Isopods Cymothoidae ectoparasites of fish from the Amazon

lurunita@gmail.com Abstract Most freshwater species of Cymothoidae are distributed in South America. They have mainly been recorded in the eastern and western regions of the Amazon River basin. However, in this ecosystem, the biodiversity of this group may be greater if the entire Amazon basin is considered. In this regard, the aim of the present study was to provide an updated list of isopod species of the family Cymothoidae that are found in fish in the Brazilian Amazon region and to report on new fish host occurrences and expanded geographical distributions for cymothoid isopods that parasitize fish in the southwestern Brazilian Amazon region. The parasites found in fish specimens were collected, fixed and identified later. We found eight species of Cymothoidae parasitizing different host fish species in the southwestern Amazon region. However, we found 14 species of Cymothoidae throughout the Brazilian Amazon region. Three additional species are thus reported here, which increases the number of species of Cymothoidae in this region to 17. These additional species are also new records for Brazil. Therefore, this study has contribute to expand the knowledge about the distribution and diversity of Cymothoidae in the Amazon


Introduction
The family Cymothoidae comprises around 40 genera and 380 species that have been described. Some species occur in both marine and freshwater environments (Ahyong et al., 2011;Smit et al., 2014). They are obligate parasites, and mostly use fish as their hosts, although some parasitize shrimps (Smit et al., 2014). Unlike other groups of parasites, isopods have a large body (~ 10 to 50 mm). Moreover, these parasites have morphological characteristics specific to parasitic life, such as buccal appendages with setae similar to the robust curved terminal and subterminal spines that serve to attach the parasite to the host tissue as well, and nails modified into powerful claws to attach to the host (Brusca et al., 2001).
The highest diversity of cymothoid isopods is concentrated in the tropics, with well-known distribution in marine environments. However, they have only been poorly studied in freshwater ecosystems (Smit et al., 2014). Most freshwater species (around 13) are distributed in South America, mainly in the Amazon River basin . These species were collected in its eastern region (around 9) and central region (around 6), which concentrate the highest cymothoid diversity (Thatcher, 1993;Magalhães et al., 2018;Oliveira et al., 2017;Esteves-Silva et al., 2020). Furthermore, the northwestern Amazon region is the only area in which the occurrence of Asotana magnifica Thatcher, 1988 has been reported, parasitizing the mouth cavity of a specimen of Serrasalmus sp. (Thatcher, 1988) ( Table 1).  The species of Cymothoidae that parasitize fish in the southwestern Brazilian Amazon region (e.g. the state of Acre) were hitherto unknown. This region shows high heterogeneity of aquatic environments and high fish diversity (Begossi et al., 1999), which are ideal for accommodating great richness of Cymothoidae. Based on this principle, it is important to develop studies that expand the knowledge about the distribution of these ectoparasite crustaceans in this region of the Amazon basin. This will not only contribute information on the biodiversity of the Neotropical region, but also can help prevent fish losses in farming systems, where these organisms are known pests (Tavares- Dias et al., 2015).
Therefore, the aim of this study was to provide an updated list of isopod species of the family Cymothoidae that are found in fish in the Brazilian Amazon region and to report on new fish host occurrences and expanded geographical distributions for cymothoid isopods that parasitize fish in the southwestern Brazilian Amazon region.

Sampling
The fish specimens were caught using gillnets of 80 m in length and 3.0 m in height, with meshes of 1.5 cm, 2.5 cm, 3.5 cm and 5.5 cm between opposite nodes. The nets were installed in the early afternoon and were kept there for 12 hours with fish collections every two hours. The two nets used each had a mesh size of 12 mm, height of 2 m and opening size of 12 m. The nets were launched ten times per collector at each collection point. A beach trawl was also used, measuring 9 m in length and 2.4 m in height, with 13 mm mesh on the wings.
Each fish was initially identified in the field and its mouth, nostrils, operculum, gills, and integument were examined macroscopically to check for the presence of isopod parasites. The fish were then stored in water-filled boxes with an oxygenator and were transported to the laboratory, where they examined for parasites using a stereomicroscope. After this, the fish were fixed in 10% formalin and were identified in accordance with the specialized literature (Silvano, 2001;Buckup et al., 2007;Queiroz, 2010). The parasites found were fixed in 70% alcohol, sent to the laboratory at Embrapa Amapá and identified in accordance with the specialized literature, as listed in Table 1.

Database review
A review was conducted in the SciELO, ISI, Scopus, Science Direct, Zoological Records, CAB Abstracts Archive, and Google Scholar databases, in order to describe the distribution of Cymothoidae species in the Brazilian Amazon region ( Table 1). The geographical coordinates of each study were ascertained using Google Earth and were used to generate a geographical distribution map of the family Cymothoidae in the Brazilian Amazon region, using the QGIS software, version 3.12.2.

Results
We collected a total of 2551 fish specimens, belonging to five orders, 23 families and 83 species. Out of the total number of fish collected, only 15 individuals (0.4%) in 10 species (12%) were parasitized by Cymothoidae (Tables 2 and 3).      Stadler, 1972; and Livoneca guianensis Van Name, 1925. All the parasites were recorded for the first time in these host fish species. Artystone bolivianensis, B. brachmanni and P. parva were recorded for the first time parasitizing fish in Brazil (Table 3).
Among the eight species of Cymothoidae collected, four were found in the Paranã River, two in the Moa River, one in the Juruá River and one in the Crôa River. Braga fluviatilis and L. guianensis were observed to parasitize the highest number of hosts, such that L. guianensis occurred in all four rivers. The mean intensity of the species of Cymothoidae were similar to each other, i.e. one parasite per fish ( Table 3).
Out of the 15 individuals of Cymothoidae collected, ten were females (73.3%). The highest prevalence of specimens were recorded for B. fluviatilis (three specimens) and L. guianensis (three specimens). Most of these isopods were found in the fish integument and mouth cavity. However, P. parva and Braga sp. occurred in the anal cavity of the hosts and B. patagonica in the gills ( Table 3).
We found a total of 34 host fish species for these species of Cymothoidae in the Brazilian Amazon region. Eight species of these isopods were found in fish in the state of Amazonas, five in the state of Amapá, four in the state of Pará, two in the state of Roraima and one in the state of Mato Grosso. Across the Brazilian Amazon region, the isopod B. patagonica showed the widest geographical distribution and largest number of host species (Table 1).

Discussion
Parasites of the family Cymothoidae are distributed throughout the Amazon River basin, in which the largest numbers of species have been reported in the central and eastern regions of this basin, where these cymothoids parasitize a great diversity of hosts belonging to the families Acestrorhynchidae, Anostomidae, Arapaimidae, Bryconidae, Curimatidae, Cynodontidae, Triportheidae, Serrasalmidae, Loricariidae, Pimelodidae, Lebiasinidae, Cichlidae, Erythrinidae and Sciaenidae (Table 1). Our results from the southwestern Amazon region, together with data from the central and eastern Amazon regions, depict an area with great richness of species of Cymothoidae, totaling 17 species recorded in fish in the Brazilian Amazon region.
This richness of cymothoid species in the Neotropical region was expected, especially in the Amazon basin (Smit et al., 2014). This ecosystem is home to a great diversity of fish, which enables occurrences of species of Cymothoidae in different aquatic environments, such as rivers, lakes and flooded forests (Winemiller et al., 2016). Recently, Tavares- Dias et al. (2015) listed eight species of freshwater Isopoda throughout Brazil. Our study, which included both bibliographic review and data collection, recorded 17 species of Cymothoidae in the Brazilian Amazon region alone. This demonstrates the importance of the Amazon region for biodiversity in the Neotropical region. However, it is essential to note that the biogeographical status of freshwater cymothoids may still be underestimated (Smit et al., 2014) because of the extensive area of the Amazon region. Many areas of this region have never been studied to assess the fauna of this group of parasites.
In the present study, not all the fish species acted as hosts for these cymothoids. The species parasitized generally have low abundance, since out of the 84 fish species analyzed, only 10 were parasitized by cymothoids, with low values for mean abundance and intensity. Immunological, behavioral, environmental and genetic characteristics may explain this complex parasite-host interaction (Poulin, 2013;Aneesh et al., 2013;Tavares-Dias et al., 2015)  and act as important factors in the selection of a host fish. Recently, it was observed that the defenses of some fish species are important for preventing colonization by cymothoids. These defenses include the ability of the host to remove the parasite using its mouth or through rapid movements with its body. In some cases, fish can eat these isopods, thereby eliminating the risk of parasitism (Alberto et al., 2009). Another factor that can influence the degree of infestation is the short period for which this parasite can survive freely in the environment. Free living requires a large energy investment, to search for a host fish, and the time that this takes can lead these parasites to death (Leonardos & Trilles, 2003).
New reports on Cymothoidae in the Amazon region can be expected, given the high diversity of environments and host fish in this region (Hata et al., 2017;Tavares-Dias et al., 2015). In this context, when research is conducted in areas where few studies had previously been conducted, new reports of known species are expected for that region, in addition to species not yet described.
The data from the present study expanded the geographical distribution of B. patagonica to the southwestern part of the Brazilian Amazon region. Braga patagonica has wide geographical distribution, occurring in different tributaries of the Amazon River basin (e.g. the middle Solimões River, Amazon River, Jari River and Matapi River). This species parasitizes a variety of hosts belonging to the families Acestrorhynchidae, Anostomidae, Bryconidae, Curimatidae, Cynodontidae, Serrasalmidae, Cichlidae, Erythrinidae and Sciaenidae ( Table 1). The generalist nature of this species of Cymothoidae demonstrates its low parasitic specificity (Tavares- Dias et al., , 2015. In general, in the Amazon region, genera of Cymothoidae such as Anphira and Vanamea have been found infesting the gill cavities of hosts, while Artystone and Riggia have been found inside holes formed by the parasite in the tegument of the fish, mostly in the pelvic region of these hosts (Table 1). However, Braga species can be found in several places in the host, such as the mouth and gill cavities, and integument surface. The ways in which the parasite becomes attached to the host fish can influence the specificity of species of Cymothoidae regarding their host choice (Hata et al., 2017). Cymothoids parasitizing the external surfaces of their hosts, such as the gill and mouth cavities, are more generalist, and can become attached to many fish species (Bruce, 1986;Hadfield et al., 2015). This was observed in the present study, in which species of the genera Braga and Livoneca were seen to infest the integument, gills and mouth cavities in many fish species.
Encapsulation in the host integument as the means of attachment is characteristic of Artystone and Riggia Junoy, 2016;Oliveira et al., 2019) and can be considered to be a host-parasite specific characteristic (Tsai & Dai 1999;Yamano et al., 2011;Hata et al., 2017). Data in the literature show that species are generalists, parasitizing fish species of the families Anostomidae, Ariidae, Characidae, Cichlidae, Heptapteridae, Loricariidae and Pimelodidae (Junoy, 2016). However, in the Brazilian Amazon region, the Artystone species that was observed can be considered to be a specialist because it was found parasitizing only Cichlidae fish (Oliveira et al., 2019). Studies on integrative, comparative and molecular biology may be the approach that should be followed in order to understand these complex interactions.
Artystone trysibia, Braga sp. and P. parva were found in the anal cavity of their hosts ( Table 2). Among the cymothoids reported in Brazil, only Riggia acuticaudata Thatcher, Lopes & Froehlich, 2002 was found parasitizing the anal cavity of its host, a specimen of Ancistrus sp. (Oda et al., 2015). Therefore, the present study suggests that because the anal cavity is an opening and these parasites have the habit of living in cavities, this site becomes an important place of refuge and the parasites may use excreted food waste in their diet.
In conclusion, through this study, the occurrence and distribution of Cymothoidae in the Brazilian Amazon are expanded, with new records of host fish and infestation sites. Therefore, this study contributes towards future studies on the diversity, distribution and specificity of cymothoids in the Amazon region, which is the most biodiverse region on the planet.