In general, neuronal injury leads of withdrawal or death of the injured neuron. The central nervous system (CNS) is especially susceptible to neuronal damage, because unlike neurons in the peripheral nervous system, neurons injured within the CNS injury rarely recover. However, the cricket exhibits a rare form of CNS recovery after injury. For example, after deafferentation of the auditory neurons, ascending neuronal dendrites recover by growing across the “midline” of the CNS. The midline is a chemical barrier that insures that neurons grow in the proper direction and connect with the proper synaptic partners during development. Therefore for this neuronal recovery to occur, the chemical composition of the neurons and/or its environment must be altered to allow this recovery to occur.
Previous research shows that the rate of dendritic growth across the midline after deafferentation differs between male and female crickets. Dendritic growth of female crickets occurs rapidly within the first few days of neuronal injury, but eventually the growth rate plateaus. Male dendritic growth after injury steadily increases over time, and reaches lengths that are twice as long as female dendrites after injury (Pfister et al., 2013). Hormones are one potential candidate for male and female dendritic growth variance. Hormone levels differ between males and females of the same species (Zera et al., 2007), and the difference could be responsible for the differential dendritic growth rates. Juvenile (JH) and ecdysteriod hormones are two hormones that could affect dendritic growth in the cricket auditory system.
Knocking down the expression of regulators and receptors of the hormones is one way to determine if these hormones do play a role in the difference in recovery between cricket conspecifics. One way to knock down protein expression is by injecting double stranded RNA (dsRNA) against a particular RNA sequence of interest. Injecting a cricket with dsRNA initiates a series of events that ultimately leads to the deletion of corresponding mRNA sequences, therefore, lowering the overall protein expression within an organism.
In order to create dsRNA, however, it is necessary to sub-clone a particular gene of interest by transforming the product into a vector. This cloning procedure has been the focus of my research this summer. Using the NCBI database, I found 4 G. bimaculatus-specific mRNA sequences. They were as follows: Allatostatin A (AllosA), Allatostatin B (AllosB), Dopaminergic Ecdysteriods Receptor (DoEcR), and Ecdysteriods B receptor (ECR). These proteins play important roles in the regulation and functionality of JHs and ecdysteriods.
Based on the sequences, appropriate primers for each sequence were found and a PCR reaction was performed with the primers. The products of the reaction were run on an agarose gel, and then extracted. The products were then ligated into a pGemT Easy vector and transformed in competent cells. Colonies with the proper inserts were selected and PCR reactions were run to verify the insert was present in the vector (Figure 1). In order to further verify whether the isolated vectors contained the desired products, the vectors were sequence at the Mount Desert Island Laboratory.
For future work, dsRNA will be created from the vectors. The dsRNA will then be injected into crickets to knockdown the expression of a particular hormonal regulator. After the knockdown I will determine if knocking down a particular gene affects the functional recovery of cricket auditory neurons after deafferentation of auditory neurons G. bimaculatus utilizing physiological recording of ascending neurons.
Faculty Mentor: Hadley Horch
Funded by the Maine Space Grant Consortium
Horch, H., McCarthy, S., Johansen, S., & Harris, J. (2009). Differential gene expression during compensatory sprouting of dendrites in the auditory system of the cricket Gryllus bimaculatus. Insect Molecular Biology, 18, 483-496.
Lorenz, J., Lorenz, M., & Hoffmann, K. (1997). Factors regulating juvenile hormone and ecdysteriod biosynthesis in Gryllus bimaculatus (Ensifera: Gryllidae). Eur. J. entomology, 94, 369-379.
Pfister, A., Horch, H., Johnson, A., & Ellers, O. (2012). Quantification of dendritic and axonal growth after injury to the auditory system of the adult cricket Gryllus bimaculatus. Frontiers in Physiology, 3, 1- 26.
Truman, J., & Reiss, S. (1988). Hormonal Regulation of The Shape of Identified Motorneruons in the Moth Manduca sexta. The Journal of Neuroscience, 8, 765-775.
Zera, A., Zhao, Z., & Kaliseck, K. (2007). Hormones in the Field: Evolutionary Endocrinology of Juvenile Hormones and Ecdysteroids in Field Populations of the Wing-Dimorphic Cricket Gryllus firmus. Physiological and Biochemical Zoology, 80, 592-606.