Laboratory longitudinal diffusion tests: 2. Parameter estimation by inverse analysis

This study focuses on the verification of test interpretations for different state analyses of diffusion experiments. Part 1 of this study identified that steady, quasi-steady and equilibrium state analyses for the through- and in-diffusion tests with solution reservoirs are generally feasible where the tracer is not highly sorptive. In Part 2 we investigate parameter identifiability in transient-state analysis of reservoir concentration variation using a numerical approach. For increased generality, the analytical models, objective functions and Jacobian matrix necessary for inverse analysis of transient-state data are reformulated using unified dimensionless parameters. In these dimensionless forms, the number of unknown parameters is reduced and a single dimensionless parameter represents the sorption property. The dimensionless objective functions are evaluated for individual test methods and parameter identifiability is discussed in relation to the sorption property. The effects of multiple minima and measurement error on parameter identifiability are also investigated. The main findings are that inverse problems for inlet and outlet reservoir concentration analyses are generally unstable and well-posed, respectively. Where the tracer is sorptive, the inverse problem for the inlet reservoir concentration analysis may have multiple minima. When insufficient measurement data is collected, multiple solutions may result and this should be taken into consideration when inversely analyzing data including that of inlet reservoir concentration. Verification of test interpretation by cross-checking different state analyses is feasible where the tracer is not highly sorptive. In an actual experiment, test interpretation validity is demonstrated through consistency between theory and practice for different state analyses.     

Anti-sense expression of putrescine N-methyltransferase confirms defensive role of nicotine in Nicotiana sylvestris against Manduca sexta

Summary. Several lines of evidence support the defensive function of nicotine production in the Nicotiana genus against a range of herbivores, but the evidence is largely correlative. To suppress nicotine production in planta and to test its defensive function, we expressed DNA of putrescine N-methyl transferase in an anti-sense orientation (AS-PMT) in N. sylvestris and fed leaf material from two lines of transformed and wild type plants to Manduca sexta larvae. Larvae consumed more leaf area and gained more mass on the foliage of plants with low PMT expression and low nicotine levels as compared to plants with high PMT expression and high nicotine levels and wild type plants. Overall, larval consumption and performance were negatively correlated with constitutive nicotine levels. We conclude that nicotine decreases the palatability of N. sylvestris leaves to the nicotine-resistant M. sexta larvae.     

Hepatopancreatic endosymbionts in coastal isopods (Crustacea: Isopoda), and their contribution to digestion

Three isopod species (Crustacea: Isopoda), commonly found in the intertidal and supratidal zones of the North American Pacific coast, were studied with respect to symbiotic microbiota in their midgut glands (hepatopancreas). Ligia pallasii (Oniscidea: Ligiidae) contained high numbers of microbial symbionts in its hepatopancreatic caeca. Numbers of endosymbionts were strongly reduced by ingestion of antibiotics. By contrast, the hepatopancreas of Idotea wosnesenskii (Valvifera: Idoteidae) and Gnorimosphaeroma oregonense (Sphaeromatidea: Sphaeromatidae) did not contain any microbiota. Results of feeding experiments suggest that microbial endosymbionts contribute to digestive processes in L. pallasii, the most terrestrial of the three isopods that we studied. The acquisition of digestion-enhancing endosymbionts may have been an important evolutionary step allowing isopods to colonize terrestrial habitats where relatively indigestible leaf litter is the primary food source. By contrast, the ability to digest phenolic compounds was most developed in one of the more marine species, suggesting that this trait may have evolved independently in isopod species that consume a phenolic-rich diet, whether in marine habitats or on land. Received: 28 August 2000 / Accepted: 8 December 2000     

Potential dietary effects on the fatty acid composition of the common jellyfish Aurelia aurita

Fatty acid composition of the natural and aquarium-reared common jellyfish Aurelia aurita was investigated. Fatty acid composition of the aquarium-reared A. aurita clearly reflected that of the diet, brine shrimp (Artemia). In the same way, fatty acid composition of the natural A. aurita was assumed to reflect those of natural diets. Samples of natural A. aurita were collected from April 1995 to September 1995 in the Seto Inland Sea, Japan, and their fatty acids were analyzed by gas chromatography and mass spectrometry. Variation of fatty acid compositions was seasonal rather than dependent on body size. Two major seasonal groups were divided by the cluster analysis of the A. aurita fatty acid composition: the April–June and the August–September clusters. The April–June cluster was characterized by high contents of the (n − 3)-fatty acids of diatom origin, accumulated via the grazing food chain. By contrast, the August–September cluster was characterized by an increase in (n − 6)-fatty acids of macroalgal origin, probably transferred via the detritus food chain. These results suggest that the diet of natural A. aurita may shift between the diatom-based food chain and the detritus-based food chain. Received: 12 April 2000 / Accepted: 1 December 2000     

Effects of salinity on endogenous rhythm of the Manila clam, Ruditapes philippinarum (Bivalvia: Veneridae)

The Manila clam Ruditapes philippinarum, an intertidal bivalve, was exposed to different salinity regimes (from 31.0–31.7‰ down to 20‰, 15‰, 10‰, 5‰), and the endogenous rhythm in its oxygen consumption was studied using an automatic intermittent-flow respirometer. When exposed to salinities reduced from 31.5‰ to 20‰ and 15‰ under otherwise constant conditions, the clams recovered a clear endogenous circatidal rhythm in their oxygen-consumption rate after having dampened periods of 12 h and 48 h, respectively. At salinities less than 10‰, however, the oxygen-consumption rate was depressed greatly at the beginning of the experiment for about 36 h and then increased to a level higher than normal, but the rhythm of oxygen consumption was not recovered. The results of this study indicate that the Manila clam, a euryhaline organism, cannot maintain a normal metabolic activity at a salinity lower than 15‰. All clams were dead after exposure at a salinity of 5‰ for 7 days. Received: 28 February 2000 / Accepted: 26 August 2000     

Possible involvement of the glycocalyx in the ichthyotoxicity of Chattonella marina (Raphidophyceae): immunological approach using antiserum against cell surface structures of the flagellate

Although the ichthyotoxic mechanism of Chattonella marina is still unknown, several lines of evidence suggest that the reactive oxygen species (ROS), such as superoxide anion (O₂^-), hydrogen peroxide (H₂O₂)_, and hydroxyl radical (·OH), produced by C. marina are involved in the mortality of fish exposed to this flagellate. Recently, we found that the cell-free supernatant prepared from C. marina, which is considered to contain the glycocalyx, showed NADPH-dependent O₂^- generation. In this study, we prepared antiserum against the crude glycocalyx of C. marina. Using indirect immunofluorescence, it was confirmed that the antiserum specifically reacted with C. marina cells. In addition to C. marina, the antiserum also reacted with other raphidophycean flagellates such as Heterosigma akashiwo, Olisthodiscus luteus, and Fibrocapsa japonica, whereas no reactivity was observed against six other flagellate species tested. These results suggest that raphidophycean flagellates have common epitopes recognized by the antiserum. Interestingly, immunohistochemical analysis of paraformaldehyde-fixed gill lamellae from yellowtail exposed to C. marina revealed that the antiserum stained the surface of gill lamellae, while no such staining pattern was observed in control gill lamellae. These results suggest that the glycocalyx may be discharged when C. marina cells are inhaled into the fishes' mouths and then come into contact with the gill surface. Based on the present results, together with our previous findings, we propose that continuous accumulation of the discharged glycocalyx on the gill surface occurs during C. marina exposure, which may be responsible for the ROS-mediated severe gill tissue damage leading to fish death.     

Regulation of ovary activation in worker honey-bees (Apis mellifera): larval signal production and adult response thresholds differ between anarchistic and wild-type bees

One-day-old anarchistic (selected for successful worker reproduction) and wild-type honey-bee workers were introduced into queenright colonies of honey-bees of two treatments. In treatment 1, all eggs and larvae were offspring of queens from an anarchistic line. In treatment 2, all eggs and larvae were offspring of wild-type queens. In both treatments, adult workers were wild type. This experimental arrangement was used to test the importance of larval genotype on ovary activation in young adult workers. After 12 days, the introduced bees were dissected to determine the frequency of ovary activation. In those colonies provided with wild-type brood, 0% of introduced wild-type bees and 16% of anarchistic bees had activated ovaries. In those colonies provided with anarchistic brood, 13% of introduced wild-type bees and 41% of anarchistic bees had activated ovaries. These results strongly support the hypothesis that selection for high levels of worker reproduction in anarchistic stocks has reduced the amount or composition of brood pheromones produced by larvae that normally signal workers to refrain from reproduction. They also suggest that anarchistic workers have a higher threshold for these signals than wild-type bees.