Caleb Paquette, Karl Joplin, Istvan Karsai, and Darrell Moore.  Dept. of Biological Sciences, East Tennessee State Univ., Johnson City, TN 37614

Many animals exhibit territorial behavior.  Successful territorial defense provides greater access to mates, nesting sites, food, and shelter.  We expect there to be fundamental differences between territorial and non-territorial animals in the way the nervous system is configured to handle encounters with conspecifics.  These fundamental differences presumably should be reflected as differences in the spatial patterning of individuals placed within groups, even under non-natural circumstances.  If this assumption is true, then we should expect that these differences could be measured under controlled, laboratory conditions.  To test this idea, we have chosen the flesh fly, Sarcophaga crassipalpis, as our model organism:  in nature, male flies appear to be territorial while females do not.  To test for potential differences in the spatial patterning between male and female flesh flies, groups of four male flies or four female flies were observed in circular and rectangular enclosures, with many replications.  Enclosures were placed under a light:dark cycle (12 hours light; 12 hours dark) and the position of each fly within the enclosure was recorded at hourly intervals throughout the light phase.  Follow-up experiments involved position measurements during the dark phase of the light cycle and during constant dark conditions over several days to determine if the degree of clustering was organized as a circadian rhythm.  The distances between each fly and its nearest neighbor in the circular enclosures were measured.  Nearest neighbor statistics were used to calculate R values, indicating the degree of clustering.  For the rectangular enclosures, tolerance of conspecifics was determined as the number of observations in which two or more flies were located in the same section of the enclosure (each section occupied one-eighth of the enclosure’s length).  Our results are in agreement with the idea that territorial individuals should have less innate tolerance for conspecifics and should also exhibit less clustering (maintain greater inter-individual distances) than non-territorial individuals.  Female flesh flies exhibited a significantly higher tolerance for conspecifics in the rectangular enclosures and were significantly more aggregated in the circular enclosures than were the male flies.  Females tended toward a random spatial distribution while males tended to locate themselves according to a uniform distribution.  Males were  significantly more aggregated (similar to the female pattern) during the dark phase than during the light phase of the light:dark cycle, suggesting that inter-individual distances are judged by visual cues.  Furthermore, under constant dark conditions over several consecutive days, the males maintained the female-like, more aggregated pattern, indicating that the behavior is not driven by a circadian clock.  We believe that the flesh fly enclosure experiments can serve as a model system for investigating the physiological, genetic, and molecular bases of territorial or aggressive behavior.

THE LONG-TERM EFFECT OF TIME-MEMORY ON FORAGER HONEY BEE RECRUITMENT.

Matthew W. Otto, Samara Miller, Guy Kramer, Jaime McManus, and Darrell Moore Department of Biological Sciences, East Tennessee State University, Johnson City, TN 37614.

The honey bee (Apis mellifera) forager has the remarkable ability to remember the time of day when food sources, such as the nectar from particular species of flowers, are most profitable.  This food-anticipatory activity (known as the honey bee time-memory) is under the control of a circadian clock and enables the forager to match its collecting behavior with the nectar presentation rhythms of flowers.   Forager honey bees also have the ability to recruit other foragers or be recruited themselves (via the famous waggle dance communication) to profitable flower patches in the environment.  While there have been considerable studies on both the time-memory and the recruitment processes, previous work has not considered any potential interactions between the two functions.  For example, does the existence of a time-memory for one food source interfere with the ability of the forager to be recruited to a different food source?  In other words, does the first time-memory need to undergo extinction before another time-memory can be established?  We have performed experiments to test two major hypotheses concerning the possible interactions between time-memory and the forager’s sensitivity to recruitment.   The experiments consisted of two groups of foragers from the same colony, each group trained to a separate food station but at the same restricted time of day for several consecutive days.  Feeding then was canceled at one station but continued for five more days at the other.  The extinction irrelevant hypothesis predicts that foragers, even those with extensive experience (and presumably very strong time-memories) at one food source, have the ability to be recruited to an alternative source as long as they are not currently occupied with foraging.  In contrast, the extinction hypothesis predicts that the time-memory for one food source should deteriorate over several days (undergo extinction) before the forager becomes susceptible to recruitment to a second food source by other foragers.   Our results show that bees with greater experience (more days of training) at a first source that is no longer producing food are significantly less likely than foragers with less experience at that source to be recruited to a second food source presented at the same time of day.  Furthermore, the ability to be recruited returns after several days, but this recovery period is longer for bees with greater experience.  These findings support the extinction hypothesis and demonstrate a long-term influence of time-memory on subsequent foraging behavior.

THE ISOLATION AND CLASSIFICATION OF MICROFLORA IN THE GUTS OF INSECTS BASED ON RDNA SEQUENCES

Amy R. Robertson, Dr. Karl Joplin and Dr. Hugh Miller III, Department of Biological Sciences, East Tennessee State University, Johnson City, TN

Microbes are often overlooked as part of an ecosystem, however their presence is vital for every kind of life.  The number of species of microorganisms is unknown and classifying them is difficult due to their small sizes, lack of morphological traits, and extreme diversity.  It is estimated that only about ten percent of bacteria have been classified and research has shown that 99% of these organisms cannot be cultured using traditional techniques (agar plates).  Carl Woese is a famous microbiologist who used molecular-based techniques to compare ribosomal DNA (rDNA) from different organisms, established three primary lines of descent—Eubacteria, Archaea, and Eukarya, and constructed life’s history with a phylogenetic tree.  The development of these molecular mechanisms allowed biologists to investigate microbe diversity.  As a result of these techniques, there have been 20 phyla identified in the Archaea and Eubacterial domains as well as a vast abundance of eukaryotic phytoplankton in the oceans.  Prokaryotic and Eukaryotic flora of the alimentary canal of animals such as insects have begun to be examined using these techniques as well. This could serve as a model system of microbial diversity and ecology.  Roaches are a possible model organism because they are large insects, easy to raise and manipulating them experimentally in a lab is simple.  It is hypothesized that through the use of rDNA sequences and molecular techniques, a vast array of organisms comprising the gut microflora will be identified, classified, and placed on the phylogenetic tree.  This would include previously unidentified organisms as well.  For this study, the Madagascar hissing cockroach, Gromphadorhina portentosa, was used as the model system.  DNA was isolated using a total DNA isolation procedure.  Eukaryotic, Archaeal, and Eubacterial specific primers with explicit restriction sites were used to amplify domain-specific rDNA fragments using PCR. The fragments were then cloned into a vector and transformed into E. coli bacteria.  Cloned plasmid DNA was sequenced at UT Knoxville.  Sequences are being compared with known ribosomal sequences using the BLAST database and the identified sequences will be recognized from the known phylogenetic tree.  The unclassified organisms will also be compared with known organisms and placed on the phylogenetic tree. 

ISOLATION AND CHARACTERISATION OF WHITE EYE GENE IN SARCOPHAGA CRASSIPALPIS

Robert Morgan, Karl Joplin, Hugh A Miller III, and Darrell Moore, East Tennessee State University, Department of Biological Sciences, Johnson City, TN 37614

The white eyed phenotype, characterized by a lack of eye pigment, is a naturally occurring mutant in some invertebrates such as Drosophila melanogaster. Thus far it has not been observed in the flesh fly Sarcophaga crassipalpis. A 569 base pair section of mRNA encoding the white (w) eye color gene from the flesh fly Sarcophaga crassipalpis has been isolated, cloned, and sequenced. A nucleotide BLAST search shows homology with other known invertebrate w sequences. RNA interference is currently being performed to create a white eye phenotype expression in S. crassipalpis.  

THE EFFECTS OF RAIN ON HONEY BEE FORAGING BEHAVIOR

Samara Miller, Matt Otto, Jaime McManus, Guy Kramer, and Darrell Moore, East Tennessee State University, Department of Biological Sciences, Johnson City, TN 37614

The honey bee time-memory (which allows forager bees to recall the time of day and the spatial location at which food rewards were previously obtained) is considered to be highly adaptive in that it allows foragers to accurately match their foraging behavior to predictable nectar secretion times of flowers in the colony’s environment.  In the absence of nectar (or sucrose solution provided by experimenters), the time-memory deteriorates over several days, much like the extinction of a conditioned response in classical conditioning.  Recent experiments in our laboratory have indicated that the presence of rain during the extinction process causes an apparent, temporary recovery of the time-memory response.   The time-memory response, assayed from the proportion of foragers continuing to reconnoiter the training station on subsequent unrewarded test days, is significantly higher on days following a rain event than on corresponding days in fair-weather experiments.   These results demonstrate that the time-memory does not simply deteriorate over time, but rather it is a malleable process and can be influenced by environmental factors.  What is the mechanism by which time-memory extinction is delayed?  In this project, we are evaluating two different hypotheses to explain this phenomenon.  First, the “memory interference” hypothesis proposes that extinction delay is accomplished by rain events because, during inclement weather, there is a severe reduction in overall hive activity which, in turn, drastically reduces the number of potential memory-interfering events caused by interactions of the forager bee with its hive-mates inside the colony.  In contrast, on fair-weather days, hive activity is bustling, thereby producing a surplus of inter-individual interactions which interfere with time-memory persistence.  Second, the “nectar regeneration” hypothesis proposes that it is the rain event itself that serves as a signal for foragers to revisit previously profitable sources.  Presumably, significant rainfall may rescue many plants from water stress and rejuvenate their ability to produce substantial volumes of nectar containing valuable concentrations of sugar.  Thus, rain itself may be a reliable indicator of an increase in food availability.   We will describe recent results from experiments designed to test predictions from these two competing hypotheses.