Two new species of Anacanthorus (Monogenoidea, Dactylogyridae) parasitizing serrasalmid fish in Brazil

Abstract During studies on fish parasites, two new species of Anacanthorus were found parasitizing serrasalmid fishes, Anacanthorus simpliciphallus sp. n. from the hybrid Piaractus mesopotamicus x Piaractus brachypomus and Anacanthorus brandtii sp. n. from Serrasalmus brandtii. Anacanthorus simpliciphallus sp. n. resembles Anacanthorus reginae in the morphology of the male copulatory organ (MCO) and accessory piece but differs from A. reginae in terms of the smaller size of the accessory piece, which corresponds approximately half the size of the MCO and by the presence of a conspicuous metraterm, with a membranous terminal region. Anacanthorus brandtii sp. n. differs from Anacanthorus scapanus by the expansion of the accessory piece, from Anacanthorus jegui by the ratio MCO (male copulatory organ) /AP (accessory piece) and by the expansion of hook shank, from Anacanthorus sciponophallus and A. reginae by the ratio MCO/AP. Anacanthorus brandtii sp. n. can be distinguished from A. reginae and A. simpliciphallus sp.n. by the size of hooks which is similar in A. reginae and A. simpliciphallus sp. n. and dissimilar in A. brandtii sp. n. The two new species also differ from each other by the expansion of shank.


Introduction
Serrasalmidae, understood by fish known as piranhas and pacus, is a diverse family of freshwater fishes belonging to the order Characiformes, that is endemic throughout tropical and subtropical South America.Piaractus mesopotamicus (Holmberg, 1887) and Piaractus brachypomus (Cuvier, 1818), popularly known in Brazil as pacu and pirapitinga, respectively, are species characteristic of tropical waters and restricted to South America (Froese & Pauly, 2023).Crossing the female of P. mesopotamicus with the male of P. brachypomus results in the hybrid known as "patinga", which has been gaining much ground in the Brazilian fish market (Ribeiro et al., 2016).

Material and Methods
One hybrid specimen of P. mesopotamicus x P. brachypomus purchased from a fish market on São Luís Island, State of Maranhão, which had been brought to the market from a fish farm established in the municipality of Matinha (3º05'13.5"S,45º02'56"W) and 168 specimens of S. brandtii captured by local fishers in Três Marias Reservoir (18º12'59"S, 45º17'34"W), Upper São Francisco River, Minas Gerais State, Brazil and sent to the "Centro Integrado de Recursos Pesqueiros e Aquicultura (CIRPA)" of the "Companhia de Desenvolvimento dos Vales do São Francisco e Parnaíba (CODEVASF)" were examined for Monogenoidea.The gills were removed and placed in vials containing hot water (~65ºC) and were shaken.Absolute ethanol was added to reach a concentration of 70%.Monogenoids were picked from the sediment and from the gill arches with the aid of a stereoscopic microscope.Some specimens were mounted in Hoyer's medium to study of the sclerotized parts and others were stained with Gomori's trichrome and mounted in Canada balsam (Humason, 1979;Boeger & Vianna, 2006).The specimens were observed using an Olympus BX 41 microscope with phase contrast and Zeiss Axioskop 2 Plus microscope with differential interference contrast, both equipped with a camera lucida for drawings.All measurements are presented in micrometers, and the range is followed by the mean in parentheses and the number of specimens measured.Identification of the authors and nomenclatural acts for the taxon was in accordance with the guidelines provided in Article 50.1 and recommendation 50A of the International Code of Zoological Nomenclature (ICZN), which specifically pertains to authorship identity.The holotype and paratypes for each parasite species were deposited in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC), Rio de Janeiro, Brazil.Etymology: The species name is from Latin (simplex=simples + phallus=penis) and refers to the morphology of the male copulatory organ.DESCRIPTION: (Based on seven specimens: six mounted in Hoyer's medium and one mounted in Gomori's trichrome).Body elongated, fusiform, 262-550 (422, n= 5) long including the haptor, by 88-145 (123, n= 5) wide at the level of germarium.Two terminal, and two bilateral well developed cephalic lobes; three bilateral pairs of head organs.Two pairs of eyes equidistant, anterior pair smaller than posterior pair, slightly closer together than posterior pair; pairs slightly close to each other; accessory granules sparse in the cephalic region.Pharynx subspherical, 20 and 27 (n= 2) in diameter; long oesophagus.Two intestinal ceca confluent posterior to the gonads, lacking diverticula.Gonads overlapping; testis dorsal to germarium, 60-100 (86; n= 4) long, vas deferens looping intestinal caeca, single prostatic reservoir pyriform.Copulatory complex comprising male copulatory organ (MCO) and accessory piece (AP).MCO tubular, heavily sclerotized, J-shaped, with slightly sclerotized walls, base with smooth margin, 65-83 (73; n= 7) long.Accessory piece with a terminal flap, non articulated to MCO base, 37-45 (41; n= 7).Ratio MCO/AP 1:0.48-1:0.58(1:0.55,n=7).Germarium 35 and 40 (n= 2) long by 40 and 45 (n= 2) wide.Metraterm conspicuous, with membranous terminal region.Genital pore and eggs not observed.Peduncle short.Haptor armed with 7 pairs of hooks (4 ventral, 3 dorsal), 2 pairs (1 dorsal, 1 ventral) of 4A's, 60-135 (93, n= 5) wide.Hooks similar in shape and size, each with truncate slightly depressed thumb, curved shaft, short point, shank proximal expansion 0.3 shank length, 20-24 (21; n= 20) long; filamentous hook (FH loop) delicate, extending as far as half of the shank.Similar 4A hooks, 9-12 (10; n= 10).Vitellaria dense, dispersed throughout the trunk, absent in the region of reproductive organs and copulatory complex.

Discussion
The new species are allocated in Anacanthorus because they possess a bilobed haptor with 7 pairs of hooks and 2 pairs of reduced hooks (4A's), lacking anchors and bars, have tandem or slightly overlapping gonads, post-ovarian testis, modified (thickened or sclerotized) distal uterine wall or metraterm and vagina is absent (Kritsky et al., 1979(Kritsky et al., , 1992)).
Anacanthorus species are exclusively parasites of Neotropical characiforms, and so far, 19 species have been reported from Bryconidae, 8 from Erythrinidae, 21 from Triportheidae, and 44 from Serrasalmidae.Kritsky & Thatcher (1974) described Anacanthorus colombianus Kritsky & Thatcher, 1974 from Salminus affins Steindachner, 1880 and also reported its presence in Oreochromis mossambicus (Peters, 1852), a cichlid fish within the order Cichliformes.According to these authors, this latter occurrence was apparently accidental.Given the absence of further records in this order, it is considered that Anacanthorus spp. is specific to characiform fishes.Species of this genus have been found in five countries in the Neotropical Region (Brazil, Bolivia, Colombia, Peru, and Venezuela), and Brazil stands out as the country with the largest number of occurrences (156) (Boeger et al., 2023).The sclerotized structures such as the copulatory complex and hooks of Anacanthorus species appear to have a high specificity in terms of morphology with regard to the host family level (Santos et al., 2019).Anacanthorus species that parasitize members of the family Serrasalmidae present the characteristic of a J-shaped MCO, an accessory piece that is not articulated to the MCO, hooks with truncated thumb and a shank with proximal dilation (Boeger & Kritsky, 1988;Kritsky et al., 1992;Van Every & Kritsky, 1992).The finding of two new species of Anacanthorus in serrasalmid hosts presenting morphological characteristics similar to those previously described on these hosts (Table 1) confirms that the lineages of the parasites from serrasalmid hosts shared those features.
Anacanthorus brandtii sp.n. is closely related to species previously described from Serrasalmus spp. as Anacanthorus scapanus, Anacanthorus jegui, Anacanthorus sciponophallus, A. reginae and A. simpliciphallus sp.n. by the morphology of copulatory complex.The new species differs from A. scapanus by the expansion of the accessory piece (subterminal in A. scapanus vs midlength expansion in the A. brandtii sp.n.), from A. jegui by the ratio MCO/AP (MCO 48 and AP 39 in A. jegui and MCO 70 vs AP 41 in the new species) and by the expansion of hook shank (0.3 and 0.4 in A. jegui vs 0.4 and 0.6 in A. brandtii sp.n.).The new species can be differentiated from A. sciponophallus and A. reginae by the ratio MCO/AP (MCO 76-82 and AP 74-79 in A. sciponophallus from different hosts, MCO 67 and AP 59 in A. reginae vs MCO 70 and AP 41 in the new species).The new species can also be distinguished from A. reginae and A. simpliciphallus sp.n. by the size of hooks which is similar in A. reginae and A. simpliciphallus sp.n. vs dissimilar in A. brandtii sp.n.The two new species also differ by the expansion of shank (0.3 in A. simpliciphallus sp.n. vs 0.4 and 0.6 in A. brandtii sp.n.).