In 1938, the Austrian ethologist Karl von Frisch made his first report on the existence of the chemical alarm signal known as Schreckstoff (fright substance) in minnows. An alarm signal is a response produced by an individual, the "sender", reacting to a hazard that warns other animals, the receivers, of danger.(Smith 1992). This chemical alarm signal is released only when the sender incurs mechanical damage, such as when it has been caught by a predator, and is detected by the olfactory system. When this signal reaches the receivers, they perceive a greater predation risk and exhibit an antipredator response. Since populations of fish exhibiting this trait survive more successfully, the trait is maintained via natural selection. While the evolution of this signal was once a topic of great debate, recent evidence suggests schreckstoff evolved as a defense against environmental stressors such as pathogens, parasites, and UVB radiation and that it was later co-opted by predators and prey as a chemical signal.
Background Chemical alarm systems have been identified in a number of different taxa, including gastropods,(Stenzler and Atema 1997; Atema and Stenzler 1977) echinoderms,(Snyder and Snyder 1970) amphibians(Hews and Blaustein 1985; Hews 1998; Lutterschmidt et al. 1994) and fishes. One of the most well-studied chemical alarm signals is schreckstoff, the use of which is widespread in the superorder Ostariophysi (e.g., minnows, characins, catfishes, etc.). About 64% of all freshwater fish species and 27% of all fish species worldwide are found in the ostariophysan superorder, which highlights the widespread use and importance of this chemical alarm system in fishes.(Nelson 1994).
Schreckstoff in ostariophysans The production of schreckstoff has been shown to be metabolically expensive and is therefore part of a conditional strategy that can only be employed by individuals with access to sufficient resources.(Wisenden and Smith 1998). One putative active ingredient in schreckstoff is hypoxanthine-3N-oxide (H3NO), which may be produced in club cells which will henceforth be referred to as "alarm substance cells".(Argentini 1976; Pfeiffer 1982). The nitrogen oxide functional group was found to be the main chemical trigger of antipredator behavior in receivers.Brown et al. (2000) Schreckstoff is a mixture, and fragments of a glycosaminoglycan, chondroitin sulfate, are able to trigger fear responses.Mathuru et al. (2012) The precursor polysaccharide is a component of mucus, and fragments are proposed to be produced during injury. Like schreckstoff obtained from skin extract, chondroitin sulfate activates a subset of olfactory sensory neurons.
Production of and responses to schreckstoff change over the course of ontogeny. For example, young brook sticklebacks (Culaea inconstans) are more likely to be caught in minnow traps that have been baited with conspecific skin extracts than adults.(Chivers and Smith 1994). This result indicates young brook sticklebacks do not make the association between schreckstoff and the potential presence of a predator as readily as adults. Whether this association strengthens over time as a result of learning or physiological development remains unclear.(Chivers and Smith 1994).
In addition to changes across ontogeny, the degree to which schreckstoff is produced varies within the breeding season. Male fathead minnows (Pimephales promelas) cease production of schreckstoff during the breeding season, but still exhibit antipredator behaviors in response to schreckstoff during this time.(Smith 1973). Schreckstoff production may be halted at this time because male fathead minnows often incur mechanical damage while building their nests. It would be detrimental to a male to produce schreckstoff while building a nest, as it would inadvertently repel females, thereby decreasing the likelihood of obtaining a mate. By ceasing schreckstoff production during the breeding season, males circumvent this problem. The cessation of alarm substance cell production appears to be controlled by androgens.(Smith 1973).
Hypotheses for the evolution of schreckstoff A number of different hypotheses have been proposed for the evolution of schreckstoff.(see Smith 1992 for a review). The first hypothesis is that the evolution of schreckstoff has been driven by kin selection. Support for this hypothesis would include evidence that individuals live in groups of closely related kin and that the release of chemical alarm signals increases the likelihood that related individuals will avoid predation. The second hypothesis, predator attraction, suggests the release of schreckstoff may attract additional predators which will interfere with the predation event, increasing the likelihood that the prey will escape and survive the attack. This hypothesis assumes predators will be attracted to schreckstoff and will interfere with one another either through competition for the captured prey or through predation of one another. It additionally assumes, despite the fact that the prey has already incurred mechanical damage, it is possible for the prey to escape and recover from the attack. Testing and validating these assumptions would provide support for the predator attraction hypothesis. The third hypothesis proposes that schreckstoff has an immune function, providing protection against pathogens, parasites and/or UVB radiation. For this hypothesis to be supported, a correlation between alarm substance cell production and the presence of pathogens and parasites would need to be observed. Direct evidence that schreckstoff inhibits the growth of aquatic pathogens and parasites would provide additional support for the immunity hypothesis. Another hypothesis is that schreckstoff is a breakdown product of mucus and club cells, induced by injury. Selection for the alarm response is primarily at the level of the receiver.
Kin selection hypothesis One of the first hypotheses for the evolution of schreckstoff centered on W.D. Hamilton’s theory of kin selection.(Hamilton 1963, 1964a, 1964b). Under the theory of kin selection, the sender of the chemical alarm signal would be willing to incur the costs of sending this signal if the benefits to related individuals were sufficiently high. In a situation where the sender of the signal is paying great costs (i.e., it releases the chemical alarm signal because it has incurred potentially mortal mechanical damage), the benefits to closely related kin would have to be great. Under the framework of kin selection, behaviors that are seemingly detrimental to the sender are selected because they benefit individuals that are likely to share alleles by common descent. In this way, the frequency of the sender's alleles in the next generation is increased by their presence in successful kin.
To apply kin selection theory to the evolution of schreckstoff, a number of conditions must be met. First, evidence must exist for the release of schreckstoff by the sender confers benefiting the receivers. Second, it must be shown that individuals in the order Ostariophysi associate mainly with family members. If either of these two assumptions is violated, then the kin selection hypothesis would not be supported.
Some evidence exists in support of the first assumption that the release of schreckstoff confers quantifiable advantages to the receivers of this chemical signal. A laboratory experimentMathis and Smith (1992) revealed that fathead minnows exposed to conspecific schreckstoff survived 39.5% longer than controls when placed in a tank with a predatory northern pike (Esox lucius). This finding suggests schreckstoff increases vigilance in receivers, resulting in a quicker reaction time following detection of the predator.
The second assumption, that individuals in the order Ostariophysi generally associate with close family members, does not appear to be supported by empirical evidence. In shoals of European minnows (Phoxinus phoxinus), no difference in relatedness was found within and between shoals,Naish et al. (1993) indicating individuals are not associating more closely with relatives than nonrelatives. Shoal composition has not been examined in all members of the ostariophysan order, and shoals composed entirely of family members may yet be discovered. Nevertheless, the finding that schreckstoff production is maintained in a species where the function is clearly unrelated to kin benefits provides strong evidence against kin selection as a mechanism for the evolution of schreckstoff.
Fathead minnows have also been found to produce fewer epidermal alarm substance cells (and therefore less schreckstoff) when in the presence of familiar shoalmates.(Wisenden and Smith 1998). The results of this study indicate one of two scenarios, neither of which is compatible with the hypothesis that schreckstoff evolved by kin selection. First, if schreckstoff evolved by kin selection, more epidermal alarm substance cells would be expected to be produced in the presence of kin than nonkin. This means familiar shoalmates in fathead minnows should be closely related kin and schreckstoff production should be increased when in shoals with familiar individuals. The study did not find this to be the case.Wisenden and Smith (1998) Second, these results indicate individuals either do not associate with kin at all or production of schreckstoff varies depending on how familiar the focal fish is with the individuals with which it shoals. In conclusion, evidence does not support the hypothesis that schreckstoff evolved because it bolstered the inclusive fitness of the sender through increased survival of kin.
Predator attractant hypothesis The predator attractant hypothesis proposes that the main purpose of schreckstoff is to attract additional predators to the area.Smith (1992) According to this hypothesis, additional predators will interact with the initial predator, and these interactions will provide the sender with an opportunity to escape. A number of conditions must be met to support this hypothesis. First, schreckstoff must attract predators. Second, subsequent predators must disrupt the predation event, thereby increasing the probability that the prey will escape. Third, the sender must be able to recover from the mechanical damage incurred during the predation event.
A 1995 studyMathis et al. (1995) provides support for the first condition that the release of schreckstoff must attract predators. This experiment revealed that schreckstoff extracted from the skin of fathead minnows attracted both northern pike (Esox lucius) and Colymbetes sculptilis predatory diving beetles. Additionally, a natural study showed that predatory fish were seven times more likely to strike a lure baited with a sponge soaked in fathead minnow skin extract than a sponge soaked in either water or skin extract from a nonostariophysan convict cichlid (which presumably does not produce schreckstoff).Wisenden & Thiel (2002)