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.

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 biological bases of invertebrate immune responses have interested scientists for decades, from the first relevant observation by E. Metchnikoff in 1882, who discovered phagocytosis while studying starfish larvae. Invertebrate immunology first began to be appre ciated as an important field in the late 1960s and 1970s. However, in the following years there was much controversy regarding the question: do invertebrates offer insight into the origin of the sophisticated immune responses of the vertebrates? There are several reasons why progress in research on invertebrate immune competence has been painfully slow. One of the main impediments to the progress, as compared to the fast development of knowledge in the vertebrate systems, was the fact that most of the studies concentrated on "whole organism" assays, mainly on grafting tissues between allogeneic partners. Only in the last few years have more and more aspects of invertebrate immunity been investigated on the cellular, biochemical and molecular levels. These studies led to discoveries of novel defense reactions, new pathways of effector mechanisms which are elicited after recognition of "nonself', and complex, sometimes highly polymorphic genetic elements that control invertebrate immune reactions. The importance of invertebrate immunity for understanding "immunology" as a whole, despite the conflicting models and hypotheses, is now much more recognized than before. Although most of the 20 phyla belonging to the inver tebrates have different modes of life, body organizations, habitats occupied, and biochemical patterns, they show striking aspects of exceptional precision for discriminating between self and nonself.

E. L. Cooper The Immunodefense System Because invertebrates are exceedingly diverse and numerous, estimates reveal nearly 2 million species classified in more than 20 phyla from unicellular organisms up to the complex, multicellular protostomes and deuterostomes. It is not surprising to find less diverse defense/immune responses whose effector mechanisms remain to be completely elucidated. Of course, I am not advocating that the few of us devoted to analyzing invertebrate immunity attempt the Herculean task of examining all these species to uncover some kind of unique response! As these two volumes will reveal, we are doing fairly well in examining in depth only the most miniscule examples of invertebrates, some of which have great effects on human populations such as edible crustaceans or insect pests. This is in striking contrast to the mass of information on the mammalian immune response which has been derived essentially from the mouse, a member of one phylum, Vertebrata, an approach, reductionist to be sure, but one that has served well both the technological and conceptual advances of immunology as a disci pline. The essential framework of immunology, the overwhelming burst of results since the 1960s, have emanated primarily from this single animal. We should not forget the thymus and the bird's bursa of Fabricius, without which we might have been slower to recognize the bipartite T /B system.