Fish stress pushes the boundaries of science
The two protagonists in this study are the European bitterling (Rhodeus amarus) and the swan-mussel (Sinanodonta woodiana). The parasitic larvae of this freshwater bivalve are technically called glochidia. These larvae, after hatching from the egg, attach their teeth, located on the edge of their shells, to the gills and fins of the fish from which they obtain their nutrition and thus harm the fish to some extent. The bitterling is a small carp-like fish found in most European still and gently flowing waters.
However, these two species were not randomly selected for this study. They are known for their characteristic coexistence under mutual parasitism. Female bitterlings lay their eggs in the gills of clams, providing a safe incubation site for the first few weeks of embryonic development. Hosting the embryos of bitterlings is costly for bivalves. In turn, the gastropod molluscs, which include those clams, develop through the obligate parasitic glochidium larval stage. The glochidia attach themselves to the fins and gills of the fish, where they are enveloped by the host tissue from which they acquire nutrients. However, glochidial parasitism can also be costly for the host fish. These effects are manifested, for example, in less efficient respiration and reduced locomotor activity of the infected fish. At high levels of infestation, glochidial parasitism can lead to slower host growth, reduced fitness, and even increased mortality.
Thus, the researchers investigated the stress response induced by glochidia of the invasive bivalve S. woodiana in R. amarus. To achieve this, they measured levels of the hormone cortisol in the bitterlings’ plasma using liquid chromatography coupled to a mass spectrometer. Cortisol is a known stress hormone secreted by the adrenal cortex. This glucocorticoid is a useful indicator of the stress response because, unlike other stress-induced substances, it is produced within minutes and its effect persists throughout the stimulation period. In this case, the bitterlings increase the blood cortisol level during larval infestation. However, the challenge with small fish such as this is collecting sufficient blood plasma. This has been solved through the contribution of Anna Janovská, who optimised the method for determining cortisol from very small amounts of plasma in the laboratory. For some samples even this was not enough, but further modification of the method helped. This involved incubating carp blood plasma with activated charcoal and adding it to the sample. The newly modified method was optimised to be sufficient even with significantly lower amounts of plasma. This enables accurate measurement of plasma steroid hormone concentrations in small fish, with the potential for use in future research.
The fish used in the research were caught in the Kyjovka river (Danube basin, south-eastern Czech Republic) and subsequently transported to the Institute of Vertebrate Biology of the Academy of Sciences of the Czech Republic in Brno, where they were placed in vats that imitated their natural environment. Following quarantine and acclimatisation, male fish were divided into experimental and control groups. The experimental group was infected with glochidia in a controlled manner. An inspection check revealed that the glochidia-infected fish had an average of 89 glochidia distributed in the gills and fins. Control fish had no glochidia. After one day, some of the fish were caught and a blood sample was taken from the tail vein. Following processing, the researchers obtained 4–35 µl of plasma from each fish, which is an extremely small amount for analysis. A second set of samples from the fish was obtained 36 hours after the attack.
The results of the study provide evidence of an immediate host response to parasitism. In addition to a consistent increase in cortisol levels in experimentally infested gentians during the first two days of infestation, the researchers found an overall increase in cortisol levels between the first and second days. This increase was independent of the treatment effect and was evident in both control and infested fish. In conclusion, cortisol is only one of multiple indicators of acute stress ‒ further studies are needed to understand how fish respond to glochidia infestation. However, elevated cortisol levels also provide survival benefits for glochidia and, marginally, for the fish themselves. It has previously been demonstrated that experimental administration of cortisol to the host significantly increased the rate of glochidial metamorphosis. In addition to increasing metabolism in response to stress, cortisol in fish also has immunosuppressive effects, such as reducing inflammation, and not only at the site of attack. Thus, resistance in fish can often be reduced to the behavioural level of actively avoiding infection. Accurate measurement of hormone levels in small fish (i.e. in small amounts of blood) is often challenging, so the newly developed method could greatly help future research. This method can be equally well applied to other steroid hormones circulating in the blood, and thus allows accurate quantification of the hormone profiles of other small species.