New species of Dermoergasilus Ho & Do, 1982 (Copepoda: Cyclopoida: Ergasilidae) parasitizing endemic cichlid Paretroplus polyactis (Bleeker) in Madagascar

Abstract Abstract Dermoergasilus madagascarensis n. sp. is described from the gills of Paretroplus polyactis, an endemic cichlid fish in Madagascar, using a combined morphological (light microscopy and SEM) and molecular approach (partial 18S rDNA, 28S rDNA, and COI sequences). The new species is characterized mainly by possessing: (i) roughly pentagonal cephalosome; (ii) antennal endopodal segments covered with slightly inflated membrane; (iii) maxillule bearing 2 equally long outer setae and a minute inner seta; (iv) interpodal sternites of swimming legs ornamented with 3–4 rows of spinules; (v) genital segment and first abdominal somite both barrel-shaped; and (vi) a caudal ramus projecting into a digitiform process with inconspicuous terminal seta and bearing 3 terminal setae. The obtained DNA sequences of Malagasy species represent the first molecular data for species of Dermoergasilus. The 28S rDNA phylogeny showed the affiliation of D. madagascarensis n. sp. to Ergasilidae and its sister relationship with cosmopolitan Ergasilus sieboldi von Nordmann, 1832. The first checklist for all species of Dermoergasilus is provided.

Until now, there are only a few parasitic crustacean records from freshwater fishes in Madagascar.Fryer (1968) questioned whether it is due to the lack of scientific interest or because of their true absence.The only record of a parasitic copepod on this island is Dermoergasilus longiabdominalis El-Rashidy and Boxshall, 2001 from Osteomugil engeli (Bleeker) (El-Rashidy and Boxshall, 2001).From other parasitic crustaceans recorded in the region only the occurrence of parasitic isopod Cymothoa borbonica Schioedte & Meinert, 1884 from the mouth of the freshwater cichlid fish Ptychochromis oligacanthus (Bleeker) is reported by (Trilles, 1975).
During the investigation of gill parasites of cichlid fishes in Madagascar, Dermoergasilus specimens were collected from the gills of Paretroplus polyactis.Description of new Dermoergasilus species was performed using morphological study (light and SEM microscopy), and a molecular study using ribosomal and mitochondrial DNA sequences (partial 18S rDNA, 28S rDNA and COI sequences).In addition, to investigate the relationship of D. madagascarensis n. sp. to other representatives of Ergasilidae, phylogenetic analyses were performed.

Parasite collection and identification
Live copepods were collected from the gills using fine needles and processed for morphological and molecular purposes, as described in Míč et al. (2023).The mounted specimens in GAP (mixture of glycerine and ammonium picrate) or pure glycerine were studied using an Olympus BX61 microscope equipped with phase contrast optics.Drawings of the copepods were made using an Olympus drawing attachment and edited with a graphic tablet (Wacom Intuos5 Touch) compatible with Adobe Illustrator and Adobe Photoshop (Adobe Systems Inc., San Jose, CA, USA).All measurements (in micrometers) were taken using digital image analysis software (Olympus Stream Motion v. 1.9.3) and are presented as the range followed by the mean (n = 10).
For scanning electron microscope analysis, 5 specimens fixed in 70% ethanol were dehydrated in an increasing ethanol grades, dried in a CPD 030 critical point drying apparatus (Bal-tec, Balzers, Liechtenstein) using liquid CO 2 , mounted on aluminium stubs with double sided adhesive discs, coated with gold in a SCD 040 sputter coating unit (OC Oerlikon Balzers Coating, Balzers, Liechtenstein) and examined in a VEGA scanning electron microscope operating at 20 kV.
The type specimens of the copepods collected in the present study were deposited in the Institute of Parasitology, Czech Academy of Sciences, České Budějovice, Czech Republic.Prevalence (percentage of infected fish) and mean intensity of infection (mean number of parasites per infected host) were calculated following Bush et al. (1997).

Molecular and phylogenetic analyses
Genomic DNA was isolated separately from each parasite specimen (or a part of its body) using DNeasy®Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.For molecular characterization, partial sequences of 1 mitochondrial gene (COI) and 2 nuclear ribosomal regions (18S and 28S rDNA) were amplified by using the primer sets listed in Table 2. PCRs for 18S and 28S rDNA were carried out in a total volume of 20 μL containing 3 μL of DNA extract, 1× PCR buffer (Fermentas), 2.5 mM MgCl 2 , 0.2 mM of each dNTP, 0.2 μM of each primer, 0.1 BSA and 1 U of Taq polymerase (Fermentas).Amplification was performed under the following conditions: 94°C for 5 min; 39 cycles of 94°C for 30 s; an annealing temperature of 52°C for 30 s; and 72°C for 1 min, with a final extension step at 72°C for 5 min.PCR for COI was carried out in a total volume of 50 μL containing 1 μL of DNA extract, 1× PCR buffer (Fermentas), 2.5 mM MgCl 2 , 0.5 mM of each dNTP, 0.5 μM of each primer, 0.1 BSA and 2 U of Taq polymerase (Fermentas).Amplification was performed under the following conditions: 95°C for 5 min; 40 cycles of 95°C for 1 min; an annealing temperature of 45°C for 1 min; and 72°C for 30 s, with a final extension step at 72°C for 7 min.The PCR amplicons were checked by electrophoresis on 1.5% agarose gels stained with Good View™ (Amplia s.r.o., Bratislava, Slovakia), and PCR products of the required length were purified using ExoSAP-IT™ (Affymetrix Inc., Santa Clara, USA), following the manufacturer's instructions.Purified products were directly sequenced using the same primers as those for PCR.DNA sequencing was carried out using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems by Thermo Fisher Scientific, Prague, Czech Republic) and a 3130 Genetic Analyzer (Applied Biosystems).The obtained sequences were assembled and edited using Sequencher software (Gene Codes Corp., Ann Arbor, MI, USA).Newly generated sequences of 18S rDNA, 28S rDNA and COI were deposited in GenBank under accession numbers PP115569 (28S rDNA), PP115568 (18S rDNA) and PP117929-PP117934 (COI).Molecular vouchers (hologenophores, paragenophores; Pleijel et al., 2008) were deposited in the Institute of Parasitology, Czech Academy of Sciences, České Budějovice, Czech Republic.
To investigate the phylogenetic position of Dermoergasilus madagascarensis n. sp., to the representatives of parasitic Cyclopoida, the sequences of 28S rDNA of the species belonging to 9 genera were retrieved from GenBank and Bold databases (for details, see Table 3).Three species of the family Lernaeidae, Lernaea cyprinacea (Linnaeus, 1758), Lamproglena chinensis Yü, 1937 and Lamproglena orientalis Markevich, 1936 were used as outgroup.Sequences were aligned using MAFFT v.7 (Katoh and Standley, 2013).Gaps and ambiguously aligned regions were removed from the alignments with Gblocks v0.91b (Talavera and Castresana, 2007) using settings for a less stringent selection.ModelFinder (Kalyaanamoorthy et al., 2017) was employed to select the most appropriate model of DNA evolution.The most suitable evolutionary model for the partial sequence of 28S rDNA was TIM3 + F + I.The phylogenetic reconstruction was performed using maximum likelihood (ML) and Bayesian inference (BI) methods.ML analyses were run using IQ-TREE (Nguyen et al., 2015) on the W-IQ-TREE webserver (Trifinopoulos et al., 2016) and nodal support for the tree was assessed through ultrafast bootstrap approximation with 1000 replicates (Hoang et al., 2018).BI analysis was carried out in MrBayes 3.2.6 (Huelsenbeck and Ronquist, 2001) using the CIPRES platform (Miller et al., 2010), the analysis included 2 simultaneous runs of Markov chain Monte Carlo for 10 6 generations, sampling every 100 generations, with a 'burn-in' of 25%.The results were checked in Tracer v. 1.7.1 (Rambaut et al., 2018) to assess chain convergence.The trees were visualized and edited in FigTree v. 1.4.3 (Rambaut, 2012).Genetic distances (uncorrected p-distance) were calculated in MEGA v. 11 (Tamura et al., 2021).

Results
Endemic cichlid P. polyactis from the Canal des Pangalanes (locality 4 in Fig. 1) was the only host species (out of 15 species examined) infected by parasitic copepods and exhibited intensity of infection ranging from 5 to 283 (mean 59) per individual fish.Overall, 20 specimens of P. polyactis were examined and the prevalence of Dermoergasilus parasites was 90%.Total prevalence of Dermoergasilus among all examined fishes in the study was 18%.
The copepod specimens collected from P. polyactis were identified as Dermoergasilus based on the diagnostic morphological characters according to Ho and Do (1982), specifically: (i) antenna, except terminal claw, covered with inflated transparent membrane; (ii) paired caudal rami each with a digitiform process; and (iii) middle segment of endopod of legs II and III possessing a single seta.Site on host: Gill filaments.
Etymology: The species was named after the type locality, Madagascar Island, from which it was first discovered.
Prosome 5-segmented, composed of cephalothorax and 3 free pedigerous somites (PS-1 to PS-4) (Fig. 2A).Cephalosome roughly pentagonal, rounded and slightly tapering anteriorly; antennules and antennae visible in dorsal view (Fig. 5A and B).Cephalic ornamentation comprising inverted T-shaped marking, sensory setae and pits with bilaterally symmetrical distribution on dorsal side.Rostrum shieldlike with 6 sensillae and 3 integumental pores (Figs.3D and 5C).PS-1 elongated, with bilateral indentations just posterior to midlength; dorsal surface with slight T-shaped and rectangular depression situated anterior and posterior, respectively, to the constricted part; dorsal ornamentation comprising circular indentations situated just posterior to cephalosome and pair of sensillae near posterior margin.PS-2 to PS-4 decreasing gradually in width posteriorly, the three together barrel-shaped.Dorsal surface of each segment possessing anteriorly arising trapezoidal plate, sensillae and pits with bilaterally symmetrical distribution.

Parasitology
Caudal rami nearly equal in length with AS-3, slightly wider than long; each projecting into tapering digitiform process (about 1.6 times longer than body of ramus) with inconspicuous terminal seta (Fig. 5E) and bearing 3 terminal setaethe innermost longest and thickest, ornamented with transversal rings of inconspicuous scales at posterior 3/4; 2 lateral setae longer than digitiform processes.Two cylindrical egg-sacs, much longer than wide (4 times), each composed of 2-4 rows of eggs (Fig. 3B).
Mouthparts (Fig 2C and D) comprising mandible, maxillule and maxilla; maxilliped absent.Mandible consisting of 3 blades (anterior, middle and posterior); anterior blade with sharp teeth on anterior margin; middle blade with sharp teeth on both margins; and posterior blade with sharp teeth on anterior margin.
Maxillule a single lobe, ornamented with rows of tiny spinules, bearing 2 equally long outer setae and minute inner seta.Maxilla 2-segmented, comprising syncoxa and basis; syncoxa small, unarmed; basis elongated, medially slightly curved, distally with numerous sharp teeth on anterior side.
Coxa of all legs unarmed; coxa of L1 with a row of spinules extending along its outer posterior margin.Basis of all legs armed with proximal outer spine, unornamented.Legs 1-4 with outer margin of both rami ornamented with rows of spinules; outer and inner margin of first endopodal and exopodal segment, respectively, of all legs partly or completely covered with bristles.

Discussion
Diversity of fish ectoparasites in native Malagasy freshwater fish has been little studied in the past.The present study was a part of large parasitological investigation performed only in 4 localities of north-western Madagascar, however, documenting unknown diversity of fish parasites in isolated freshwater region with endemic fish fauna (i.e., Madagascar), the pattern which was previously shown for endemic freshwater fish in other regions i.e., Peri-Mediterranean and Middle East (Benovics et al., 2017(Benovics et al., , 2021;;Rahmouni et al., 2017;Řehulková et al., 2020;Nejat et al., 2023).Prior to this study, 12 valid species of Dermoergasilus were known, including 1 species, specifically D. longiabdominalis, in mugilid hosts in Madagascar.Two Dermoergasilus species were previously reported on cichlid hosts in India and Iraq.The first species, D. amplectens, was recorded on a number of fish species and over a wide geographic range, including Pseudetroplus maculatus, an endemic cichlid of southern India and Sri Lanka.The second species, D. cichlidus, was described from Coptodon zillii, a non-native cichlid in Iraq.Dermoergasilus madagascarensis n. sp.represents the third species of the genus reported on cichlids and the second species of the genus revealed in Madagascar and a single known species currently known only from endemic Malagasy cichlids (i.e., P. polyactis).
Even though questioned in the past (Gussev, 1987;Kabata, 1992;El-Rashidy and Boxshall, 2001), Dermoergasilus still remains valid.From the 3 morphological characters proposed by Ho and Do, 1982 only 1 clearly differentiates this genus, which is a digitiform process on each paired caudal rami.The other 2 characters seem to be ambiguous.The inflated transparent membrane is quite a vague morphological character, and some species of Dermoergasilus do not have it well developed (e.g.D. curtus or D. intermedius).The membrane could be an ancestral trait that is being lost during the evolution, from clearly visible balloon-like inflation in D. amplectens to barely noticeable cuticle in D. curtus.Moreover, there are some Ergasilus species with some kind of hyaline membrane on antenna.For example, the membrane on antenna of Ergasilus megacheir (Sars, 1909) appears to be very similar to that of D. curtus.The middle segment of endopod of legs II and III possessing a single seta is even less persuasive character, since at least 10 Ergasilus species (e.g.E. tumidus Markevich, 1940, E. briani Markevich, 1933, E. gibbus von Nordmann, 1832, E. gobiorum Markevich & Sukhnenko, 1967 etc.) also possess this character (Ho et al., 1992;Kabata, 1992).There are other morphological traits present in most of the species of Dermoergasilus, e.g.long first free abdominal segment, similar morphology of leg 5, falciform seta on legs, some species even share the same spine-seta and antennal formula.However, neither of them can clearly distinguish Dermoergasilus from other members of Ergasilidae but could indicate their possible close relationship and a common ancestry.
Based on the literature review, D. madagascarensis n. sp. shares the same spine and setal formula with 6 other species of the genus.Future studies using molecular analyses should focus on this aspect and verify, if species with the same armature of swimming legs are phylogenetically related.Many of these species were recorded from mugilid hosts in Indian region.It is possible that they have the common origin, and the divergence of the species is associated with geographical isolation of Madagascar, drifting away from the Indian peninsula 96-65 Mya (Vences et al., 2009).El-Rashidy and Boxshall (2001) suggested that a mugilid as a host is a plesiomorphic character for Dermoegasilus, and that the ancestor of this group of parasites also occurred on a mugilid host.Acquiring hosts of other fish families could be a result of the adaption to the conditions in the new environment, which are cichlids in this case.However, only molecular data from D. curtus, D. longiabdominalis and D. semiamplectens reported from mugil hosts in India, China, Madagascar, Philippines and Myanmar would shed more light on the origin of D. madagascarensis n. sp. and clarify its relationship with other Dermoergasilus and Ergasilus species.
Scanning electron microscopy (SEM) is the method providing the appropriated visualization of some morphological structures, in our study, specifically sensory setae and pits, and also the minute seta on the digitiform process in D. madagascarensis n. sp., while the latter character was not visible under the light microscope.It is highly likely that some morphological characters might be overlooked in older descriptions of Ergasilidae, in which authors did not use SEM.
Specimens preserved in ethanol faint brown in colour, with blue spot in eyespot and sometimes in cephalothorax.Male: Unknown Remarks Dermoergasilus madagascarensis n. sp.represents another species of Dermoergasilus, besides D. curtus (El-Rashidy and Boxshall, 2001) and D. intermedius (Kabata, 1992), that have antennae with only slightly inflated cuticular membrane.All other known Dermoergasilus spp.possess a conspicuous balloon-like inflated membrane covering all or only the first (in D. semicoleus) antennal endopodal segment.In D. curtus, however, the cuticular membrane covers only the inner surface of the first endopodal segment of the antenna, whereas in D. intermedius and D. madagascarensis n. sp. the membrane ensheathes all endopodal segments.The new species differs further from D. curtus mainly by having: (i) a pentagonshaped cephalosome (vs bullet-shaped cephalosome); (ii) second endopodal segment of the antenna without a minute seta (vs with a minute seta proximally on inner margin of the segment); (iii) interpodal plates ornamented with 3-4 rows of spinules (vs 1 row of spinules); (iv) genital segment with 1 medial row of spinules (vs 3 posterior rows of spinules); (v) urosomites without folded membrane; and (vi) 2 lateral caudal setae longer than the digitiform

Table 2 .
List of primers used for PCR amplifications of mitochondrial and nuclear markers in the present study

Table 3 .
List of parasitic copepods used for phylogenetic analyses and calculation of p-distances, including their host species, collection locality, and accession numbers for partial 18S, 28S rDNA and COI sequences from database GenBank and Bold (indicated with *)

Table 3 .
(Continued.) Newly generated sequence is given in bold.