Occurrence of concavities on the lorica of two species of Testudinella ( Rotifera , Monogononta , Testudinellidae )

Testudinella is a taxon of Rotifera broadly distributed in Brazil. In a recent collection in marginal lakes connected to the Paranapanema River (São Paulo, Brazil), we found specimens of Testudinella mucronata and T. patina with concavities on their lorica. The objective of this study was to describe the concavities observed in individuals of both species and discuss their possible causes. Plankton samples were collected at two sampling stations, in the pelagic (PZ) and littoral (LZ) zones of the Panema and Coqueiral lakes; both of these lakes are ultra-oligotrophic and not polluted. For T. mucronata, 4% of individuals with concavities were found in the PZ of Coqueiral lake and 50% in the LZ of Panema lake. In T. patina, the proportion of specimens with concavities corresponded to 34% in the LZ of Panema lake and 17% in PZ of Coqueiral lake. In this study, we discussed that low water temperatures, predation events and/or recent hatching are factors that may justify the concavities in the lorica of specimens of our work.

Many monogonont rotifers are well known to respond to environmental factors with morphological variations in their body.This phenomenon is known as polymorphism and is usually caused by kairomones from predators, variations in water temperature and food availability (see review of Gilbert 2017;2018).In general, polymorphism in rotifers has great relevance because morphological variations can influence life history, population dynamics and ecological interactions (Gilbert, 2017).Moreover, laboratory bioassays demonstrated that environmental pollution of metals can change the size and morphology of rotifers (Ríos-Arana et al. 2007, Xue et al. 2017).
During a study conducted in two marginal lakes to the Paranapanema River (São Paulo State, Brazil), we collected specimens of T. patina and T. mucronata with the presence of concavities on the lorica.In this work, we describe the cases and discuss possible causes of the presence of these concavities.

Data collection and analysis
Samples were collected in April 2017 (rainy season), from two sampling stations, in the pelagic (PZ) and littoral zones (LZ) of each of the studied lakes.The water surface temperature was measured with a Toho Dentam thermometer, pH with a Micronal B-380 pH meter and the electrical conductivity with a Hach Mod.2511 conductivimeter.The dissolved oxygen concentration of the water was determined by the method of Winkler, modified by Golterman et al. (1978).The total phosphorus and nitrogen concentrations were determined according to Strickland & Parsons (1960) and Mackereth et al. (1978), respectively.
The chlorophyll-a concentration was determined using the 90% acetone extraction method (Golterman et al. 1978).The trophic state index was calculated to classify the trophic level in both zones of the lakes (Cunha et al. 2013).
Rotifers were collected with a plankton net (50 μm mesh size), by means of a vertical trawl in the water column.The filtered volume (V) was calculated by the formula V = π.r 2 .d,where r is radius of plankton net opening and d is the distance traveled by the net.Water sampling was conducted in order to result a filtered final volume of 100 L. Subsequently, the organisms were fixed with a 4% formaldehyde solution saturated with sugar.The counting of sampled individuals was carried out in a minimum of four subsamples of 1 mL, in a Sedgewick-Rafter chamber under an optical microscope; abundance was expressed in terms of individuals.m - .The organisms were identified based on specialized literature (Koste 1978, Sharma 1990, Shiel 1995).
In order to estimate the frequency of individuals with concavities, two categories were established based on the external morphology for Testudinella mucronata (individuals were sorted based on the presence and absence of concavities; see Figure 2).Three categories were designated for T. patina (absence of concavities; Type 1 -presence of a concavity on the lorica; Type 2 -more than two concavities on the lorica; Figure 3).This nomenclature (i.e., Type 1 and Type 2) was used for Cladocera (see Melo et al. 2017) and adapted herein to apply to Rotifera as well.
A student's t-test was used to verify significant differences between limnological variables and zones of each lake.Normality assumptions were performed with the Shapiro-Wilk test.We used the level of statistical significance of 95% (p<0.05).Statistical analyses were performed in R software (R Development Core Team, 2016).

Results
In general, high mean values of water temperature and electric conductivity were recorded in Panema lake, while low dissolved oxygen concentrations were found in Coqueiral lake (Table 1).In both lakes, the pH was neutral.Total phosphorus and nitrogen concentrations were low in both lakes.In relation to the limnological variables between the compartments of the lakes (pelagic and littoral zone), there was no significant difference between both lakes (p>0.05).The trophic status indexes showed that the lakes are composed of ultra-oligotrophic waters, in both the pelagic and littoral areas (Table 1).
Concavities were observed in individuals of both species of rotifers.In the case of Testudinella mucronata, several concavities were observed on the lorica of the individuals (Figure 2).In both lakes, the densities of affected organisms reached 59 ind.m -3 in littoral zone of the Panema lake and 33 ind.m -3 (pelagic zone) in Coqueiral lake (Figure 4).For this species, 4% of individuals with concavities were found in the PZ of Coqueiral lake and 50% in the LZ of Panema lake.In T. patina, the concavities were classified as Type 1 and Type 2 (Figure 3).The total density of specimens with Type 1 and Type 2 concavities for this taxon were 24 ind.m -3 in Coqueiral lake and 118 ind.m -3 in Panema lake (Figure 5).The total proportion of organisms with both types of concavities observed in T. patina corresponded to 34% in LZ of Panema lake and 17% in the PZ of Coqueiral lake.Chlorophyll-a (µg.L -1 ) 0.69 ± 0.17 0.21 ± 0.08 0.19 ± 0.01 0.10 ± 0.03

Discussion
Morphological alterations in zooplankton may be caused by human impacts on aquatic environments (e.g., Montú and Gloeden 1982, Dias et al. 1999, Souza et al. 2011, Melo et al. 2017).In Rotifera, for instance, Zurek (2006) recorded deformations on the spines of Keratella cochlearis (Gosse, 1851) caused by the exposure to sulfides or derivatives present in the water from a sulfur mine impoundment.Ecotoxicological tests have also shown that heavy metals and pesticides induce morphological changes in the rotifer Plationus patulus (Müller, 1786) (Ríos-Arana et al. 2007) and Brachionus calyciflorus Pallas 1766 (Alvarado-Flores et al. 2015).In our study, concentrations of phosphorus and total nitrogen in lakes were around 7 µg.L -1 and 355 µg.L -1 , respectively.When comparing these values with the reference conditions for nitrogen (300-350 µg.L -1 ) and total phosphorus (10-15 µg.L -1 ) established in subtropical reservoirs with different levels of enrichment (Cunha et al. 2012;Cunha & Calijuri 2011), we concluded that the lakes studied here are relatively nutrient depleted.In addition, the trophic Biota Neotrop., 19(2): e20180633, 2019 http://dx.doi.org/10.1590/1676-0611-BN-2018-0633http://www.scielo.br/bnstatus index showed that the lakes were classified in ultra-oligotrophic category, suggesting that the trophic state of the environments is not related to the presence of concavities in Testudinella.While we did not performed metal analyses, we believe that metals are not the cause of the concavities.Both lakes, despite their riverine connections, are not near any pollution sources and their environments are ultra-oligotrophic.
On the other hand, there are reports of polymorphism in some Rotifera species of the Brachionidae family (Stemberger & Gilbert 1984, see Gilbert 1999).This phenomenon can be induced by several environmental factors, including temperature variations (e.g., Gilbert 2018, Ge et al 2018).For example, some studies report that the temperature is responsible for lengthening spines of lorica in B. falcatus (Zacharias, 1898) and K. cochlearis (Bielañska-Grajner 1995;Ahad & Rao 2017).At low temperatures, the population growth rate of K. cochlearis decreases, resulting in the occurrence of organisms with longer caudal spines (Lindström & Pejler 1975).In the present study, there was no significant difference in limnological variables between the pelagic and littoral zones of the lakes.However, our study didn't include the temporal scale.The occurrence of concavities may also be due to the colder riverine influx, since both lakes are connected to the river channel.Studies carried out in lacustrine environments in the same region related the influence of the lateral influx of water from the river on the limnological variables and planktonic community (Casanova et al. 2009;Henry et al. 2011).
Lastly, concavities may be related to the stage of development.De Smet (2009) suggests that the presence of concavities (called by this author as "flattening") on the lorica of T. elliptica (Ehrenberg, 1834) is related to the development of the vitellarium.It is likely that the same occurrence is applicable to the organisms of both species of Testudinella.We do not discard the possibility that individuals in this study may be newly hatched.
In summary, here we described the presence of concavities in T. mucronata and T. patina, suggesting three possible causes for their formation: (i) temperature; (ii) predation; (iii) or recent hatching.However, additional studies are needed to investigate which of these possibilities are responsible for presence of concavities in our exemplars.

Figure 1 .
Figure 1.Localization of the sampling stations (red circle) in two marginal lakes on the Paranapanema River, São Paulo, Brazil.

Figure 2 .
Figure 2. Testudinella mucronata with absence (a) and presence of concavities (b).The arrows indicate the locations of the concavities.

Figure 3 .
Figure 3. Testudinella patina with absence (a) presence of Type 1 (b) and Type 2 concavities (c).The arrows indicate the locations of the concavities.

Figure 4 .
Figure 4. Density of individuals (ind.m -3 ) with absence and presence of concavities in Testudinella mucronata collected in two marginal lakes (São Paulo State, Brazil).PZ = pelagic zone and LZ = littoral zone.

Figure 5 .
Figure 5. Density of individuals (ind.m -3 ) with absence and presence of concavities of Type 1 and Type 2 in Testudinella patina collected in two marginal lakes (São Paulo State, Brazil).PZ = pelagic zone and LZ = littoral zone.