Monogenea: Diplozoidae) Exposed to Aqueous Aluminium Beric M

Gilbert and Avenant-Oldewage Parasites & Vectors (2016) 9:420 DOI 10.1186/s13071-016-1706-z RESEARCH Open Access Hatchability and survival of oncomiracidia of Paradiplozoon ichthyoxanthon (Monogenea: Diplozoidae) exposed to aqueous aluminium Beric M. Gilbert and Annemariè Avenant-Oldewage* Abstract Background: Monogenea is a diverse group of ectoparasites showing great potential as sentinel organisms for monitoring environmental health. Exposure to metals negatively affects infrapopulations of monogeneans and exposure to aluminium has been found to negatively impact the survival of gyrodactylids. Methods: Samples of infected host fish, the smallmouth yellowfish Labeobarbus aeneus (Cyprinidae), were collected from the Vaal Dam, South Africa and transported back to the laboratory in dark 160 l containers. Eggs of the monogenean Paradiplozoon ichthyoxanthon infecting L. aeneus were collected and exposed to varying concentrations of aluminium along with a control group in static tanks. The eggs were checked every 24 h and hatching commenced 13–14 days after exposure. Water samples were taken from exposure tanks and acidified for analysis of Al levels with inductively-coupled plasma mass spectrometry. Results: Hatching of eggs was variable between exposures, and in 30 μg Al/l and 60 μg Al/l was found to occur before eggs in control beakers, whereas, exposure to 120 μg Al/l delayed hatching and reduced hatchability. Survival of hatched oncomiracidia was concentration dependent and negatively correlated with aluminium concentrations. Lowest survival was recorded for 60 μg Al/l and 120 μg Al/l where all larvae died shortly after or during hatching. Normal development of embryos of P. ichthyoxanthon within eggs exposed to all doses of aluminium indicates that the egg shell is moderately impermeable to metals and inhibits movement of aluminium across the shell and interacting with developing embryos. Conclusions: Higher larval mortality rate in 120 μg/l exposure can be related to aluminium crossing the egg shell in the late stages and causing death of unhatched yet fully developed embryos, possibly due to changes in the permeability of the egg shell as embryos neared developmental completion. Accelerated death of oncomiracidia after hatching indicates sensitivity toward high concentrations of aluminium. Keywords: Heavy metals, Larval development, Metal accumulation, Toxicity, Pollution, Parasite indicators * Correspondence: [email protected] Department of Zoology, University of Johannesburg, Johannesburg, South Africa © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gilbert and Avenant-Oldewage Parasites & Vectors (2016) 9:420 Page 2 of 11 Background that aluminium exposure would have on the hatchability of The sensitivity of parasites in general toward metals is the eggs and survival of oncomiracidia of P. ichthyoxanthon. poorly documented, complicating the interpretation of field studies and obscuring the identification of sensitive Methods taxa which can serve as bioindicators for monitoring en- P. ichthyoxanthon egg collection vironmental degradation [1, 2]. Studies have suggested Labeobarbus aeneus infected with P. ichthyoxanthon that some parasites are useful biological indicators of en- were collected from the Vaal Dam using gill nets and vironmental health [3]. Most of these studies have fo- maintained on UJ Island (26°52′33.62″S, 28°10′25.76″E) cused on the use of parasites as accumulation indicators, in 160 l holding tanks containing aerated borehole water. especially endoparasites [4–6]. Monogenean parasites From here the fish were anaesthetised with 100 ml 2- have been shown to be sensitive toward metal pollution Phenoxy Ethanol (Sigma-Aldrich, Steinheim, Germany) [7] and changes in infection biology of this group have in 160 l water and transported back to the Parasitology been identified for a number of different species [8–10]. Laboratory at the University of Johannesburg. In the la- In terms of pollution indication, all investigations thus boratory fish were kept in the dark and eggs were col- far have focused on infection level alterations with few lected daily when water changes were done with reverse studies documenting accumulation patterns in this osmosis (RO) water. According to MacDonald & Jones group. Exposure to pollution by metals [11, 12], acidifi- [24] laying of eggs by diplozoids increases during periods cation [7, 13] and pulp and paper mill effluent [14, 15] of darkness. Parasite eggs were collected by filtering have been found to negatively affect prevalence, mean water through a Visser Sieve apparatus [25] (25 μm intensity and abundance of monogeneans, while the mesh). Filtrate was collected from the funnels in 500 ml opposite has been found to occur in the presence of hy- beakers and then aliquots of filtrate were examine in a drocarbons and eutrophication [16, 17]. In terms of glass Petri dish with a Zeiss DV4 stereomicroscope. Eggs metal toxicity toward monogeneans, a number of studies of P. ichthyoxanthon were identified, isolated and col- have documented the potential use of aluminium as a lected with a triple zero Camel’s hair paintbrush and parasiticide for the treatment of adult gyrodactylid infec- placed into a clean Petri dish containing aerated RO tions in fishes under laboratory conditions [18–20]. water. Collected eggs were kept in fresh, aerated RO These studies have noted that exposure to elevated alu- water for 24 h before exposure experiments were minium concentrations is able to completely eliminate commenced. infections with little toxicity toward the hosts. However, no investigations have documented the toxicity of alu- Exposure bioassays minium or other heavy metals toward diplozoid mono- Static bioassays were performed using varying nominal geneans under laboratory conditions. concentrations of aluminium (0–120 μg/l). A stock solu- The present work forms part of an investigation on the tion of 10 mg Al/l was made up by weighing out 1.07 g responses of the diplozoid, Paradiplozoon ichthyoxanthon, aluminium nitrate (Sigma-Aldrich, St Louis, United toward environmental pollution and, therefore, its use as a States of America) and dissolving in 500 ml RO water. sentinel organism in the Vaal River system. An earlier field The 10 mg/l Al stock was aerated for 24 h before expos- investigation [10] had documented the local extinction of ure experimentation commenced to negate any possible this parasite at a site in the Vaal River, below the Vaal effects that Al may have on the dissolved oxygen levels River Barrage, where elevated levels of metals and poor of the RO water. Exposure media were made up by seri- water quality have been identified for a number of years ally diluting 10 mg/l stock solution with fresh aerated [21, 22]. Dissolved aluminium concentrations at the Vaal RO water to a final volume of 150 ml per beaker. Expos- River site, where the parasite is absent, were 23 times ure assays were prepared in triplicate in acid washed higher compared to levels at the Vaal Dam where parasites glass beakers and three eggs were placed into each of are present [10]. Further to this, a study was done to de- the replicates. Exposure levels of aluminium were se- scribe the microhabitat biology of the parasite in the Vaal lected according to the environmental relevance to the Dam in relation to the physical water quality variables at Vaal Dam and Vaal River site assessed in Gilbert and the site, where changes in microhabitat selectivity were Avenant-Oldewage [10], and slightly higher for compari- the result of seasonal changes and were not driven by son to previous studies conducted on exposure of a water quality [23]. This study is therefore a continuation number of species of Gyrodactylus to varying aluminium of this work and aimed to describe the effects of exposure levels [18–20]. Reference to metal concentrations in the of eggs and larval stages of P. ichthyoxanthon to alumin- study is done on the basis of nominal concentrations at ium. Larval stages of the parasite were selected as this de- the beginning of the exposure. Experiments were per- velopmental stage is responsible for transmission of formed in environments with constant photoperiod and parasites to new hosts. Therefore, we examined the effects room temperature at 14 h light and 10 h dark, and 23 °C, Gilbert and Avenant-Oldewage Parasites & Vectors (2016) 9:420 Page 3 of 11 respectively. Exposure tank temperatures were therefore for Windows (Statistical Package for the Social Sciences, maintained at a temperature of 20.8 °C (± 0.21). Eggs and SPSS Inc., USA). Homogeneity of the data for alumin- physical water quality variables (vis. pH, temperature, con- ium analysis was tested using a Shapiro-Wilk test. ductivity, dissolved oxygen and oxygen saturation) were Trends in temporal variances of metal levels in exposure measured every 24 h to observe if any noticeable changes in assays during the course of the experiment were development of the embryos and water quality occurred. A assessed using a Kruskal-Wallis test. Comparison of the total of 55 eggs of P. ichthyoxanthon were exposed to vary- differences between concentrations over the period of ing concentrations of aluminium for a period of 19 days, the study were assessed using a one-way analysis of vari- namely: RO water (control); 7.5 μgAl/l;15μg Al/l; 30 μg ance (ANOVA) and Tukey tests. Spearman’s correlation Al/l; 60 μg Al/l and 120 μgAl/l.Exposureoftheeggswas was used to determine the relationship between time continued until hatching had stopped to determine if the and the levels of aluminium eggs and larvae were ex- levels of metal affected the hatchability of the eggs as well as posed to.

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