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An investigation of the cellular mechanism for ammoniagenesis in a freshwater teleost, the channel catfish, provided the basis for research performed in this thesis. Activities and subcellular localizations of enzymes associated with nitrogen metabolism were measured in catfish liver. Glutamate dehydrogenase (the enzyme responsible for ammonia generation during transdeamination) and AMP deaminase (the ammonia releasing enzyme of the purine nucleotide cycle) were both present in quantities sufficient to account for ammoniagenesis by the catfish. Neither glutamine synthetase nor carbamyl phosphate synthetase (enzymes responsible for "detoxication" of intramitochondrially generated ammonia in uricoteles and ureoteles) were detectable in catfish liver mitochondria. The role of the purine nucleotide cycle was investigated through the use of heavy isotope tracer studies. If the purine nucleotide cycle were operative in fish liver, incubation of hepatocytes with ('15)N alanine should result in the incorporation of the amino nitrogen of alanine into the 6-amino function of AMP (an intermediate in the purine nucleotide cycle). No significant incorporation of the heavy isotope was observed in either the total adenine nucleotide pool or the smaller AMP pool. Experiments using hepatocytes from both the rat (a ureotele) and the chicken (a uricotele) likewise did not show incorporation. End-products isolated from hepatocyte preparations of all three animals, however, exhibited marked incorporation (over 80 percent) of the heavy isotope. Based on these results, a major contribution by the purine hucleotide cycle to ammoniagenesis in fish was ruled out. Preliminary studies utilizing isolated mitochondria examined the alternative pathway for ammoniagenesis, transdeamination. Isolated catfish mitochondria liberated ammonia from glutamate, glutamine, and alanine, although the mitochondrial rates were lower than those obtained using hepatocytes. Various metabolic inhibitors (bromofuroate, an inhibitor of glutamate dehydrogenase; and aminoxyacetate, which inhibits transminases) were used to determine their effect on the capability of mitochondria to produce ammonia. The results obtained in these studies imply a central role for glutamate dehydrogenase during both oxidation and ammonia release from amino acids. Data presented in this thesis effectively eliminate the purine nucleotide cycle as a viable mechanism for catabolizing amino acids in catfish liver. If transdeamination is the pathway utilized in fish during ammoniagenesis, the process of ammonia release from the mitochondria may be different than the mechanism used by ureoteles or uricoteles.

The restriction endonuclease MboII recognizes a nonpalindromic, pentanucleotide DNA sequence and cleaves both strands of the DNA eight bases downstream, leaving a single 3' protruding base. MboII is one of a group of restriction enzymes which has its site of cleavage removed from its recognition sequence. Insertions of DNA sequences into the MboII cleavage site will not disrupt the recognition site allowing further additions to be performed. Several synthetic oligonucleotides containing MboII sites in position for DNA manipulations. These oligonucleotides have been cloned into a plasmid vector which has no MboII sites. The plasmids constructed with the synthetic oligonucleotides allow a DNA sequence to be inserted into the MboII site, without disrupting the MboII recognition sequence. Following isolation of the recombinant plasmids, further insertions have been performed, thus juxtaposing DNA sequences without the need for compatible restriction enzyme sites. In addition, a small DNA fragment has been constructed. This sequence consists of the lac operator region, so that it may be easily screened, surrounded by two MboII recognition sequences. This DNA fragment has been randomly inserted into plasmid DNA using DNAase I. The MboII recognition sequences face outward and will cleave into surrounding DNA, removing up to 14 base pairs. This allows specific deletions to be performed, as well as insertion of DNA fragments into the cleaved DNA. Because of the mode of action of MboII, the entire sequence can be removed from the region of insertion. Some of the plasmids constructed in this thesis had unusual regions surrounding the origin of replication. This area was much smaller than had been previously reported necessary for stable replication. The stability of the plasmids in different strains was found to vary and they had a much higher copy number than pBR322 in exponentially growing cells, but did not amplify to the same extent as pBR322. The plasmids were found to be compatible with pBR322.

During normal embryogenesis in Rana pipiens the primordial germ cells (PGCs) undergo a displacement from deep within the ventral endoderm of the Stage 17 tailbud embryo to the dorsal endodermal crest of the hatched Stage 20 larva; eventually they exit the endoderm and populate the paired gonadal primordia. This study examines the migratory properties of PGCs and the mechanisms of their displacement by temporally uncoupling PGC migration from the morphogenetic events that normally accompany it. The methodology consisted of reciprocally transplanting germ cell-containing endoderm regions between Stage 17 embryos and Stage 20 larvae. The hosts were UV-irradiated to prevent their contribution to the gonadal germ cell population. Migration from the donor graft was determined by direct gonadal germ cell counts at host Stage 25. Normal Stage 20 PGCs transferred to 2 1/2 day younger Stage 17 hosts were able to colonize the gonadal ridges of the heterochronic graft larvae. The number of gonocytes present was approximately 20% of the unoperated control number. These data and the histological study of the heterochronic larvae (Subtelny and Penkala, 1984), demonstrate that PGCs can be experimentally retained within the endoderm for at least 2 1/2 days and are still able to migrate to the gonadal ridges. Reciprocal transfers of Stage 17 PGCs into Stage 20 hosts resulted in colonization of the host gonadal ridges 2 1/2 days earlier than would normally have occurred. The proportion of PGCs occupying the host gonadal ridges was significant, but restricted. Nevertheless, the results show that (1) intraendodermal PGCs can colonize gonadal ridges at least 2 1/2 days earlier than they normally would and (2) they do so in the absence of the main events of embryogenesis that occur during intraendodermal migration (Stages 17 to 20). Under conditions of temporally disengaged morphogenesis, the PGCs in either form of heterochronic graft emerge from the endoderm during a restricted time of development of the host, corresponding to Stage 22+/23-. This emergence is associated with the movements of mesodermal cells of the host lateral plates involved in dorsal mesentery formation. These findings support the hypothesis that morphogenesis plays a major role in the migration of PGCs from the endoderm to the gonadal ridges. (Abstract shortened with permission of author.)

Group living is intriguing because animals that live together incur automatic costs but gain no automatic benefits. Chief among these costs are increased competition for opportunities to reproduce, and increased parasite and disease transmission. I investigate some factors that favor group living in Polistes wasps in the face of these costs, and two arenas of competition to reproduce within groups. Reviewing my own work and the literature, I show that groups withstand predation better than single females, suggesting that predation is a fundamental cause of group nesting. Group nesting females are shown to have emerged from the same nest and to nest near their natal-nest site. This suggests that relatedness and dispersal are important factors. On balance, the evidence indicates that queens do not manipulate their progeny to predispose some of them to becoming subordinate group members. Reproductive conflict is expected among females that have cooperated in starting a new nest since the queen lays most of the eggs. During the period in which the first workers emerge, subordinate nest foundresses of P. exclamans did not change their behavior in ways which would increase their chances of laying eggs. They continued to work for the colony and suffered higher mortality after worker emergence than they had before. This continued altruism may be favored because it increases the otherwise low probability that nests of this species have of producing offspring. Intense reproductive conflict is also expected when a queen dies and workers vie to become the new queen. I simulated queen death by removing queens from 20 nests of P. instabilis. The result was consistent among the 3 study sites which ranged from tropical to temperate; one of the oldest workers became the new queen. All new queens had ranked second in the dominance hierarchy to the original queens. Age was more important than size in determining dominance, and the identity of the new queen. Large females that probably could win high rank by fighting may be prevented from doing so by the likelihood that this would cause widespread fighting among nestmates thereby greatly lowering the fitness of the new queen.

Parvalbumins are low molecular weight, water soluble sarcoplasmic proteins that are found in abundance in the fast twitch muscles of fish and amphibia. They are further characterized by a high affinity for calcium, acidic nature, polymorphism, unusual amino acid composition, unique ultraviolet absorption spectrum and high antigenicity. Parvalbumins are believed to serve in the rapid removal of calcium ions from troponin C during the relaxation phase of fast twitch muscle. Parvalbumins are species-specific for each species of fish in terms of these physico-chemical characteristics. In view of this specificity and of the protein's considerable antigenicity, it was proposed to employ the parvalbumin from red drum (Sciaenops ocellata) as the basis for an immunological field test to identify the red drum species from muscle samples. The ability to identify red drum from only a sample of muscle tissue would allow game law enforcement personnel to screen contraband fish fillets on site. A field test as described would greatly strengthen enforcement efforts of parks and wildlife personnel. The major component parvalbumins were isolated from red drum and spotted seatrout (Cynoscion nebulosus) by gel filtration and ion-exchange chromatography, and antisera were produced in two rabbits against the red drum parvalbumin. This antisera was then examined for cross-reactivity against the parvalbumin from spotted seatrout and against the myogens of the following species of fish: sand seatrout (Cynoscion arenarius), black drum (Pogonias cromis), southern kingfish (Menticirrhus americanus), freshwater drum (Aplodinotus grunniens), vermillion snapper (Rhomboplites aurorubens), sheepshead (Archosargus probatocephalus) and southern flounder (Paralichthys lethostigma). The antisera were examined by double diffusion in gels, by interfacial ring testing, and by enzyme-linked immunosorbent assay (ELISA). In double diffusion in gels, the anti-red drum parvalbumin antiserum reacted specifically with its homologous antigen when the antiserum used was the five-week post primary inoculation antiserum. As the antiserum matured, following secondary and tertiary inoculations with antigen, the specificity lessened and the antiserum reacted in varying degrees with the heterologous antigens. With ELISA, reactivity of the antiserum was demonstrated against all antigens tested, regardless of the stage of the antiserum.

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