Many metazoans convert the reproductive modes presumably depending upon the environmental conditions and/or the phase of life cycle, but the mechanisms underlying the switching from asexual to sexual reproduction, and vice versa, remain unknown. We established an experimental system, using an integrative biology approach, to analyze the mechanism in the planarian, Dugesia ryukyuensis (Kobayashi et al., 1999). Worms of exclusively asexual clone (OH strain) of the species gradually develop ovaries, testes and other sexual organs, then copulate and eventually lay cocoons filled with fertilized eggs, if they are fed with sexually mature worms of Bdellocephala brunnea (an exclusively oviparous species). This suggests the existence of a sexualizing substance(s) in sexually mature worms. Random inbreeding of experimentally sexualized worms (acquired sexuals) produces an F1 population of spontaneous sexuals (innate sexuals) and asexuals in a ratio of approximately 2:1. All regenerants from various portions of innate sexuals become sexuals. In the case of acquired sexuals, head fragments without sexual organs regenerated into asexuals though regenerants from other portions became sexuals. Thus, we conclude that neoblasts, the totipotent stem cells in the planarians, of acquired sexuals remain “asexual” and the worms require external supply of a sexualizing substance for the differentiation of sexual organs and gametes. On the other hand, some, if not all, neoblasts in innate sexuals are somehow “sexual” and do not require external supply of a sexualizing substance for the eventual differentiation of themselves and/or other neoblasts into sexual organs and gametes. It is also shown that sexuality in acquired sexuals is maintained by the putative sexualizing substance(s) of their own. The sexualization is closely coupled with cessation of fission, and the worms seem to have an unknown way of controlling the karyotype. Our integrative approach integrates multiple fields of study, including classic breeding, regeneration, and genetics experiments, as well as karyotyping, and biochemical and molecular biological analyses; none of which would have revealed much about the intricate mechanisms that regulate sex and fission in these animals.

Volvox and its relatives provide an exceptional model for integrative studies of the evolution of multicellularity and cellular differentiation. The volvocine algae range in complexity from unicellular Chlamydomonas through several colonial genera with a single cell type, to multicellular Volvox with its germ–soma division of labor. Within the monophyletic family Volvocaceae, several species of Volvox have evolved independently in different lineages, the ultimate cause presumably being the advantage that large size and cellular differentiation provide in competing for limiting resources such as phosphorous. The proximate causes of this type of evolutionary transition are being studied in V. carteri. All volvocine algae except Volvox exhibit biphasic development: cells grow during a motile, biflagellate phase, then they lose motility and divide repeatedly during the reproductive phase. In V. carteri three kinds of genes transform this ancestral biphasic program into a dichotomous one that generates non-motile reproductive cells and biflagellate somatic cells with no reproductive potential: first the gls genes act in early embryos to cause asymmetric division and production of large–small sister-cell pairs; then lag genes act in the large cells to repress the biflagellate half of the ancestral program, while regA acts in the small cells to repress the reproductive half of the program. Molecular-genetic analysis of these genes is progressing, as will be illustrated with regA, which encodes a transcription factor that acts in somatic cells to repress nuclear genes encoding chloroplast proteins. Repression of chloroplast biogenesis prevents these obligately photoautotrophic cells from growing, and since they cannot grow, they cannot reproduce.

The monospecific light organ association between the Hawaiian sepiolid squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri has been used as a model for the study of the most common type of coevolved animal-bacterial interaction; i.e., the association of Gram-negative bacteria with the extracellular apical surfaces of polarized epithelia. Analysis of the squid-vibrio symbiosis has ranged from characterizations of the harvesting mechanisms by which the host ensures colonization by the appropriate symbiont to identification of bacteria-induced changes in host gene expression that accompany the establishment and maintenance of the relationship. Studies of this model have been enhanced by extensive collaboration with microbiologists, who are able to manipulate the genetics of the bacterial symbiont. The results of our studies have indicated that initiation and persistence of the association requires a complex, reciprocal molecular dialogue between these two phylogenetically distant partners.

Cladistic biogeographic methods remain susceptible to the confounding effects of “pseudo-congruence” and “pseudo-incongruence” because they were not designed to incorporate information on the absolute timing of the diversification of lineages. Consequently, results from cladistic biogeographic studies are difficult to interpret and cannot be confidently attributed to any particular cause. We illustrate these points with concrete examples, paying special attention to recent work on the biogeography of the Northern Hemisphere, and outline ways in which topological and temporal information might be better integrated. The development of historical biogeography over the last few decades provides general insights into the nature of integration through the life of a discipline.

Integrative Biology is exemplified by a diversity of recently established collaborations to study the genetic diversity of the European rabbit, Oryctolagus cuniculus. Molecular markers were developed and used to investigate the link between wild population decreases or domestication procedures and possible losses of genetic diversity. Simultaneously, a European programme was launched for the management of genetic resources. The Integrative Biology approach shows that changes in genetic diversity are often buffered by the flexibility of rabbit reproductive systems. It appears, also, that all domestic animals belong to a subset of the wild genetic pool of their species without major loss of diversity despite exposure to severe viral infections. Consequently, management of genetic resources for production purposes and conservation or protection of declining Iberian wild populations require different approaches and measures.

Comparative immunology, derived from zoology and immunology, examines immune systems during evolution. We now know that invertebrates have molecules that share homology with some of those in vertebrates. Acquired immunity first appeared in the vertebrates, but before then innate immune systems had been successfully defending invertebrates and plants against microbial infections for hundreds of millions of years. The germline-encoded receptors of innate systems are relatively limited in diversity and unable to make fine distinctions between closely related structures. Nevertheless, they can recognize certain chemical features shared by groups of microorganisms (e.g., pattern recognition receptors) but not by the host, such as lipopolysaccharide of Gram-negative bacterial cell walls. This capability enables innate immunity to detect the presence of an infection, if not the precise cause—it is thus a biological rather than a structural distinction. Because of its evolutionary success, innate immunity is no longer considered primarily a stopgap measure, a temporary expedient for host defense. It no longer seems to matter that there is an absence of genetic-recombination mechanisms to generate neither specificity nor ‘memory’, because first and second exposures to a microbial substance elicit similar responses. Comparative immunology has enriched the parent field of immunology.

During the evolutionary transition to Metazoa, cell-cell- as well as cell-matrix recognition molecules have been formed, which made a further step in evolution possible, the establishment of an immune system. Sponges [Porifera] represent the oldest still extant metazoan phylum and consequently testify to major features of the common metazoan ancestor, the Urmetazoa. Most studies with respect to evolution and phylogeny in sponges have been performed with the marine demosponges Suberites domuncula and Geodia cydonium. These animals possess effective defense systems against microbes and parasites which involve engulfment of bacteria into specific cells, but also signal transduction pathways which actively kill bacteria. Among those is the LPS-mediated pathway, with the stress-responsive kinases. In addition, sponges are provided with an interferon-related system, with the (2–5)A synthetase as controlling enzyme. Transplantation studies have been performed on tissue, as well as at the cellular level (“mixed sponge cell reaction assay”) which demonstrate the complex molecular strategy by which sponges respond to allogeneic- and/or autogeneic signals. Among the molecules involved in histo(in)compatibility response of sponges, cytokines e.g., the allograft inflammatory factor 1, have been identified which control rejection of allografts. Furthermore, transcription factors, with Tcf-like factor as an example, have been identified which very likely control gene expression during histocompatibility reactions. The immune reactions in sponges can be modulated by FK506, a drug which has been successfully used as immunosuppressant in humans. One further surprising finding is the fact that G. cydonium has several molecules containing polymorphic Ig-like domains of the variable type. It is concluded that the successful evolutionary transition to the Metazoa, with the sponges as the oldest still extant phylum, and the subsequent rapid radiation into the other metazoan phyla, became possible because of the acquisition of modular molecules, involved in cell adhesion and the immune system.

Long before vertebrates first appeared, protists, plants and animals had evolved diverse, effective systems of innate immunity. Ancestors of the vertebrates utilized components of the complement system, protease-inhibitors, metal-binding proteins, carbohydrate-binding proteins and other plasma-born molecules as humoral agents of defense. In these same animals, immunocytes endowed with a repertoire of defensive behaviors expressed Toll-like receptors. They made NADPH oxidase, superoxide dismutase and other respiratory burst enzymes to produce toxic oxygen radicals, and nitric oxide synthase to produce nitric oxide. Antimicrobial peptides and lytic enzymes were in their armory. Immune responses were orchestrated by cytokines. Furthermore, genes within the immunoglobulin superfamily were expressed to meet a variety of needs possibly including defense. However, recombination activating genes played no role. With the acquisition of one or more transposases and the resulting capacity to generate diverse receptors from immunoglobulin gene fragments, the adaptive (lymphoid) arm of the immune system was born. This may have coincided with the elaboration of the neural crest. Naturally, the role of the adaptive arm was initially subservient to the defensive functions of the pre-existing innate arm. The strong selective advantages that stemmed from having “sharp-shooters” (cells making antigen-specific receptors) on the defense team ensured their retention. Refined through evolution, adaptive immunity, even in mammals, remains dependent upon cells of the innate series (e.g., dendritic cells) for signals driving their functional maturation. This paper calls for some fresh thinking leading to a clearer vision of the origins and co-evolution of the two arms of modern immune systems, and suggests a possible neural origin for the adaptive immune system.

Production of antimicrobial peptides and proteins is an important means of host defense in eukaryotes. The larger antimicrobial proteins, containing more than 100 amino acids, are often lytic enzymes, nutrient-binding proteins or contain sites that target specific microbial macromolecules. The smaller antimicrobial peptides act largely by disrupting the structure or function of microbial cell membranes. Hundreds of antimicrobial peptides have been found in the epithelial layers, phagocytic cells and body fluids of multicellular animals, from mollusks to humans. Some antimicrobial peptides are produced constitutively, others are induced in response to infection or inflammation. Studies of the regulation of antimicrobial peptide synthesis in Drosophila have been particularly fruitful, and have provided a new paradigm for the analysis of mammalian host defense responses. It now appears that the general patterns of antimicrobial responses of invertebrates have been preserved in vertebrates (“innate immunity”) where they contribute to host defense both independently and in complex interplay with adaptive immunity.

The involvement of circulating hemocytes as the principal cellular effector mediating molluscan immune responses is well established. They participate in a variety of internal defense-related activities including microbial phagocytosis, multicellular encapsulation, and cell-mediated cytotoxicity reactions that are presumed to be initiated through foreign ligand binding to hemocyte receptors and subsequent transduction of the binding signal through the cell resulting in appropriate (or in some cases, inappropriate) hemocyte responses. At present, however, although functional evidence abounds as to the existence of hemocyte “recognition” receptors, few have been characterized at the molecular level. Similarly, signal transduction systems associated with various receptor-mediated hemocyte functions in molluscs are only beginning to be investigated and understood. This review examines what is currently known about the molluscan hemocyte receptors and the putative signal transduction pathways involved in regulating their cellular behaviors/activities. The cumulative data implies the presence of various hemocyte-associated receptors capable of binding specific carbohydrates, extracellular matrix proteins, growth factors, hormones, and cytokines. Moreover, receptor-ligand interactions appear to involve signaling molecules similar to those already recognized in vertebrate immunocyte signal transduction pathways, such as protein kinases A and C, focal adhesion kinase, Src, Ca² and mitogen-activated protein kinase. Overall, the experimental evidence suggests that molluscan immune responses rely on molecules that share homology with those of vertebrate signaling systems. As more information regarding the molecular nature of hemocyte recognition receptors and their associated signaling molecules is accumulated, a clearer picture of how hemocyte immune responses to invading organisms are regulated will begin to emerge.

Because tunicates rely on innate immunity, their hemocytes are important contributors to host defense. Styela clava, a solitary ascidian, have eight hemocyte subtypes. Extracts of their total hemocyte population contained multiple small (2–4 kDa) antimicrobial peptides. When purified, these fell into two distinct families that were named styelins and clavanins.

Styelins A-E are phenylalanine-rich, 32 residue peptides with activity against marine bacteria and human pathogens. They show considerable sequence homology to pleurocidins, antimicrobial peptides of the flounder, Pseudopleuronectes americanus. Styelin D, one of the five styelins identified by peptide isolation and cDNA cloning, was remarkable in containing 12 post-translationally modified residues, including a 6-bromotryptophan, two monohydroxylysines, four 3,4-dihydroxyphenylalanines (DOPA), four dihydroxylysines and one dihydroxyarginine. These modifications enhanced Styelin D's bactericidal ability at acidic pH and high salinity. A novel histochemical stain for DOPA suggested that Styelin D was restricted to granulocytes.

Clavanins A-E are histidine-rich, 23 residue peptides that are C-terminally amidated and most effective at acidic pH. Clavaspirin is a newly described family member that also has potent cytotoxic properties. By immunocytochemistry, clavanins were identified in the granules of five eosinophilic granulocyte subtypes and in macrophage cytoplasm.

Transmission and scanning electron micrographs of methicillin-resistant Staphylococcus aureus (MRSA) and E. coli that had been treated with Styelin D and clavaspirin suggested that both peptides induced osmotic disregulation. Treated bacteria manifested cytoplasmic swelling and extrusion of cytoplasmic contents through their peptidoglycan cell wall. The diverse array of antimicrobial peptides in S. clava hemocytes constitutes an effective host defense mechanism.

It is widely recognized that humoral and phagocyte-associated lectins constitute critical components of innate immunity in vertebrates and invertebrates. Their functions include not only self/non-self recognition but also engaging associated effector mechanisms, such as complement-mediated opsonization and killing of potential pathogens. One of the unresolved questions concerns the diversity in recognition capacity of the lectin repertoire, particularly in those organisms lacking adaptive immunity. In this paper, we discuss evidence suggesting that lectin repertoire in invertebrates and protochordates is highly diversified, and includes most of the lectin classes described so far in vertebrate species, as well as associated effector pathways.

The prototypic forms of teleost novel immune-type receptors (NITRs) consist of a variable (V) region, a unique V-like C2 (V/C2) domain, a transmembrane region and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibition motifs (ITIMs). NITRs encode diversified V regions in large multigene families but do not undergo somatic rearrangement. Studies in four different bony fish model systems have identified a number of different organizational forms of NITRs. Specifically, NITR genes encode N-terminal ectodomains of the V-type but otherwise vary in the: total number of extracellular immunoglobulin domains, number and location of joining (J) region-like motifs, presence of transmembrane regions, presence of charged residues within transmembrane regions, presence of cytoplasmic tails, and/or distribution of ITIM(s) within the cytoplasmic tails. V region-containing NITRs constitute a far more complex family than recognized originally and currently include individual members that potentially function through inhibitory as well as activating mechanisms. The genomic organization of the NITR gene cluster as well as the structural diversity and overall architecture of the NITR proteins is reminiscent of genes encoded at the mammalian leukocyte receptor cluster (LRC); however, there presently is no functional evidence to support an orthologous relationship between NITR and LRC gene products. Comparisons of the predicted structures of the NITRs have identified several short regions of sequence identity and a novel cloning strategy has been devised that selects for secretory and transmembrane proteins that encode these short motifs. Using this approach, related genes termed immune-type receptors (ITRs) have been identified in cartilaginous fish. Taken together, these studies indicate that leukocyte regulatory receptors, including those that mediate natural killer function, might have emerged early in vertebrate evolution and that the NITR/ITR genes represent a new and potentially highly significant link between innate and adaptive immune responses.

The antigen receptors on cells of innate immune systems recognize broadly expressed markers on non-host cells while the receptors on lymphocytes of the adaptive immune system display a higher level of specificity. Adaptive immunity, with its exquisite specificity and immunological memory, has only been found in the jawed vertebrates, which also display innate immunity. Jawless fishes and invertebrates only have innate immunity. In the adaptive immune response, T and B-lymphocytes detect foreign agents or antigens using T cell receptors (TCR) or immunoglobulins (Ig), respectively. While Ig can bind free intact antigens, TCR only binds processed antigenic fragments that are presented on molecules encoded in the major histocompatibility complex (MHC). MHC molecules display variation through allelic polymorphism. A diverse repertoire of Ig and TCR molecules is generated by gene rearrangement and junctional diversity, processes carried out by the recombinase activating gene (RAG) products and terminal deoxynucleotidyl transferase (TdT). Thus, the molecules that define adaptive immunity are TCR, Ig, MHC molecules, RAG products and TdT. No direct predecessors of these molecules have been found in the jawless fishes or invertebrates. In contrast, the complement cascade can be activated by either adaptive or innate immune systems and contains examples of molecules that gradually evolved from non-immune functions to being part of the innate and then adaptive immune system. In this paper we examine the molecules of the adaptive immune system and speculate on the existence of direct predecessors that were part of innate immunity.

Although the capacity of cells to respond to environmental challenges such as oxidative damage are ancient evolutionary developments that have been carried through to modern higher vertebrates as “innate” immunity, the characteristic immune response of vertebrates is a relatively recent evolutionary development that is present only in jawed vertebrates. The vertebrate “combinatorial” response is defined by the presence of lymphocytes as specific antigen recognition cells and by the complete panel of antibodies, T cell receptors, and major histocompatibility complex molecules all of which are members of the immunoglobulin family. Its emergence in evolution was an extremely rapid event (approximately 10 million years) that was catalyzed by the horizontal transfer of recombinase activator genes (RAG) from microbes to an ancestral jawed vertebrate. RAGs occur in jawed vertebrates, but have not been found in invertebrates and other intermediate species. We propose that antigen recognition capacity contributed by this novel combinatorial mechanism gave jawed vertebrates the ability to recognize the entire range of potential antigenic molecular structures, including self components and molecules of infectious microbes not shared with vertebrates. The contrast within the vertebrates is striking because the most ancient extant jawed vertebrates, sharks and their kin, have the complete panoply of T-cell receptors, antibodies, MHC products and RAG genes, whereas agnathans possess cells resembling lymphocytes but ostensibly lack all of the molecules definitive of combinatorial immunity. Another vertebrate innovation may have been the utilization of nuclear receptor superfamily, in the regulation of lymphocytes and other cells of the immune lineage. Unlike, RAG, however, this superfamily occurs in all metazoans with the exception of sponges.