Long-time readers of this blog remember that, some years ago, I did a nifty little study on the Influence of Light Cycle on Dominance Status and Aggression in Crayfish . The department has moved to a new building, the crayfish lab is gone, I am out of science, so chances of following up on that study are very low. And what we did was too small even for a Least Publishable Unit, so, in order to have the scientific community aware of our results, I posted them (with agreement from my co-authors) on my blog. So, although I myself am unlikely to continue studying the relationship between the circadian system and the aggressive behavior in crayfish, I am hoping others will. And a paper just came out on exactly this topic – Circadian Regulation of Agonistic Behavior in Groups of Parthenogenetic Marbled Crayfish, Procambarus sp. by Abud J. Farca Luna, Joaquin I. Hurtado-Zavala, Thomas Reischig and Ralf Heinrich from the Institute for Zoology, University of Gottingen, Germany: Crustaceans have frequently been used to study the neuroethology of both agonistic behavior and circadian rhythms, but whether their highly stereotyped and quantifiable agonistic activity is controlled by circadian pacemakers has, so far, not been investigated. Isolated marbled crayfish (Procambarus spec.) displayed rhythmic locomotor activity under 12-h light:12-h darkness (LD12:12) and rhythmicity persisted after switching to constant darkness (DD) for 8 days, suggesting the presence of endogenous circadian pacemakers. Isogenetic females of parthenogenetic marbled crayfish displayed all behavioral elements known from agonistic interactions of previously studied decapod species including the formation of hierarchies. Groups of marbled crafish displayed high frequencies of agonistic encounters during the 1st hour of their cohabitation, but with the formation of hierarchies agonistic activities were subsequently reduced to low levels. Group agonistic activity was entrained to periods of exactly 24 h under LD12:12, and peaks of agonistic activity coincided with light-to-dark and dark-to-light transitions. After switching to DD, enhanced agonistic activity was dispersed over periods of 8-to 10-h duration that were centered around the times corresponding with light-to-dark transitions during the preceding 3 days in LD12:12. During 4 days under DD agonistic activity remained rhythmic with an average circadian period of 24.83 ± 1.22 h in all crayfish groups tested. Only the most dominant crayfish that participated in more than half of all agonistic encounters within the group revealed clear endogenous rhythmicity in their agonistic behavior, whereas subordinate individuals, depending on their social rank, initiated only between 19.4% and 0.03% of all encounters in constant darkness and displayed no statistically significant rhythmicity. The results indicate that both locomotion and agonistic social interactions are rhythmic behaviors of marbled crayfish that are controlled by light-entrained endogenous pacemakers. I think the best way for me to explain what they did in this study is to do a head-to-head comparison between our study and their study – it is striking how the two are complementary! On one hand, there is no overlap in methods at all (so no instance of scooping for sure), yet on the other, both studies came up with similar results, thus strengthening each other’s findings. You may want to read my post for the introduction to the topic, as I explain there why studying aggression in crayfish is important and insightful, what was done to date, and what it all means, as well as the standard methodology in the field. So, let’s see how the two studies are similar and how the two differ: 1) We were sure we used the Procambarus clarkii species. They are probably not exactly sure what species they had, so they denoted it as Procambarus sp. , noting in the Discussion that it was certainly NOT the Procambarus clarkii , which makes sense as our animals were wild-caught in the USA and theirs in Germany. As both studies got similar results, this indicates that this is not a single-species phenomenon, but can be generalizable at least to other crayfish, if not broader to other crustaceans, arhtropods or all invertebrates. 2) We used only males in our study. They used only females. In crayfish, both sexes fight. It is nice, thus, to note that other aspects of the behavior are similar between sexes. 3) We used the term ‘aggression’. They use the term ‘agonistic behavior’, which is scientese for ‘aggression’, invented to erase any hints of anthropomorphism. Not a bad strategy, generally, as assumed aggression in some other species has been later shown to be something else (e.g., homosexual behavior), but in crayfish it is most certainly aggression: they meet, they display, they fight, and if there is no place to escape, one often kills the other – there is no ‘loving’ going on there, for sure. 4) The sizes of animals were an order of magnitude different between the two studies. Their crayfish weighed around 1-2g while ours were 20-40g in body mass. This may be due to species differences, but is more likely due to age – they used juveniles while we used adults. Again, it is nice to see that results in different age groups are comparable. 5) We did not measure general locomotor activity of our animals in isolation. We, with proper caveats, used aggressive behavior of paired animals as a proxy for general locomotor activity, and were straightforward about it – we measured aggressive behavior alone in a highly un-natural setup. As Page and Larimer (1972) have done these studies before, we did not feel the need to replicate those with our animals. The new study, however, did monitor gross locomotor activity of isolated crayfish. Their results, confirming what Page and Larimer found out, demonstrate once again that activity rhythms are a poor marker of the underlying circadian pacemaker (which is why Terry Page later focused on the rhythm of electrical activity of the eye, electroretinogram – ERR – in subsequent studies) in crayfish. Powerful statistics tease out rhythmicity in most individuals, but this is not a rhythm I would use if I wanted to do more complex studies, e.g., analysis of entrainment to exotic LD cycles or to build and interpret a Phase – Response Curve . Just look at their representative example (and you know this is their best): You can barely make out the rhythm even in the light-dark cycle (white-gray portion of the actograph) and the rhythms in constant darkness (solid gray) are even less well defined – thus only statistical analysis (bottom) can discover rhythms in such records. The stats reveal a peak of activity in the early night and a smaller peak of activity at dawn, similarly to what Page and Larimer found in their study, and similar to what we saw during our experiments. 6) They used an arena of a much larger size than ours. We did it on purpose – we wanted to ‘force’ the animals to fight as much as possible by putting them in tight quarters where they cannot avoid each other, as we were interested in physiology and wanted it intensified so we could get clearly measurable (if exaggerated) results. Their study is, thus, more ecologically relevant, but one always has to deal with pros and cons in such decisions: more realistic vs. more powerful. They chose realism, we chose power. Together, the two approaches reinforce and complement each other. 7) As I explained in my old post – there are two methodological approaches in this line of research: Two standard experimental practices are used in the study of aggression in crustaceans. In one, two or more individuals are placed together in an aquarium and left there for a long period of time (days to weeks). After the initial aggressive encounters, the social status of an individual can be deduced from its control of resources, like food, shelter and mates. In the other paradigm, two individuals are allowed to fight for a brief period of time (less than an hour), after which they are isolated again and re-tested the next day at the same time of day. They used the first method. We modified the second one (testing repeatedly, every 3 hours over 24 hours, instead of just once a day). What they did was place 6 individuals in the aquarium, a couple of hours before lights-off, then monitor their aggressive behavior over several days. What they found, similar to us, is that the most intense fights resulting in a stable social hierarchy occur in the early portion of the night: Once the social hierarchy is established on that first night, the levels of aggression drop significantly, and occasional bouts of fights happen at all times, with perhaps a slight increase at the times of light switches: both off and on. Released into constant darkness, the pattern continues, with the most dominant individual initiating aggressive encounters a little more often during light-transitions then between them. The other five animals had no remaining rhythm of agonistic behavior: they just responded to attacks by the Numero Uno when necessary. In our study we tried to artificially elevate the levels of aggression by repeatedly re-isolating and re-meeting two animals at a time. And even with that protocol, we saw the most intense fights at early night, and most conclusive fights, i.e., those that resulted in stable social hierarchy, also occuring at early nights, while the activity at other time of the day or night were much lower. The goals of two studies differed as well, i.e., we asked somewhat different questions. Our study was designed to provide some background answers that would tell us if a particular hypothesis is worth testing: winning a fight elevates serotonin in the nervous system; elevated serotonin correlated with the hightened aggression in subsequent fights, more likely leading to subsequent victories; crayfish signal dominance status to each other via urine; melatonin is a metabolic product of serotonin; melatonin is produced only during the night with a very sharp and high peak at the beginning of the night; if there is more serotonin in the nervous system, there should be more melatonin in the urine; perhaps melatonin may be the signature molecule in the urine indicating social status. In order to see if this line of thinking is worth pursuing, we needed to see, first, if the most aggressive bouts happen in the early night and if the most decisive fights (those that lead to stable hiararchy) happen in the early night. This is what we found, indicating that our hypothesis is worth testing in the future. They asked a different set of questions: Is there a circadian rhythm of locomotor activity? They found: Yes. Is there a circadian rhythm of aggression? They found: Yes. Do the patterns of general activity and aggressive activity correlate with each other? They found: Yes. Does the aggression rhythm persist in constant darkness conditions? They found: Yes. Do all individuals show circadian rhythm of aggression? They found: No. Only the most dominant individual does. The others just defend themselves when attacked. Is there social entrainment in crayfish, i.e., do they entrain their rhythms to each other in constant conditions? They found: No. All of them just keep following their own inherent circadian periods and drift apart after a while. Is there a pattern of temporal competitive exclusion, i.e., do submissive individuals shift their activity patterns so as not to have to meet The Badassest One? They found: No. All of them just keep following their own inherent circadian periods. So, a nice study overall, the first publication I know of that attempts to connect the literature on circadian rhythms in crayfish to the literature on aggressive behavior in crayfish. Except…. Read the rest of this post… | Read the comments on this post…

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Circadian Rhythm of Aggression in Crayfish [A Blog Around The Clock]