Effect of Temperature on Allelopathic Interactions Between Copepods (Copepoda) and Rotifers (Rotifera)

Abstract

The role of abiotic factors in the allelopathic interactions between copepods and rotifers is poorly documented. Temperature has a marked effect on the metabolism of zooplankton. Therefore, the release of allelochemicals by copepods and the response of rotifers to them may change as temperatures increase. Here, we tested the effect of two temperatures (20 and 25 °C) on the population growth of Brachionus havanaensis cultured on a conditioned medium (CM) of Arctodiaptomus dorsalis and Eucyclops sp. The CM was obtained daily, separately, for the males and females of both copepod species at a density of 0.1 ind. mL⁻¹ for 24 h prior to experiments. In the controls and CM treatments, higher temperatures had a stimulatory effect on the population growth and the rate of population increase (r) of B. havanaensis. At 25 °C, the CM from the females and males of A. dorsalis caused >30% increase in r, but for the CM from Eucyclops sp., this effect was lower (<21%). At 20 °C, the r in the controls and CM treatments was not significantly different. The morphometry of B. havanaensis varied depending on the treatments. Compared to controls, longer loricae were recorded in the CM of male A. dorsalis at both temperatures. However, in the CM of female calanoids, longer rotifers were observed only at higher temperatures. At 20 °C, the CM from Eucyclops produced smaller loricae. The relationship between the lorica length and width of rotifers showed a linear relation but the slope differed among the treatments.

1. Introduction

In waterbodies with zooplanktivorous fish, the zooplankton community is mainly composed of copepods and rotifers [1]. Copepods, through chemical communication, interact among themselves and with other zooplankton species, especially rotifers [2]. Cyclopoid adults feed on rotifers, while the naupliar stages of both calanoids and cyclopoids adversely affect planktonic rotifers via exploitative competition [3]. Chemically mediated interactions in freshwater ecosystems may benefit rotifers as they regulate their reproductive rates depending on the predator type [4]. Species of Brachionus such as B. calyciflorus and B. bidentatus minimize or even escape invertebrate predators such as Asplanchna spp. by developing defensive spines, induced by predator-released kairomones [5,6]. Copepods are known to use infochemicals throughout their life cycle for reproduction, mate recognition [7], food discrimination [8] and allelopathic interaction with other zooplankton species [9].

While experimental evidence of chemically mediated effects within zooplankton groups is increasing, the role of some common abiotic factors like temperature needs to be considered in laboratory tests for a better understanding of chemical communication in aquatic ecosystems. Changes in temperatures have an impact on the metabolic and population growth rates in zooplankton [10,11]. Further, due to global warming, the interactions between zooplankton in aquatic ecosystems might change their community composition [12,13]. For example, in corals, species recruitment can be determined through allelopathic effects, and in combination with temperature, there is a change in the survival and resilience of the organisms [14].

The impact of the interaction of fish kairomones and temperature on cladocerans has been documented [15]. For example, higher temperatures increase the percentage of long-spined phenotypes in daphniids and reduce ephippia production [16,17]. While many works on rotifers have focused on predators such as Asplanchna and prey species such as brachionids and lecanids, the interactions between allelochemicals and abiotic factors such as temperature are few. For example, some data are available on the impact of temperature on the morphological changes in Keratella tropica in the presence of Daphnia and Asplanchna [18]. These results showed that temperature had an additive effect on the response of K. tropica to the presence of Daphnia and Asplanchna. While the population growth rate does increase with temperature alone, allelochemicals modulate them by inducing morphological changes, as in the case of K. tropica. Predators such as Asplanchna can also drastically change the morphology of their prey, namely Brachionus and Keratella [18]. The induction of spines in rotifers by invertebrate predators such as copepods can be detected from field-collected samples or laboratory experiments [19]. Laboratory evaluations permit the predator-dependent quantification of prey responses. For example, it has been shown that calanoids induce changes in the survivorship and reproductive variables of rotifers, but not cyclopoids [9]. The morphology of field-captured prey rotifers cannot be definitively attributed to predators because of other overriding factors such as temperature and competition, and hence their response is collectively treated as cyclomorphosis [16].

Most predatory zooplankton species continuously release kairomones and other allelochemicals into the medium. This predator-released conditioned medium (CM) is known to cause changes in the morphology, body size and life history variables of different rotifer genera, including Brachionus, Keratella, Lecane and Plationus [18,19,20,21]. At a given temperature, infochemicals produced by copepod species may have a mild or no effect on the interacting species [9,21,22]. The aim of this work was, therefore, to quantify the effect of temperature on the allelopathic and morphometric responses of the rotifer Brachionus havanaensis to the conditioned medium from a calanoid (Arctodiaptomus dorsalis) and a cyclopoid (Eucyclops sp.) copepod.

2. Materials and Methods

All zooplankton species were obtained from Lake Xochimilco (Mexico City, Mexico) and cultured on synthetic, moderately hardwater medium at 23 ± 1 °C (after EPA medium). The EPA medium was prepared by dissolving 96 mg of NaHCO₃, 60 mg of CaSO₄, 60 mg of CaSO₄ and 4 mg of KCl in 1 L of distilled water [23]. The herbivores Brachionus havanaensis, Arctodiaptomus dorsalis and nauplii of both A. dorsalis and Eucyclops sp. were fed daily on the single-celled green alga Chlorella vulgaris (hereafter as Chlorella) exclusively. The microalga was mass cultured at 18 °C on defined medium (Bold’s basal medium [24]). The batch-cultured alga in the log phase was routinely harvested, centrifuged and resuspended in a small volume of distilled water. Freshly harvested Chlorella was quantified and later used as food for zooplankton to ensure consistent nutritional quality. For feeding the predatory cyclopoid Eucyclops sp. adults, we cultured another brachionid rotifer, Brachionus calyciflorus. Eucyclops sp. was fed daily on a mixture of B. calyciflorus at a density of 1 ind. mL⁻¹ and Chlorella at 1 × 10⁶ cells mL⁻¹. All zooplankton cultures and experimental jars were maintained in a 12:12 h L:D photoperiod. The pH of the medium did not vary considerably (7.0–7.4) within 24 h. The culture medium of zooplankton was 100% replaced every 2 days in mass cultures but daily in test jars.

2.1. Population Growth Study

The population growth experiments of B. havanaensis were conducted at two temperatures (20 and 25 °C). The copepod conditioned medium was obtained separately for the males and non-gravid females of each copepod species (A. dorsalis and Eucyclops sp.). To prepare the CM, we maintained copepods for 24 h at a density of 0.1 ind. mL⁻¹ in EPA medium with Chlorella at a low concentration (0.1 × 10⁶ cells mL⁻¹), just to keep the copepods active. After this period, the copepods were separated with a 60 µm mesh and transferred to fresh medium and algae. The resultant medium was filtered through a 0.45 µm Millipore filter to exclude bacteria and algal fragments [25].

For population growth experiments on B. havanaensis, we added 20 rotifers (≥24 h old), randomly, to each of the 36 (2 temperatures × 2 copepod species × 2 sexes × 4 replicates plus 4 controls) test jars containing 20 mL of CM or only EPA medium for the controls. All the test jars received Chlorella at a concentration of 1 × 10⁶ cells mL⁻¹. Following the initiation of population growth experiments, we estimated the density of rotifers in each test jar daily. Later, the living rotifers were transferred to new test jars containing the appropriate CM with food. The experiments were discontinued when the rotifer densities in all test jars showed a declining trend.

2.2. Morphometric Data

During the population growth study of B. havanaensis, we also measured the body sizes of samples of rotifers from the early exponential growth phase (day 12, for all treatments). For body size measurements, we only used adult rotifers. For this, four ovigerous females were randomly collected per replicate from each treatment. They were then fixed in 4% formalin for morphometric analysis. Rotifers were individually photomicrographed using a Nikon Eclipse Ni microscope (Tokyo, Japan), equipped with a MOTIC S6 camera. Image acquisition and subsequent measurements were performed using the MOTIC Image Plus 3.0 software. The following morphometric variables were measured: total body length, body width and posterior spine length (short and long spines) (Figure 1). We also measured the anterior spine lengths, but these were excluded from analysis due to the lack of a significant association with body length.

From the population growth data, we derived the rate of population increase, r d⁻¹, by log natural transformation [26,27]. To assess if there were significant differences in the peak population abundances and the rate of population increase associated with the treatments, we used a two-way ANOVA and a post-hoc Tukey Test. For morphometric variables, we derived standard regression equations (SigmaPlot 11, Palo Alto, CA, USA).

3. Results

The population growth of Brachionus havanaensis in the controls at 20 °C had a long lag phase of about 6 days before reaching the exponential phase of about 10 days and later entered the retardation phase. On the other hand, at 25 °C, the population had a shorter lag phase of 4 days but had a long exponential phase of about two weeks (Figure 2). The day of peak population densities and maximal population abundances of B. havanaensis were significantly lower at 20 °C compared to those at 25 °C (p < 0.001;

Table 1. Peak density and population growth rates of Brachionus havanaensis in the controls and grown on the conditioned medium (CM) of females (F) and males (M) of Arctodiaptomus dorsalis (Ad) and Eucyclops sp. (Eu) at 20 and 25 °C.

Treatments	Peak Density (ind. mL−1)		Growth Rate (r) Day−1
	20 °C	25 °C	20 °C	25 °C
Control	70 ± 0.74 a	100 ± 2.21 c	0.268 ± 0.006 a	0.289 ± 0.002 c
Ad F	78 ± 0.68 a	114 ± 2.4 d	0.259 ± 0.001 a	0.343 ± 0.004 d
Ad M	70 ± 2.49 a	94 ± 6 c	0.240 ± 0.005 a,b	0.324 ± 0.004 d
Eu F	68 ± 2.1 a,b	100 ± 2.3 c	0.287 ± 0.012 a	0.321 ± 0.006 d
Eu M	61 ± 1.34 a	122 ± 5.84 d	0.282 ± 0.003 a	0.342 ± 0.0052 d

The data shown are the mean and standard errors based on four replicates. For a given variable in the controls and copepod CM at both temperatures, superscripts with the same letters are not statistically significant (p > 0.05).

).

Regardless of the origin of the conditioned medium (copepod species or male and female), the growth curves of B. havanaensis were similar at 20 °C, with a longer initial lag phase and a short exponential phase, compared to those at 25 °C. In terms of the peak population abundances at 20 °C, there were no significant differences among the controls and treatments with CM from the males and females of A. dorsalis or that of Eucyclops sp. However, at 25 °C, the peak densities from the CM of female A. dorsalis and males of Eucyclops sp. had a significant effect, in comparison to the controls (p < 0.05; Table 1).

Regardless of treatments, the rate of population increase, r, for rotifers was significantly higher at 25 °C compared to that at 20 °C. At the higher temperature, the r in CM treatments was significantly higher than the controls (p < 0.05; Table 1). At 25 °C, CM from the females and males of A. dorsalis caused a 32 and 35% increase in r. On the other hand, at this temperature, CM from the females and males of Eucyclops sp. resulted in a 12 and 21% increase in r, respectively. At 20 °C, the r in the controls and CM treatments was not significantly different (p > 0.05).

Data on the morphometry of B. havanaensis showed changes in the total lorica length and width in relation to the copepod conditioned medium. Compared to the controls, significantly longer loricae were recorded in treatments with CM from male A. dorsalis at both 20 and 25 °C (p < 0.05; Figure 3). However, in the CM of female calanoids, significantly larger rotifers were observed only at 25 °C. At 20 °C, the CM from Eucyclops resulted in smaller loricae.

The relationship between the lorica length and width was linear (Figure 4). However, the slope differed depending on the treatment. In controls, the mean body length and width (mean ± SE, in µm) of B. havanaensis were 269 ± 3 and 103 ± 1, respectively. Longer individuals (293 ± 3) were observed in treatments with the CM of A. dorsalis, compared to the controls or with CM from Eucyclops.

4. Discussion

It has been suggested that, in some cases, the production of infochemicals increases with temperature, but there may not be a concomitant increase in the response of the organisms [28]. For example, in the presence of fish kairomones at low temperatures, Daphnia produces more diapause eggs than at higher temperatures, where an increase in growth rates is a better strategy to escape predation [17]. This might change according to the response measured or the origin of the infochemical, as in the case of Chaoborus, where an increase in temperature enhances the production of more spined phenotypes of Daphnia [16]. While the response of rotifers to copepods seems to be mild in comparison with other predators such as Ambystoma [29], some authors suggest that these mild to null responses in the rotifers to copepods are related to the body size of predators [22].

In this study, we observed that at a higher temperature, Brachionus havanaensis saw an increase in its population growth rate with the CM from Arctodiaptomus dorsalis and Eucyclops sp., in comparison with a lower temperature where the effect was non-significant. For species of zooplankton, it is known that an increase in temperature causes an increase in r [30,31]. However, the CM from both the sexes of Eucyclops sp. and from males of A. dorsalis enhanced the population growth rate, which suggests that there are species-level differences in the allelochemicals from copepods on rotifers. Further, temperature impacts these interactions. For example, kairomones change the antipredation strategies of rotifers and cladocerans as the temperature rises [18,32].

Brachionus havanaensis is consumed by predatory copepods, including small taxa such as Eucyclops [3]. B. havanaensis can modify its responses, depending on the predator, by changing its morphology or the demographic characteristics; in the indirect presence of Mesocyclops pehpeiensis, the growth rate of B. havanaensis was greater than the controls [21,29]. We also observed in our study that in the CM treatments of male and female A. dorsalis or Eucyclops, the growth rates of B. havanaensis were elevated. Many species of cyclopoids produce a mild to no effect on their prey species, and some authors suggest low responses to chemical cues happen when the predator is not voracious [21,22]. This corresponds to the results observed in this work, where the increase in r was about 12–35%. Further, it has been shown that changes in the morphology of prey due to kairomones from voracious predators, especially from the family Asplanchnidae (Rotifera), are associated with a reduced net reproductive rate and generation time, and an increase in prey’s rate of population and their morphometry [19]. The impact of these copepod species on B. havanaensis needs to be studied further.

We found that the CM of Eucyclops sp. males provoked higher growth rates at both temperatures compared to the CM from the corresponding females. In many cyclopoids, females are more voracious than males [33], but the effects of CM obtained separately from males and females on the population growth rates of prey is not definitively established [34]. We did not measure the quantity of CM from the males and females of either species of copepod [33,35,36]. Previously, a life table demography study reported that the CM of A. dorsalis had a negative effect on the survival and reproduction of rotifers [9]. While we found an increase in the population growth of B. havanaensis with both male and female-conditioned medium in this study, this might indicate that even if there is a negative effect on r in the first generation, the next generation might have another tradeoff. Compared to cohort life table studies, in population growth experiments, several generations are simultaneously present; therefore, individuals of different generations may have different tradeoffs [34]. This requires further studies.

The results show that temperature had a great effect on B. havanaensis by increasing the population growth rates. While the allelopathic response of rotifers to the conditioned medium also increased with temperature, the effects continued to be mild on r, which is consistent with the literature. In the case of A. dorsalis, the sex-dependent effect was higher at higher temperatures (32–35% on r), while for cyclopoids this effect was lower (12–21%) at comparable conditions. It is not known if these differences explain the impact of calanoids and cyclopoids on the population abundances of brachionid rotifers. Field studies are needed to assess the impact of cyclopoids or calanoids on the community structure of rotifers.

Morphometric variations in prey brachionid rotifers cultured on predators’ conditioned medium have been reported in the literature. For example, a meta-analysis of field data on the relation between spine induction in loricate rotifers and invertebrate predators showed that prey species respond differently depending on the predator type [33]. Our data further showed that this response is also temperature-dependent. For example, when cultured on CM from calanoids, larger individuals of B. havanaensis were obtained at both 20 and 25 °C. However, when cultured on cyclopoid conditioned medium, the lorica length of the brachionids was significantly smaller than that cultured at 25 °C under a similar treatment. The fact that significantly larger individuals of B. havanaensis were obtained in CM from A. dorsalis compared to Eucyclops also suggests that this prey rotifer can adjust its responses in relation to the predator type, as shown previously in field data [33] and experiments [29].

The relationship between the length–width of the lorica in rotifers is dependent on various factors, including the presence or absence of predators and diet quality [34]. Although the lorica width is directly related to body length, this relation is not always proportional, even under stress-free culture conditions [33]. This is because depending on the stage of population growth, lorica length–width relations vary, as shown in several strains of Brachionus plicatilis. Relating body size changes to population abundances also requires careful consideration of different factors, such as intra-specific competition and food limitations [33]. In this work, it is further influenced by the predator’s allelochemicals. Therefore, the interpretation of brachionid prey growth rates and morphometry involves the allelopathic role of predators conditioned medium.

5. Conclusions

Our data showed that temperature had a significant effect on B. havanaensis by increasing the population growth rates. While the allelopathic response of rotifers also increased with temperature, the effect on the population growth rates continued to be mild. For the calanoid species used in this study, the sex-dependent effect is clear, where this effect on the population growth rates was up to 35%. For cyclopoids, this effect on the population growth rates of B. havanaensis was about 40% lower. The lorica length of brachionids exposed to the conditioned medium of male A. dorsalis at both 20 and 25 °C was greater. The relationship between the lorica length and width showed a linear relation, but the slope differed depending on the treatment. These differences might explain the impact of cyclopoid or calanoid-dominated waterbodies on the population abundances of rotifers. However, field studies are still needed to assess the impact of copepods on the community structure of rotifers.