Immunity has been studied for years in both invertebrates and vertebrates comparing similarities and differences. Even though invertebrate immune systems are less complex than those found in vertebrates, the similarities observed have aided in furthering the research of the more complex immune systems. There have been many invertebrates contributing to important research findings, but recent research with Periplaneta americana, the American cockroach, has pointed to some interesting analogies with the complex mammalian immune system. The American cockroach immune system displays memory and specificity, after immunization with a deactivated bee phospholipase A2 (BPA2) antigen, similar to the mammalian adaptive immune system. Further experimentation has shown that the immunization is mediated by a 102kDa inducible humoral protein. To identify the genes involved in this immunity, a cDNA library was created from BPA2 immunized roaches. Out of 16 total clones, 11 of these were potentially differentially expressed during the immune response. Northern blot analyses and/or semi-quantitative RT-PCR were performed on nine of the eleven clones to further narrow the potential genes involved. One of these clones was shown to be up-regulated in the immune response, yet the gene transcript was not complete. Two additional clones, not previously confirmed, as well as this clone were going to be subjected to further experimentation. Due to some problems in past experiments with immunization, another control was going to be implemented. A lethal level of the active form of the antigen BPA2 was going to be injected in a small population of experimental and control roaches during the harvest period to determine immunity. Also, a time course study was going to be developed in order to determine the best time to harvest the mRNA for future genomics experiments. These experiments were unable to be completed at the present time due to an opportunistic pathogen Escherichia blattae, which contaminated the entire roach colony.

The evolution of immunity has been difficult to unravel, particularly when considering the relationship between invertebrates and vertebrates. Innate (nonspecific) antimicrobial factors, first found in insects, were once thought to be limited to invertebrate immunity. However, recent research has discovered that these factors, and their gene regulation, are employed in vertebrate immunity as well. While immunologists now agree that both of these groups share aspects of innate immunity, few are willing to consider adaptive (specific) immunity can occur in invertebrates. Nevertheless, it has been demonstrated that an inducible humoral protein in the American cockroach displays immunological memory and specificity, which are hallmarks of adaptive immunity, against soluble protein antigens such as bee phospholipase A2 (BPA2). Furthermore, this response has been linked with a 102kDa protein subunit in the hemolymph of immune animals as revealed by SDS-PAGE analysis. Molecular characterization of this response had been initiated with the creation of three? phage cDNA libraries (A-C) made from cockroaches injected with BPA2. The libraries were tested for cDNA insertion and length by PCR analysis. Library C had the best results, with every plaque tested possessing an insert with an average length of 0.9 kb. Therefore, library C was used in a differential screening, yielding 25 plaques (with multiple phage/plaque) appearing to be upregulated in BPA2-injected animals compared to saline controls. A secondary screening of these 25 plaques was completed yielding 20 plaques with the desired single clone/plaque. Restriction digests helped identify 4 duplicate clones, leaving 16 clones to pursue. Six of these were successfully subcloned, sequenced, and analyzed with BLAST 2.0. Three clones were artifacts (16S rRNA), while the other three were of interest. Clone 1had no homology to any protein in the NCBI database, clone 2 was identified as clone PL25 (an American cockroach lectin-related protein), while clone 11 possessed some identity (39%) with vitellogenin from the American cockroach. Northern blot analysis, of these 3 clones and any clones of interest derived from the remaining 10 clones needing to be sequenced, should confirm proteins upregulated in an immune response against BPA2. Characterization of these sequences should be useful in helping to understand the adaptive immune response in the American cockroach. The results will also be valuable for comparing this response to the one characterized in vertebrates, and for stimulating research regarding the possibility of adaptive immunity in other long-lived invertebrates as well.

The study of insect immunity has traditionally focused on innate immune responses, under the longstanding assumption that insects are incapable of generating an anticipatory immune response. Research in our lab has focused on a specific, adaptive response in Periplaneta americana, the American cockroach. This response was shown to be specific and exhibit immunological memory in survivorship studies. Protein factors in cell-free immune hemolymph demonstrated the ability to precipitate antigen and confer immunity on nav̐e roaches. One reduced protein (102 kDa) was implicated in the immune response due to its enhancement in immune hemolymph and association with antigen binding. The goals of the research presented in this dissertation were to characterize the 102 kDa protein, identify and characterize other proteins involved in antigen binding, and to illuminate the relationship between reduced antigen binding proteins and native bands capable of antigen binding. In order to identify the 102 kDa protein, a combination of preparative electrophoresis and MALDI mass spectrometry was employed. A positive match was achieved between the 102 kDa protein and a 40-45 kDa region of vitellogenin from the American cockroach. Affinity chromatography using immunizing antigen was used to determine other proteins involved in antigen binding, including reduced proteins at 115 kDa and 95 kDa. Isolation and MALDI mass spectrometry analysis of these proteins yielded a positive match for the 115 kDa protein with vitellogenin-2 from the American cockroach, but no significant match for the 95 kDa protein. Schiff's staining and enzymatic deglycosylation of the antigen binding proteins, 115, 102, and 95 kDa, indicated that all three were glycosylated. Analysis of native protein bands indicated the presence of the immune associated proteins in native bands 2 and 4 (the native bands capable of binding antigen). The above research strongly indicates a role for a vitellogenin-like protein in the adaptive immune response to soluble protein antigen in the American cockroach, and links the native antigen-binding bands with reduced proteins at 115, 102, and 95 kDa.