First assessment of the metazoan parasite community of Gymnogeophagus balzanii (Cichliformes: Cichlidae) from Pantanal wetlands, State of Mato Grosso do Sul, Brazill

ABSTRACT Gymnogeophagus is a genus of fish from the family Cichlidae present in freshwaters from Southern South America, and the knowledge on its parasite communities is rather scarce. Therefore, the present study evaluated, for the first time, the parasite community of G. balzanii. Fish were collected in the Pantanal wetlands, State of Mato Grosso do Sul, Brazil and analysed for metazoan parasites. Ten taxa (larvae and adult) were found including Nematoda (3), Digenea (5), Copepoda (1) and Monogenea (1). All taxa were aggregated within the host population, few showed high prevalence and all had low abundance. The parasite community was composed by few specialists and several generalist and characterised by low richness, diversity and, consequently high dominance of few species and low evenness. These characteristics are typical of isolationist parasite communities. The predominance of larval forms indicates that G. balzanii occupies a lower position within the food chain, acting as an important link for trophic relations. Host length and weight were strong determinants in the parasite community, making fish sex also important since males were significantly longer and heavier than females. Generally, the parasite fauna did not influence host body condition, indicating no apparent debility. Presence of Clinostomum metacercariae is important for human health issues.


Introduction
Cichlidae is a high-diversity family of actinopterygians that currently includes 1745 nominal species of freshwater fish reported in the Middle East (four species), Iran (one species), Madagascar (17 species), Sri Lank and South India (three species), as well as in Africa (900 species) and in the American continent (389 species), which have the greatest diversities (Kullander 2003;Froese and Pauly 2022). Regarding South America, the current number of cichlid species reported is around 290 (Kullander 1998;Froese and Pauly 2022), including the genus Gymnogeophagus that occurs in the southern part of the continent (Froese and Pauly 2022), and is of special interest for parasitological studies, since parasites of these fish still are rather poorly known. They are important members of ecosystems (including threatened ones), and are commercially used as ornamental fish and a food resource (Říčan et al. 2019;Froese and Pauly 2022).
The scarcity of parasitological studies in Gymnogeophagus is illustrated by the fact that, of the 20 valid species assigned to the genus, only three were analysed for parasites and most of the approaches were taxonomic or based on species surveys (Mansur 1999;Alberto et al. 2001Alberto et al. , 2009Kohn et al. 2007;Malta et al. 2018;Montes et al. 2020). In Brazil, only G. balzanii has been reported as a host, in a study describing a new species of nematode (Malta et al. 2018). Additionally, Montes et al. (2020) recently reported some metacercariae in the same fish species. Therefore, it is possible to assume that, as observed within the genus, G. balzanii has a poorly understood parasite fauna, especially pertaining to the ecology and the community structure of these organisms, which has never been studied.
Parasites are important components of ecosystems, with crucial ecological roles including implications for biodiversity conservation (Poulin 1999(Poulin , 2014Marcogliese 2004). Thus, it is valuable to expand knowledge of these organisms, especially in high biodiversity hotspots as represented by the Pantanal wetlands (Polaz et al. 2014). This biome supports a rich and abundant ichthyofauna (Britski et al. 2007;Corrêa et al. 2009), which are potential hosts of a rich fauna of parasites, little known so far .
The present study focuses on analysing the community structure of metazoans parasitising G. balzanii from Pantanal wetlands, State do Mato Grosso do Sul, Brazil, and correlates the parasitological descriptors with host features.

Host and parasite sampling
Thirty-one specimens of G. balzanii were collected using gillnets, during September 2015, in a lake next to the highway MS-184, in Pantanal wetlands, municipality of Corumbá, State of Mato Grosso do Sul,Brazil (19°28′S;57°02′W). Fish were kept alive in containers filled with lake water, oxygenised by pumps prior to parasitological analysis in the laboratory of the 'Base de Estudos do Pantanal' from the Universidade Federal de Mato Grosso do Sul. Fish were anaesthetised and euthanised individually in an eugenol bath, photo-documented, measured (standard length [SL], given in mm), weighed (given in g) and analysed for parasites. Gills, opercula, nostrils, mouth, body surface and eugenol bath liquid were searched for ectoparasites using a stereomicroscope; similarly, the endoparasites were investigated in all internal organs and body cavity. Parasites found were processed for taxonomic identification following standard techniques (Eiras et al. 2006) and identified according to Cressey and Collette (1971), Moravec (1998), Anderson andChabaud (2009) andNiewiadomska (2002).
Hosts were identified according to Britski et al. (2007); the nomenclature and classification were updated following Froese and Pauly (2022). All procedures involving animals were permitted by the 'Sistema de Autorização e Informação em Biodiversidade' (acronym SisBio, licence number 54895) and by the 'Colégio Brasileiro de Experimentação Animal' (CONCEA 2013).

Ecological and statistical analyses
The relative condition factor (Kn = W o /W e ) was calculated based on the ratio between the observed weight (W o ) of a fish at a given length and the expected weight of the same fish at the same length (W e ), in which the weight is given in g and the length in cm (Le Cren 1951). The W e was estimated by the regression W e = a + bSL, where SL = standard length; the coefficients a (intercept) and b (angular coefficient) were estimated using the least square method using the equation logW o = loga + b(logSL); Kn of hosts was compared with the standard value (Kn = 1.00) using the Student's t-test (Kurup and Samuel 1987;Zar 2010).
According to Bush et al. (1997) the population descriptors of prevalence and abundance were calculated. The following community descriptors were also calculated according to Rohde et al. (1995), Magurran (2004) and Poulin (2014): (1) Species richness: absolute number of smallest taxonomic entities within a community.
(2) Frequency of dominance: percentage of infracommunities in which a parasite species was numerically dominant, indicating possible well-established species.
(3) Berger-Parker dominance index (D): a measure of the numerical importance of the most abundant species within a community, which can be related to the parasite community diversity where more dominant communities are less diverse and tend to be isolationist (according to Holmes 1987;Esch et al. 1990). (4) Brillouin index (HB): a measure for evaluating diversity, which is adequate at the infracommunity level of parasites, as well as at component community level when given as a mean value.This measure is not affected by low species richness. (5) Pielou species evenness (J'): indicates how close in abundance each species within a community is, negatively related to dominance, and illustrating if the number of individuals from different species are equivalent.
Distribution of parasites within the host population was evaluated by the discrepancy index (DI) (Poulin 1993). The community status for the parasite species was attributed according to the conception of Zander et al. (2000), in which species with mean abundance (MA) < 0.2 were considered rare; those with 0.2 < MA < 0.6 were considered satellite; those with 0.6 < MA < 2.0 were considered secondary species; and those with MA > 2.0 were considered central species.
A preliminary analysis was performed to evaluate the relationships among host length, weight and sex. The analysis was based on multiple linear regression models using weight as dependent, and length and sex as independent variables; two models were constructed: one with interaction and one without interaction between the independent variables. The best-fit model was chosen based on the Akaike information criterion (AIC) using the stepwise bidirectional elimination method (Harrell 2001;Mark and Goldberg 2001). Linear regression models were also used to verify possible relationship between Kn (dependent) and parasite abundance (independent). Adjustment of the linear models to the observed data was adequate, as the residuals were normally distributed (Shapiro-Wilk test) and showed homoscedasticity when pertinent (Bartlett test) (Zar 2010).
Since linear regression models were not well adjusted when parasitological data was included, analyses with parasitological descriptors were performed using multiple generalised linear models (GLMs). The Poisson regression model was used when parasite abundance, species richness or HB were tested as dependent variables, as they are all quantitative (numeric) variables. A logistic model was used when prevalence was the dependent variable, as it is a categorical (qualitative) variable. Host length and sex were used in all models as independent variables; weight was not used since it was collinear with length (see results). Models were built with and without interaction among the independent variables and the best-fit chosen as previously described. Odds ratio (OR) and confidence interval (CI of 95%) for the GLMs were estimated in order to confirm the presence and evaluate the nature of interaction between dependent and independent variables, where 0 < OR < 1 indicates negative association, OR = 1 indicates lack of association and OR > 1 indicates positive association (in case of host sex, the category of reference was 'male'); if 1 was included in the CI, the model was considered not significant even if p < 0.05 (Dohoo et al. 2003).
All statistical analyses were performed in R software using RStudio interface (RStudio Team 2020; R Core Team 2021) with significance of p < 0.05.
A total of 10 different ecto and endoparasites (six species and four morphotypes) were recovered from G. balzanii, including adult and larval forms of Nematoda, Digenea, Monogenea and Copepoda. Details of parasite taxa, site of infection and parasitological descriptors can be found in Table 1. Details of the morphological diagnosis of the parasite taxa found here are presented in Supplementary Material 1. All hosts were parasitised by only one taxon of parasite, except by one fish that harboured two; the overall mean abundance was 25.61 ± 29.91 (1-91).
The nematode Sprentascaris andersoni was by far the most abundant, dominant and prevalent, followed by an unidentified species of dactylogyrid monogenean; both taxa were allocated central species community status (Table 1). Additional central species status was attributed to one morphotype (M2) of Neascus and one morphotype (M1) of Dipolstomum metacercariae; these parasites showed intermediate prevalence values within the community (Table 1). Although a different morphotype (M1) of Neascus metacercaria and the copepod Acusicola sp. showed higher prevalence than Neascus M2 and Diplostomum M1, their mean abundances were low, and consequently they were considered satellite species (Table 1). Other taxa considered satellites in the community were the metacercariae of Clinostomum sp. and Diplostomum M2. The only secondary and rare species were the nematode larvae of Brevimulticaecum sp. and Contracaecum sp., respectively. All parasites were highly aggregated within the host population (DI > 0.8), S. andersoni was the least aggregated but still with a high value of discrepancy index (0.65, see Table 1 for details). The mean diversity, species evenness and richness of the parasite community were 0.22 ± 0.36 (HB), 0.24 ± 0.35 (J') and 1.94 ± 1.34, respectively; D dominance index was 0.86 ± 0.24.
Due to low sampling, Contracaecum sp. and Diplostomum M2 were excluded from the following statistical analyses (both prevalence and abundance were rather low and they were present only in male hosts), except analyses including diversity, richness, evenness and dominance.
The multiple GLMs performed to correlate the prevalence and abundance of parasites with host sex (with males as reference category) and length indicated positive and strong interaction among sex and length, as noted in the linear regression run for host data. These variables interacting and alone indicated positive correlation with both prevalence and abundance of S. andersoni, Brevimulticaecum sp. and Neascus M2, as well as with the prevalence of Acusicola sp., in which males showed higher parasitological descriptors than females. Conversely, the abundance of Diplostomum M1 was negatively correlated with host length and sex (reference category males), in which females harboured more parasite individuals than males. These results are detailed in Table 2.
Parasite diversity was also higher in male hosts (HB = 0.45 ± 0.48) than in females (HB = 0.09 ± 0.19) and positively correlated with fish body length and weight, with or without interaction among sex (reference category males), length and weight (p < 0.04; OR = 1.31; CI = 1.22-1.38). The same results were observed regarding the parasite richness, which was 2.81 ± 1.67 in males and 1.45 ± 0.83 in females and positively correlated with length and weight (p = 0.01; OR = 1.94; CI = 1.17-3.22). However, no host variable was correlated with species evenness J' and dominance D (p > 0.3).
Fish Kn was tested against the overall parasite abundance and that of each taxon separately, using linear regression models; it showed relatively weak and positive correlation with the abundance of S. andersoni (p = 0.005; r 2 = 0.20) and that of Acusicola sp. (p = 0.032; r 2 = 0.11). The Kn was also tested against HB, J', D and richness, but their models were not significant (p > 0.57).  Abbreviations: CI, confidence interval of 95% for odds ratio; NA, not applicable; OR, odds ratio; p, probability value of the inferential statistics.

Discussion
Based on the present findings, the genera of nematodes Brevimulticaecum and Contracaecum, digeneans Diplostomum and Neascus, the crustacean Acusicola and the Monogenea represent new host records for the fish G. balzanii. In addition to the fact that there are only two parasitological studies on this host (Malta et al. 2018;Montes et al. 2020), the present results support the suggestion that G. balzanii has a poorly known parasite fauna and, at the same time, represent an important step towards a better comprehension of these organisms.
Although the community of parasites in G. balzanni was numerically dominated by adult individuals of the endoparasite S. andersoni and of the ectoparasite dactylogirid monogenean, larval forms of several different taxa were also represented. In fact, of the 10 different parasites found in the present study, 70% (7/10) were in larval stages. For example, the nematode Brevimulticaecum, which is found as adults mainly in aquatic carnivorous reptiles (e.g. crocodilians) and as infective larvae (L3) mainly in amphibians (Anderson 2000), attained the status of secondary species in the parasite community of G. balzanii, indicating that this fish may be an important intermediate host for Brevimulticaecum within Pantanal wetlands environmental patches (lakes). The same is noted for Diplostomum and Neascus metacercariae. Moreover, the representativeness of infective larval forms in the parasite community indicates that G. balzanii represents an important link for infection transmission within the local trophic chain and occupies a lower position in the food web. Similar results have been documented in other cichlid species from Brazil (Tavares-Dias et al. 2014, 2017a. Environmental features commonly influence parasite community assemblages; in aquatic habitats, this influence tends to be more intense, especially on ectoparasites that are in direct contact with the external environment (Rohde et al. 1995;Marcogliese et al. 2006;Chapman et al. 2015). Not only are micro-scale physicochemical variables important in these processes, but so are large-scale features such as size and limitation of the environment (Tinsley 2002). The present study was conducted in a small lake that, due to its spatial limitation, may favour both intermediate and definitive host crowding and, consequently, the spread of parasite infestations as well as of digenean free-swimming stages followed by active transmission (i.e. miracidium and cercaria). This may explain the highest prevalence and abundance, and the central status showed by monogeneans and the dominant metacercariae (i.e. Diplostomum M1 and Neascus M2). It should be mentioned that, despite the low abundance of Acusicola sp., Neascus M1 and Clinostomum sp., their prevalences were consistent within the community.
On the other hand, the uneven dominance of the nematode endoparasite S. andersoni may be explained not only by the space-limited environment favouring the crowding of its possible intermediate/paratenic hosts, but by the fish diet. The transmission of species of Raphidascaris is not fully elucidated, but it firstly involves an aquatic invertebrate, e.g. planktonic copepods and chironomid larvae (Anderson 2000), which are important diet components of Gymnogeophagus spp. (Yafe et al. 2002;Moreno et al. 2017).
Since the early 1970s, host features have been widely documented as important driving forces in the structure of parasite communities (see Crofton 1971;Price 1990;Timi and Poulin 2003;Santoro et al. 2020). Regarding fish hosts, these features are mainly related to diet and body size, which can differ according to ontogeny and/or sex (González et al. 2001;Timi and Poulin 2003;Tadiri et al. 2016;Santoro et al. 2020). The present results showed a strong influence of host sex on the parasite community structure, which may be related to the fact that male individuals are often larger and heavier. These individuals mostly harboured parasites in higher prevalence and abundance. Larger (and heavier) hosts have more energetic requirements, consequently ingesting intermediate/paratenic hosts with higher frequency, and using larger foraging areas, thus facilitating encounters with parasites; somewhat analogous to the theory of island biogeography (MacArthur and Wilson 2001). Moreover, larger individuals have more physical space for parasites to occupy (Poulin and George-Nascimento 2007;Amarante et al. 2016, but see Morand and Poulin 1998) and are often older, and older hosts may have more parasites (Zelmer and Arai 1998). The present results disagree with previous studies showing a lack of correlation among parasite descriptors and size, weight and sex of cichlid host species from the Brazilian Amazon (Tavares-Dias and Neves 2017;Tavares-Dias et al. 2017a, 2017bBorges et al. 2019) and Atlantic Forest (Paraguassú et al. 2005). Such obervations indicates that general laws in parasite ecology cannot yet be formulated, since deep analyses using meta-databases are complicated to perform (see Poulin 2007), and the current knowledge on parasites of Neotropical fish remains proportionally incipient in relation to their high biodiversity potential.
Similarly, diversity and species richness were positively correlated with host size and weight, and were consequently higher in male individuals. However, the mean values of these descriptors can be considered low and seem to reflect a pattern suggested by other studies on parasite community ecology of cichlid fish in South America (Paraguassú et al. 2005;Oliveira et al. 2017;Tavares-Dias and Neves 2017;Tavares-Dias et al. 2017a, 2017bBorges et al. 2019). Species evenness was also low, indicating marked inequality in parasite abundance among taxa (see Table 1), which contributes to a high dominancy by one or few groups, as observed for S. andersoni that was highly dominant over the other parasites. These results corroborate the high mean value of the dominance index (D).
The parasite community of G. balzanii was characterised by low richness and diversity, with few taxa showing high prevalence and the parasite abundances generally low, similar to what has been found for other cichlids from Brazil (Paraguassú et al. 2005;Oliveira et al. 2017;Tavares-Dias and Neves 2017;Tavares-Dias et al. 2017a, 2017bBorges et al. 2019). These characteristics, along with high degree of aggregation within the host population, as also demonstrated here, are typical of isolationist (or non-interactive) communities (see Holmes 1987;Esch et al. 1990). Therefore, patterns of dominance and status observed in the present study are probably related to host specificity (as may be the case for monogeneans and S. andersoni) as well as particular features related to the environment and the fish (as may be the case for metacercariae), rather than direct interference (interaction) among parasites.
All individuals were parasitised by at least one parasite taxon and their Kn (1.1 ± 0.43) was not different from the standard value (1.00), indicating a good body condition. In fact, Kn was positively correlated with the abundance of S. andersoni and with that of Acusicola sp. However, according to the regression coefficients the correlation was weak, suggesting the influence of other factors (probably host length, weight and sex) on Kn values. Based on these results, it is possible to assume that most components of the present parasite community cause no significant loss of body condition to the host. However, it should be highlighted that the abundance of Diplostomum M1 metacercaria was negatively associated with host length and weight, as well as higher in females. This may represent a strategy of the parasite to reduce the intermediate host health, making it more susceptible to predation by the definitive host. Such a strategy is well known in life cycles of digeneans (Poulin 1995(Poulin , 2010Marcogliese 2004) and may be the case for Diplostomum M1, since these metacercariae were found infecting fish brains.
The presence of Clinostomum metacercariae found in G. balzanii is of particular interest regarding public health issues, since parasites of this genus have been reported as fishborne zoonotic agents (Kitagawa et al. 2003;Hara et al. 2014;Lee et al. 2017) and, even though G. balzanii is not usually consumed by humans, it indicates that the infective forms are present in the environment and may be present in other important fish for human consumption. However, it might also be a Clinostomum species that cannot infect humans.
The present results shed light on the complex interactions among G. balzanii, its parasite fauna and the environment, representing the first step towards a clearer comprehension of the patterns and processes influencing both parasite community and host population. Moreover, it provides better knowledge of the G. balzanii parasite fauna.

Disclosure statement
No potential conflict of interest was reported by the authors.