﻿Two new species of Curvianchoratus (Monogenoidea, Dactylogyridae) parasitizing Psectrogasteramazonica (Characiformes, Curimatidae) and a new record for Curvianchoratussingularis in the Tocantins River, Maranhão, Brazil

﻿Abstract Several studies have demonstrated parasitism by monogenoids in characiform fish in the Neotropics. During studies on the helminth fauna of curimatids from the Tocantins River, specimens of Psectrogasteramazonica Eigenmann & Eigenmann, 1889 were examined and species of Curvianchoratus Hanek, Molnar & Fernando, 1974 were found. Species of the genus are characterized mainly by the complex shape of haptoral anchors with a modified dorsal anchor, composed by two subunits, dorsal-median and dorsal. To date, two species of Curvianchoratus are known to parasitize curimatid fishes: the type species Curvianchoratushexacleidus Hanek, Molnar & Fernando, 1974 and Curvianchoratussingularis (Suriano, 1980). During examination of specimens of P.amazonica collected in the Tocantins River, Embiral, Imperatriz, Maranhão State, Brazil, two new species of Curvianchoratus were found and are described herein. Curvianchoratuspsectrogasterisp. nov. and Curvianchoratusdominguesisp. nov. are characterized by possessing the male copulatory organ formed by a long cirrus and a claw-shaped accessory piece, connected to the base of the male copulatory organ by a ligament. The new species differs from the two known congeneric species mainly by the morphology of the dorsal-median and dorsal subunits of the dorsal anchor. Curvianchoratuspsectrogasterisp. nov. also differs from other species of the genus by the absence of the ventral bar and Curvianchoratusdominguesisp. nov. by the size and shape of the ventral bar. An amendment to the diagnosis of Curvianchoratus is provided to accommodate the new species. The present study increases the number of Curvianchoratus species to four and extends the occurrence of the genus to the Tocantins-Araguaia Basin.


Introduction
Characiformes is one of the main orders of Ostariophysi, which occur exclusively in freshwater, with the greatest diversity and species richness concentrated in the Neotropical Region. These fish represent the historical connection between Africa and South America, which can be proven through their distribution and the presence of fossil records Malabarba 2010, 2014). Among the families of the order, Curimatidae comprises eight extant genera and more than 110 species, living in diverse freshwater habitats from Costa Rica to Argentina (Melo et al. 2018).
Psectrogaster Eigenmann & Eigenmann, 1889 is a morphologically diverse lineage of Curimatidae, distributed in streams, rivers, and still waters throughout the drainage basins of the Orinoco River, Essequibo River, Amazon River, Paraguay-Parana River, and some of the rivers of northeastern Brazil (Vari 1989). Psectrogaster amazonica Eigenmann & Eigenmann, 1889, popularly known in Brazil as "branquinha" (Santos et al. 1984) is a freshwater fish that lives in tropical waters in the Tocantins and Amazon River basins (Vari and Röpke 2013;Froese and Pauly 2022). This species presents benthopelagic behavior, with the formation of large shoals, and has a maximum length of 19 cm, being one of the largest species of the genus (Vari 1989). The species is one of the most common and abundant along the Tocantins River in midwest, north and northeast Brazil and is the basis of an intensive fishery in this area. Individuals belonging to this species primarily feed on organic detritus, benthic organisms, and periphyton (Santos et al. 1984).
Several studies have demonstrated parasitism by monogenoids in Curimatidae fish in the Neotropics (Table 1). However, research involving the helminth fauna of P. amazonica is still incipient, with only two monogenoids described for this host so far (Freitas et al. 2021). Other studies regarding this fish species refer to larval development, the distribution of larvae in a natural breeding area, hematological parameters, and branchial histopathological alterations (Nascimento and Araújo-Lima 1993;Carvalho et al. 2009;Ponte et al. 2017;Bezerra et al. 2020;Pereira et al. 2020).
Monogenoidea Bychowsky, 1937 are, in general, ectoparasites that mainly infect fish species . They have a simple and direct life cycle and, consequently, may present a high reproduction rate in these hosts (Lapera et al. 2017). Monogenoids are among the main components of the parasitic fauna of freshwater fish that occur in the Neotropics, most of them belonging to the family Dactylogyridae Bychowsky, 1933 (Cohen et al. 2013).
In Brazil, C. singularis has been reported in the rivers of the state of São Paulo parasitizing Curimatidae fishes (Vieira et al. 2013;Dias et al. 2017).
During studies on the helminth fauna of curimatids from the Tocantins River, specimens of P. amazonica were examined and species of Curvianchoratus were found. The present paper presents an emended diagnosis of Curvianchoratus, with the proposal of two new species and a new geographical record for C. singularis.

Material and method
From July 2018 to April 2022, 136 samples of P. amazonica (average 14 cm in standard length and average 62 g in weight) were collected in the Tocantins River, close to the urban perimeter of Imperatriz, in the village of Embiral, state of Maranhão, Brazil (5°27'50''S, 47°33'48''W) (Fig. 1). Fish were caught with the aid of local fishermen using gill nets, or a hook and line and were examined asites immediately after capture at the Nucleus of Advanced Morphophysiological Studies at UEMASUL. The gills were removed and placed in vials containing hot water (65 °C) in order to detach the parasites from the gill filaments. Later, absolute ethanol was added to reach a concentration of 70%. Monogenoids were collected from the sediment and gill arches in the laboratory with the aid of a stereoscopic microscope. Specimens were mounted in Hoyer's medium to study the hard parts, such as the copulatory complex and haptoral sclerites and some were stained with Gomori's trichrome to study the internal organs of the parasite (Kritsky et al. 1978). Measurements are presented in micrometers; range values are followed by mean and number of structures measured in parentheses. Sclerotized structures, such as bars and anchors were measured according to the scheme shown in Fig. 2. The parasitological indices were calculated as proposed by Bush et al. (1997), followed by the standard deviation. The authorship of the taxa followed the recommendation of Article 50.1 of the International Code of Zoological Nomenclature (ICZN), which deals with the identity of the authors. 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. The images were captured with a SONY MPEG Movie EX DSC-S75 digital camera. Type specimens were deposited in the Coleção Helmintológica do Instituto Oswaldo Cruz (CHIOC) in Brazil.

Results
In the present study, a total of 87 monogenoid specimens were collected, being: 68 Curvianchoratus psectrogasteri sp. nov., eight Curvianchoratus dominguesi sp. nov. and 11 Curvianchoratus singularis. Emended diagnosis. Tegument smooth, thin. Body elongated; divisible into cephalic area, trunk, peduncle, and haptor. Terminal and lateral cephalic lobes developed or not; head organs and cephalic glands present. Eyespots present or absent. Mouth subterminal, midventral, prepharyngeal. Pharynx muscular; esophagus short. Intestinal caeca two, not presenting diverticula. Common genital pore midventral near the level of intestinal bifurcation. Gonads intercaecal, overlapping or not; testis dorsal to germarium. Seminal vesicle large, dilated portion of vas deferens; prostatic reservoir single. Copulatory complex comprising male copulatory organ (MCO) and accessory piece; MCO consisting of sclerotized coiled tube with counterclockwise rings, which make a short turn on itself near the base; cirrus and accessory piece articulated basally. Accessory piece with bifurcated distal portion, pincer-shaped. Ovary pretesticular. Vagina medial or sinistral. Vitellaria well developed, coextensive with intestinal ceca, absent from regions of other reproductive organs. Peduncle short, haptor subquadrate. Ventral bar present or absent. Dorsal bar absent. Ventral anchor of dactylogyrid type. Dorsal anchor modified, composed by two subunits, dorsal-median and dorsal, forming a full circle, the shaft of the dorsal subunit passes over the superficial root of the dorsal-median subunit anchor. Hooks 14; 5 pairs ventral, 2 pairs dorsal, with inflated base and slender shank. Parasites of curimatid fishes.
Remarks. Curvianchoratus was proposed to accommodate Curvianchoratus hexacleidus , a parasite of the characiform S. argentea from the Arouca River, Trinidad ). The main characteristic of Curvianchoratus is the haptor with a modified dorsal anchor, composed of two subunits, which is a unique shape for the genus. The absence of bars in one of the species described here, made it necessary to expand the original diagnosis of the genus to include the characteristics of the new species described below, as well as to detail some morphological characters. Etymology. The specific name of the species is derived from the generic name of the host.
Remarks. Curvianchoratus psectrogasteri sp. nov. is allocated in the genus mainly by the morphology of the copulatory complex, composed by a tubular cirrus and a pincer-shaped accessory piece, connected to the base of the cirrus by a ligament and by the presence of the greatly modified dorsal anchor, composed by two subunits: dorsal-median and dorsal. The new species differs from the two known species by the absence of the ventral bar and by the morphology of the dorsal-median subunit (strongly curved, with the shank and tip visibly separated; a robust shank ornamented with a thorn at the base and crested-shaped deep and superficial roots in the new species vs. a stickle shaped end inserted in the cavity of the shaft in C. hexacleidus and C. singularis) and by the morphology of the dorsal subunit (elongated, with a broadly circular central region in the new species vs. elongated, with deep root in C. singularis and without in C. hexacleidus). Type locality. Embiral, rural zone (5°27'50"S, 47°33'48"W), municipality of Imperatriz, Maranhão State.
Remarks. Curvianchoratus dominguesi sp. nov. is similar to the other species of the genus mainly by the morphology of the dorsal anchor, the copulatory complex, which is composed by a long cirrus and a pincer-shaped accessory piece, connected to the base of the cirrus. The new species is similar to C. singularis and C. hexacleidus by the presence of the ventral bar. The new species differs from all congeneric species by the shape of the ventral anchor (formed by a shaft with two subunits in the new species vs. well-developed roots in C. hexacleidus and moderately developed deep root and well-developed superficial root in C. singularis and C. psectrogasteri sp. nov.), the morphology of the ventral bar (short and robust, anvil-shaped ventral bar in C. dominguesi sp. nov. vs. serpentine-shaped in C. hexacleidus, M-shaped in C. singularis and absent in C. psectrogasteri sp. nov.) and by the ratio of the subunits of the dorsal anchor (dorsal subunit is 30% larger than the dorsal-median subunit in C. dominguesi sp. nov. vs. the approximately same size of the subunits in the other three species of the genus).
Specimens deposited. Voucher CHIOC 39990, 39991, 39992. Remarks. Suriano (1980) described the species as Notodiplocerus singularis and the original drawings are simplified lacking important details of the morphology of these unique anchors that characterize the genus. In the description provided by Rossin and Timi (2016), the morphology of the dorsal anchor complex and copulatory complex was described in detail and illustrated by means of drawings and photographs. The morphology of the present material is in agreement with the material studied by Rossin and Timi (2016).

Discussion
Monogenoideans represent one of the main components of the helminth fauna present in freshwater fish in the Neotropics, most of which are dactylogyridean parasites of teleost fish (Boeger and Vianna 2006;Cohen et al. 2013;Braga et al. 2014). The great heterogeneity of hydrographic basins present in Brazil reflects a rich and diversified monogenoid fauna in this region (Tavares- Dias et al. 2022). Despite taxonomic efforts, the diversity of monogenoid parasites of freshwater fishes from South America remains poorly known . According to Melo et al. (2018), most of the information regarding monogenoidean diversity in the Neotropical Region is limited to medium-sized fish species, while the parasitic fauna of small-sized species, especially Characiformes and Siluriformes, is not yet described.
Studies on the parasite diversity of freshwater fish from the Tocantins-Araguaia Basin have gradually increased in recent years [see Fehlauer and Boeger (2005) In this sense, the proposal of two new species of Curvianchoratus contributes to the knowledge of parasite fauna of the region and enlarges the morphological characters used to describe the unique species that ventral bar is absent (Curvianchoratus psectrogasteri sp. nov.), which is contrary to the known species of the genus. Although the presence of the bar is an important characteristic, we adopted a conservative approach when proposing a new species of this genus, because C. psectrogasteri exhibit the complex morphology of the dorsal anchor, characteristic of Curvianchoratus.
With the description of the two new species in the present study, Curvianchoratus now encompasses four species, all parasitizing curimatid fishes. Curvianchoratus singularis is herein reported in P. amazonica from the Tocantins River, expanding the occurrence of the species to the Tocantins-Araguaia Basin. The finding of three species of the genus in P. amazonica confirms the high specificity of the genus to neotropical curimatid hosts. On the other side, Psectrogaster amazonica harbors five monogenoidean species, two from a generalist monogenoidean genus, Urocleidoides (parasites from three different fish orders) (Freitas et al. 2021) and three from the specialist genus Curvianchoratus.

Conflict of interest
No conflict of interest was declared.

Ethical statement
Permission to collect the hosts was given by the Instituto Chico Mendes de Conser-  Carine Almeida Miranda Bezerra: Collected the hosts, and processed the methodology of collect of parasites; substantial contribution in the concept; contribution to data analysis and interpretation; contribution to manuscript preparation; Simone Chinicz Cohen: Substantial contribution in the concept and design of the study; contribution to data analysis and interpretation; contribution to data collection; contribution to manuscript preparation; contribution to critical revision, adding intelectual content. Yuri Costa de Meneses: Processed the methodology to prepare of parasites; contribution to critical revision, adding intelectual content. Helyab Gabriel Chaves Neres Collected the hosts, and processed the methodology of collect of parasites. Diego Carvalho Viana: Project administration.
Marcia Cristina Nascimento Justo: Substantial contribution in the concept and design of the study; contribution to data analysis and interpretation; contribution to manuscript preparation; contribution to critical revision; Writing original draft; prepared figures.