be distinguished from testes.

Ortlepp (1933) disagreed with this view following the isolation of over a hundred small cestodes (2.0 – 6.5 cm long) from the small intestine of a cat in Pretoria. Examination of the hermaphrodite segments of these tapeworms revealed a similar testes distribution as described by Baer (1926). Ortlepp's material is not available anymore, but the helminthological collection of the Onderstepoort Veterinary Institute stores a glass container with the label: S325: Joyeuxia fuhrmanni; wild cat; small intestine; Kaalplaats (Limpopo Province), Onderstepoort; 7/6/1937. These dipylidiid cestodes were up to 54 mm long and consisted of 78 to 110 segments. Although the collector is not mentioned, it was most probably also R. J. Ortlepp who worked as helminthologist at Onderstepoort Veterinary Institute from 1930 to 1954.

Southwell (1930) mentioned J. gervaisi from fishing cats (Prionailurus viverrinus Bennett) and Asian palm civets (Paradoxurus hermaphroditus Pallas) from a Zoological garden in Calcutta. The seven-line long, not very specific paragraph described these worms as a one to four cm long cestodes with a 150 μm long neck that was not sharply divided from scolex. The small rostellum bore 8 – 12 rows of hooks. No information was given regarding the distribution of testes and egg capsules nor the cirrus length. Another mention of Setti's cestode can be found in a description of morphological abnormalities in D. caninum by Honigberg (1944) but does not provide any important morphological details.

Hudson (1934) reported J. fuhrmanni from a serval in Kenya without any further description and the last reported finding of J. fuhrmanni was from domestic cats in South Africa by Barker et al. (1989) without providing further morphological details.

In Setti's drawings, as well as in our material, mature proglottids were more or less rectangle-shaped, broader than long and slightly convex. Ovaries were shifted towards the centre, leaving a distinct space to the cirrus pouch. The long cirri, a key feature according to Baer (1926), were not noticed in the type material of J. fuhrmanni studied by Witenberg (1932). These structures, which are well represented in Setti's drawings of both mature and gravid proglottids, were perceived in our material as everted delicate long cirri and were noticed in live cestodes as well as in most of the segments during fixation and processing.

Mettrick and Beverley-Burton (1961) gave a description of J. fuhrmanni from a serval from Southern Rhodesia (today: Zimbabwe). This cestode had a length of up to 94 mm and a rostellum covered with 17 to 19 rows of hooks. The tapeworm had fan shaped ovaries and 270 μm long cirri (Table 1). In the same paper, the authors described J. paucitestis Mettrick et Beverley-Burton, 1961 as a new species from a rusty spotted genet (Genetta maculata Grey) hunted at Lake Kariba in the Zambezi Valley of Southern Rhodesia (today: Zimbabwe). Joyeuxiella paucitestis measured only 20 mm in length. Its scolex had a 50 to 80 μm long rostellum bearing 10 rows of small hooks. An obvious differential feature was the elongated shape of mature segments. As in the case of J. fuhrmanni, testes did not occur in front of the vasa deferens. Cirri reached a length of 140 μm.

Based on examination of cestodes from felids: servals from South Africa and Belgian Congo, African wild cat (F. lybica Forster) and domestic cats from South Africa and Genetta rubriginosa (Grey) from Southern Rhodesia), Jones (1983) gave an extended diagnosis for J. fuhrmanni and resurrected the status of this cestode. According to this, single egg capsules can also be present externally to the longitudinal excretory vessels but the extremely long cirri, one of the key morphological features according to Baer (1924) were not mentioned. Also, Jones (1983) synonymized J. fuhrmanni with J. paucitestes although in the latter, mature segments were clearly longer than wide. Jones (1983) declared J. gervaisi a species inquirenda because of the unavailability of a deposited sample.

Beside J. fuhrmanni, Jones (1983) recognized J. pasqualei and J. echinorhynchoides as the only valid species of the genus Joyeuxiella. Table 1 summarizes morphometrical data on J. gervaisi and J. fuhrmanni according to different authors. Jones (1983) regarded also Dipylidium sp. of Kofend (1917) as synonym of J. fuhrmanni.

In a preliminary communication, Kofend (1917) merely mentioned the finding of a single cestode in a serval from the Sudan. According to Kofend (1917) the worm resembled D. echinorhynchoides. Dipylidium sp. however, differed from D. echinorhynchoides based on the arrangement of the rostellar hooks. A more detailed description was given six years later (Kofend, 1923). According to this, the cestode was 1.5 to 6 cm long with a rostellum armed with 16 to 18 rows of hooks. The description however, did not mention the arrangement, size and number of testes and the length of the cirrus. The uterus in gravid proglottids appeared as a dorsal cavity with branches in all directions and end eggs were also situated externally to longitudinal excretory vessels. A drawing of a mature segment of Kofend's Dipylidium sp. by Witenberg (1932) showed that the testes in this species were present also in the anterior part of the proglottide. For these reasons, the allocation of Dipylidium sp. of Kofend (1917) to J. fuhrmanni remained doubtful. Neither Witenberg (1932) nor Jones (1983) mentioned the enormous cirrus length of J. fuhrmanni. One reason for this might have been that these authors examined stained mounts. During the fixation, staining and embedding processes, these extremely thin structures often break and get lost.

It is interesting that the ‘J. fuhrmanni’ type was so far only detected in Africa and in the United Arab Emirates. In a study of parasites of feral cats in Dubai, both J. pasqalei and J. fuhrmanni were detected with an overall prevalence of 65.8 % (Schuster et al., 2009). A further study revealed that 11 out of 13 cestode infected cats in an untreated control group in an anthelminthic efficacy trial of a new dewormer for cats harbored J. fuhrmanni while 13 out of 13 cats were infected with J. pasqualei (Schuster et al., 2016). J. fuhrmanni was not mentioned in cat surveys carried out in other countries of the Middle East, like Qatar (Abu-Madi et al., 2010), Kuwait (Abdul-Salam & Baker, 1990; El Azazi et al., 2016), Iraq (Al-Rammahi et al., 2014; Al-Rubae et al., 2015) or Iran (Dalimi et al., 2006; Changizi et al., 2007; Esmaeilzadeh et al., 2009; Arabi & Hooshyar 2009; Borij et al., 2011). The mention of J. gervaisi in India by Southwell (1930) needs further confirmation. A more recent publication of cat parasites in Sudan named Joyeuxiella spp. in 60 % of feral cats trapped in Khartoum and figure 2 of this paper pictures mature proglottids with genital pores shifted to the anterior end with heavily coiled vasa deferens (Mohammed et al., 2021).

Key to species of Joyeuxiella Fuhrmann, 1935

Rostellum cylindrical, hook blades longer than bases, number of alternating rows of rostellar hooks: 20-30…………………… ………………………………………………. J. echinorhynchoides

- Dichotomous rostellum consisting of a cylindrical base and conical armed top. Hook blades shorter than bases, number of alternating rows of rostellar hooks less than 20……………………………………..2

Summarizing all these facts, it became clear that J. gervaisi is a valid species that shares the main morphological features with its junior synonym, J. fuhrmanni, e.g. small strobila length, long cirri, heavily coiled vasa deferentia at the anterior end of mature segments and positioning of egg capsules between the longitudinal excretory vessels.