Year 1: Ontogeny of aggressive behavior in flesh flies. (Brief Description) Territorial behavior in Sarcophaga crassipalpis can be observed for pairs of males exposed to simplified laboratory conditions in which there are no resources to defend except for space. We have described a repertoire of 16 stereotyped behaviors that male flies exhibit under these conditions. Also, we have developed a 16 x 16 behavioral transition matrix for males 1, 2, 3, 4, and 6 days of age post-eclosion. Our results demonstrate that male flesh flies radically reorganize both their aggressive and, surprisingly, their non-aggressive behaviors in parallel with age-related increases in agonistic interactions (Moore, Paquette, Seier, & Joplin, in preparation). Between 3 and 4 days of age, male-to-male agonistic encounters change from low intensity sparring and avoidance to full-fledged combat. Why should non-aggressive behaviors also be reorganized, especially if agonistic encounters occur relatively infrequently? We will construct behavioral transition matrices and determine which transitions occur with frequencies greater than would be expected by chance. We then will need to determine if different matrices, representing different experimental conditions (e.g., different ages, males vs. females, experienced males vs. isolated males, etc.) are different from one another.
Year 2: Influence of biogenic amines on aggressive behavior. (Case Study) What are the influences of biogenic amines (including serotonin & octopamine, known to influence aggressive behaviors in arthropods) on the expression of aggressive behaviors in flesh flies? We will extend the basic ethology studies from year 1 by first measuring the natural levels of biogenic amines throughout the 24-hour day to determine if there are daily variations in the levels of these neurohormones. Previous work has shown that the effects of many biologically active compounds depend upon the time of application during the circadian cycle. Flies reared in LD 12:12 conditions will be isolated from one another beginning at adult eclosion. The daily cycle will be sampled at eight separate times of day (4 times within the photophase, 4 times within the scotophase), using 10 animals at each sample time. This baseline study will be performed twice, once with flies of day 1 of age, known to be non-aggressive, and once with flies of day 4 of age (which we have determined are fully aggressive). Octopamine and serotonin are used as standards for identification of peaks and to determine the concentration of the different amines. Internal standards added to the samples will allow us to determine actual concentrations of biogenic amines and to monitor any changes that may occur to the endogenous biogenic amines in the samples. We will use our newly acquired HPLC-EC which can detect femtomolar levels of these neurohormones. The results of these baseline studies will serve as a foundation upon which we can determine the appropriate times of day at which to (1) administer agonists and antagonists and (2) make behavioral observations.
To manipulate the system experimentally, octopamine and serotonin (as well as various agonists and antagonists) will be dissolved in dimethylformamide (DMF) and applied exogenously to the thorax at various times during the daily cycle. We have established that flesh flies tolerate the DMF and application does not appear to alter behavior. In attempting to determine if there is a lethal limit to biogenic amine application, we have found that octopamine applied at 10 times reported levels (i.e.100μg/μl) is not lethal (but, anecdotally, does affect overall activity levels). The results of our baseline study will help inform us as to the most appropriate times of day in which to administer the biogenic amines, their agonists and antagonists.
Our goal is to determine the role of serotonin and octopamine on the ontogeny of flesh fly behavior. Can the non-aggressive state be converted to the aggressive state by simply elevating or blocking particular amines? How do biogenic amines influence the relative proportions of aggressive and non-aggressive behaviors? Do the networks of behavioral transitions become reorganized? Are some behaviors de-emphasized while others are up-regulated? Comparisons of behavioral transition matrices will be necessary to determine the influences of biogenic amines on the reorganization of the behavioral repertoire.
Year 3: Agent-based modeling of spatial distribution. (Brief Description) Male flesh flies, when placed in groups of four individuals in a confined enclosure, maintain a significantly greater degree of spatial separation among themselves than do groups of females: males tend toward a uniform spatial distribution whereas females tend to be clustered (Paquette et al. 2008). We hypothesize that this spacing among the males (but not females) reflects the territorial behavior observed in males (but not females) in nature (Arnett 2000). If true, then the tendency for males to separate themselves spatially, even in a simplified laboratory environment, indicates the existence of a fundamental neural substrate controlling this behavior. Alternatively, the inter-individual spacing among males may not be inherent but rather the result of previous aggressive encounters. The environment will be manipulated to determine how different parameters change the dynamic. For example, we may (1) change the size of the arena, (2) the number of flies within the arena, (3) add a female to a group of 4 males or vice versa, (4) compare males that have and have not had previous aggressive encounters, and (5) test males of different ages.
We will use agent-based modeling to begin to determine the rules by which individuals maintain their spatial separation. Graph-theoretic modeling may facilitate interpretations of the experimental results