Project 2
Behavioral and Biochemical Analyses of Sex
Identification Pheromones in Crickets
(PIs: B.
Rence, J. Lokensgard)
Literature reports differ as to the
extent to which the cricket species Teleogryllus
commodus and Acheta domesticus
utilize volatile pheromones and contact chemoreception as aids to sex
recognition of conspecific individuals, and as to the extent to which females
and males may differ in their use of these two methods. The main hypothesis of this project is that Acheta, which is also reported to use a
volatile aggregation pheromone, may use a smaller volatile molecule in sex
identification, while Teleogryllus
commodus may rely almost exclusively on non-volatile chemoreception based
on antennal contact. Consequently, the
project objectives were to replicate previous studies and design new ones to
prove the volatile or non-volatile nature of the sex identification pheromones.
Summary of Research Results
Daniel Weingrow spent the bulk of the summer 2002 redoing
three types of previous behavioral bioassays that had been used to reveal the
nature of the sex identification pheromones in crickets. Two of these were essentially maze studies in
which the male cricket was put in a chamber to make choices between various
volatile odor sources. The two previous
studies used slightly difference mazes and methods of pheromone delivery and
one had concluded that the pheromone was volatile while the other indicated it
was non-volatile. Daniel's exacting
replication of these two studies showed a fatal flaw in both. Neither had controlled for the presence or
absence of the male spermatophore in the test males. Having a spermatophore in place is essential
for the male to display sexual behavior and without it he will only perform
aggressively toward both males and females.
Daniels replication
of the third methodology, one where the male cricket is tickled on his antennae
with recently severed antennae of either adult males or females showed that
indeed the spermatophore must be present for the male to react with courtship
behavior to the female antenna; if the spermatophore is absent the male
responds aggressively to both male and female antennae. When the two previous
maze studies were replicated with males who had spermatophores in place there
was a random choice between chambers containing males or females, or between
chambers containing either sex and no crickets.
These data prove that the pheromone is non-volatile. Further confirmation of this result comes
from the inability to record any perceptible electroantennograms from antennae
of male crickets when the odor of either males or females were delivered. The sex
discrimination pheromone is communicated to the males by contact
chemoreceptive means as is indicated by the clear sexual or aggressive
reactions to antennal touch from respectively females or males.
A multi-chamber experimental apparatus was assembled to
test for the presence of volatile pheromones. Clean, dry air was moved through
two closed chambers in which were placed male crickets, female crickets, or no
crickets at all. Air from these chambers passed into a larger chamber in which
an individual “test animal” was placed. During experiments in which Daniel was
observing the behavior of a test animal, James Stark inserted a solid-phase
micro-extraction (SPME) fiber into the path of the air flow to sample for any
volatile compounds being swept from the multiple-cricket chambers into the test
chamber. He later desorbed the SPME fiber in the injection port of a gas
chromatograph, and analyzed the resulting effluent by mass spectrometry
(gc-ms). There was little or no evidence of any volatile pheromone, but the
experiments will be repeated during the summer of 2003.
In order to collect information bearing on tactile
communication methods, James also sampled the cuticular hydrocarbons of a
number of crickets by immersing individual legs, wings, antennae, or whole
crickets in solvents (principally hexane and dichloromethane), then analyzing
the extracts by gc-ms. He collected a
large data base of chromatograms and mass spectra, representing well over 60
compounds, which we are cataloging in terms of their abundance, and to some
extent location (antennae, wings, legs), and the differences in their
occurrence on female and male crickets. The analysis of these data will guide a
planned set of experiments, to take place during the summer of 2003, employing
electroantennograms to ascertain, to the extent possible, what are the roles of
these compounds in sex identification.