Antibody response of bluegill sunfish during development of acquired resistance against the larvae of the freshwater mussel Utterbackia imbecillis.

The larvae of freshwater mussels in the order Unionoida are obligate parasites on fishes, on which they metamorphose into juveniles. Bluegill sunfish (Lepomis macrochirus) acquire resistance against glochidia of the freshwater mussel Utterbackia imbecillis after 2 infections. In order to study the systemic and mucosal antibody response associated with acquired resistance, sera from experimentally infected fish were collected at 10-d intervals during 4 sequential infection periods and from naïve fish. Enzyme-linked immunosorbant assays (ELISA) revealed that fish exhibited a humoral and mucosal antibody response around day 20 after the 1st infection which was followed by second antibody response beginning at day 60 (day 20, 3rd infection) that persisted until the end of the collection period. Western blots of glochidial proteins probed with the sera revealed that the profile of proteins recognized by antibodies produced by fish changed over the course of multiple infections. Serum collected from fish at day 20 (peak of primary response) contained antibodies against approximately 39 and 91 kDa proteins. Immunohistochemical studies on whole-mount glochidia probed with serum from these fish demonstrated that the antibodies recognize granular structures located between the larval mantle and shell. Serum collected from fish during the secondary antibody response (days 60-80) bound additional protein bands in Western blots. Those antibodies recognized other cells of the larval mantle, most prominently in a ciliated region that contains the primordia of the gills and organs of the juvenile and adult mussel.

Encapsulation of attached ectoparasitic glochidia larvae of freshwater mussels by epithelial tissue on fins of naive and resistant host fish.

To metamorphose into juveniles and subsequently mature into adults, the glochidia larvae of freshwater mussels in the order Unionoida must temporarily parasitize the gills, fins, or other external structures of fish. Once attached to the fish, the glochidium is encapsulated by host fish epithelial tissue. The migration of epithelial cells of the bluegill sunfish Lepomis macrochirus over glochidia of Utterbackia imbecillis was examined by time-lapse video microscopy, and the morphology was examined by scanning electron microscopy. Initially, the leading edge epithelial cells migrating over the larvae became rounded and the cells moved as a sheet until the attached glochidium was completely covered. Cyst formation on host fish that had been repeatedly exposed to mussel larvae was significantly delayed and morphologically irregular compared to that on naïve fish. Cyst formation on other species of fish that are less successful as hosts was examined. In general, it took longer for glochidia to become encapsulated on these less suitable potential hosts. The delay and irregularities in cyst formation on resistant fish and nonhost fish species may result in increased mortality and reduced success of metamorphosis of glochidia.

Cross-resistance of largemouth bass to glochidia of unionid mussels.

We tested whether host fish that acquired resistance to glochidia of one mussel species were cross-resistant to glochidia of other species. Largemouth bass (Micropterus salmoides) were primed with 4-5 successive infections of glochidia of Lampsilis reeveiana. The percentage of attached glochidia that survived and transformed to the juvenile stage (transformation success) was compared between primed fish and naïve controls. Transformation success of L. reeveiana, Lampsilis abrupta, Villosa iris, and Utterbackia imbecillis was significantly lower on primed fish (37.8%, 43.5%, 67.0%, and 13.2%, respectively) than on control fish (89.0%, 89.7%, 90.0%, and 22.2% respectively). Immunoblotting was used to analyze the binding of serum antibodies from primed fish with glochidia proteins. Antibodies bound to glochidia proteins of similar molecular weight from L. reeveiana and L. abrupta. Bound proteins of V. iris differed in molecular weight from those of the Lampsilis species. There was no binding to specific glochidia proteins of U. imbecillis or Strophitus undulatus. Our results indicate that host-acquired resistance can extend across mussel genera and subfamilies and might involve both specific and nonspecific mechanisms. Understanding the specificity of acquired resistance of hosts to glochidia could enhance understanding of the evolutionary and ecological relationships between mussels and their host fishes.

Behavioral aspects of host recognition by the symbiotic water mite Unionicola formosa (Acarina, Unionicolidae).

The occurrence and specificity of host recognition behavior of adult and nymphal Unionicola formosa and the capability of adult mites to recolonize various mussel species were examined. Adult U. formosa aggregated on excised tissue from their host mussel, Anodonta imbecilis, in preference to that of two other species of mussels. Nymphs also exhibited an aggregation response to host tissue. A radioisotope (51Cr) technique was used to monitor the recolonization behavior of U. formosa. Adult female mites preferentially re-entered A. imbecilis rather than the sympatric mussel A. cataracta. The specificity of this behavior parallels the distribution of this water mite among potential bivalve hosts in the southeastern U.S. Host recognition by U. formosa may contribute to re-establishing contact with a host after accidental separation and probably helps to maintain mite-mussel symbioses. Whether or not larval U. formosa employ similar host recognition behavior while selecting a potential host has not as yet been determined.

Acquired resistance of bluegill sunfish Lepomis macrochirus to glochidia larvae of the freshwater mussel Utterbackia imbecillis (Bivalvia: Unionidae) after multiple infections.

The effects of multiple infections on the host-parasite relationship between bluegill sunfish (Lepomis macrochirus) and parasitic glochidial larvae of the freshwater mussel Utterbackia imbecillis were examined. Naïve, young-of-the-year bluegills were infected with glochidia and placed in individual observation chambers. Each day, water was drained from each chamber and the numbers of dead glochidia, live glochidia, partially metamorphosed glochidia, and fully metamorphosed juvenile mussels were counted. The same fishes were infected a total of 4 times. After 2 infections, the fish began to exhibit evidence of acquired resistance to glochidia. During the third and fourth infections, this resistance was clearly evidenced by the marked increase in the percentage of dead and live glochidia shed during the first 5 days of the infection and by the significant decrease in the success of metamorphosis. The total number of glochidia that successfully attached to the fish decreased significantly during the fourth infection relative to the first. The number of larvae attached to the host fish was positively correlated with the size of the fish during the first infection but was negatively correlated during all subsequent infections. Variance to mean ratios indicated that larvae were aggregated among host fishes during the infections. This study has important implications in propagation and conservation efforts of this endangered group of organisms.

Persistence of host response against glochidia larvae in Micropterus salmoides.

Host fish acquire resistance to the parasitic larvae (glochidia) of freshwater mussels (Unionidae). Glochidia metamorphose into juvenile mussels while encysted on host fish. We investigated the duration of acquired resistance of largemouth bass, Micropterus salmoides (Lacepède, 1802) to glochidia of the broken rays mussel, Lampsilis reeveiana (Call, 1887). Fish received three successive priming infections with glochidia to induce an immune response. Primed fish were held at 22-23 degrees C and were challenged (re-infected) at intervals after priming. Metamorphosis success was quantified as the percent of attached glochidia that metamorphosed to the juvenile stage and were recovered alive. Metamorphosis success at 3, 7, and 12 months after priming was significantly lower on primed fish (26%, 40%, and 68% respectively) than on control fish (85%, 93%, and 92% respectively). A second group of largemouth bass was similarly primed and blood was extracted. Immunoblotting was used to detect host serum antibodies to L. reeveiana glochidia proteins. Serum antibodies were evident in primed fish, but not in naive control fish. Acquired resistance of host fish potentially affects natural reproduction and artificial propagation of unionids, many of which are of conservation concern.