Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
K'Lynn Spivey - Benedict College
The dorsal longitudinal flight muscles (DLMs) of the adult house cricket, Acheta Domesticu, undergo active programmed cell death (PCD) leading to the loss of ability to fly. Histolysis of the DLMs is triggered by Juvenile Hormone (JH) and requires expression of new proteins. The overall purpose of our research was to determine if JH affects the expression pattern of Apolipohorin III, a protein thought to be involved in lipid transport, to be a regulator of immune response and to be up-regulated in insect PCD. We utilized real time quantitative reverse transcriptase polymerase chain reaction (qRT PCR) in conjunction with agarose gel electrophoresis to establish protocols for analysis of the expression of transcripts of apolipophorin in the ovary, DLMs, fat body and femoral leg muscle of crickets undergoing the normal course of histolysis and from crickets ligated to prevent circulation of JH. We have also obtained transcriptome sequencing from total RNA submitted for gene expression analysis (NovoGene) of control and degenerating DLMs. cDNA was prepared with New England Biolab?s reverse transcription system using AMV enzyme with both random hexamer and oligo-dT primers. Gene specific primers were designed based on the published sequence for Acheta domesticus Apolipophorin III, and PCR amplification was performed to assess the presence of transcripts for Apolipophorin III in the cDNA derived from the tissues. PCR reactions revealed a 273 base-pair product of the expected size that was expressed at various levels in the tissues examined. The amplification product was submitted for DNA sequence analysis and the sequence was compared (98% identity) to published gene records using the NIH MegaBlast analysis tools. Preliminary analysis focused on ovary and DLM tissue suggests that apolipophorin and ribosomal RNA are expressed by each tissue. Cycle threshold (CT) values and quantification of ethidium bromide stained PCR amplification product showed no significant differences in levels of apolipophorin transcripts in a subset of cDNA prepared ovaries and DLMs of Day 1 and 3 crickets. Transcriptome sequencing revealed the presence of transcripts for Apolipophorin III identical to the published house cricket sequence and highly homologous for another cricket, Gryllus Bimaculatus. Transcripts were identified from non-degenerating as well as degenerating DLMs in our PCR studies and in our transcritptome analysis. Further analysis will include qPCR with samples collected from a more complete experimental design to allow for direct comparison of muscles and JH status. Overall, our results suggest that expression of apolipophorin in tissues of the cricket is consistent with a role for the protein that is not restricted to lipid transport and that Apolipophorin III could be actively involved in flight muscle histolysis.Not Submitted
Funder Acknowledgement(s): Dr. Samir Raychoudhury, Dean Benedict College, School of STEM, Principal Investigator HBCU-UP NNSF Grant # HRD-1436222.
Faculty Advisor: Rush Oliver, Rush.Oliver@benedict.edu
Role: I performed the dissections, the RNA extractions, helped design and order primers, performed both RT-PCR and Real-time PCR. I helped review results for the transcriptome sequencing using NCBI blast and clustal alignment tools. I also maintained the crickets for the experiments.