We found that the sampling of tissues that do not result in the death of the fish, such as scale and fin tissue, may be substituted for muscle tissue in stable isotope analysis (SIA) of fishes. Comparisons were made between the values of δ13C and δ15N found in muscle tissue with the corresponding scale tissue of three sunfish species (bluegill Lepomis macrochirus, pumpkinseed L. gibbosus, and redbreast sunfish L. auritus) and with caudal fin tissue of slimy sculpin Cottus cognatus. The fish showed strong linear correlation in δ13C values between their nonlethally sampled scale or fin tissue and their muscle tissue (combined sunfish: r = 0.97; slimy sculpin: r = 0.84). Sunfish δ13C values were higher in scale tissue than in muscle tissue and required a correction factor for converting the scale values to the muscle values (regression equation: y = 1.1673x + 1.0531). Slimy sculpin δ13C fin and muscle values were similar and did not require a correction factor. The correlation of δ15N values between the tissues was also strong in both sunfish (r = 0.94) and slimy sculpin (r = 0.90). A correction factor was needed to convert δ15N values from scale to muscle in the three sunfish species (y = 0.8504x + 2.6698) and from fin to muscle in slimy sculpin (y = 1.2658x − 3.3234). Results of this study and other literature support the use of nonlethally sampled tissues for SIA of fish. These methods may be used for investigations of rare and endangered species and also allow for analysis of archived fish scales.

Tobacco (Nicotiana tabacum) pith explants were grown on manganese containing medium. At moderate concentration (10 millimolar), manganese selectively inhibited chlorophyll synthesis, resulting initially in growth of white callus. Several weeks later the white callus turned brown due to the accumulation of a pigment identified as protoporphyrin IX by its elution profile using high performance liquid chromatography, by its absorption spectrum, and by its fluorescence properties. At a concentration of 100 millimolar manganese the pigment accumulated without growth of the explant.Manganese has a vital role in plants as an essential cofactor for 02 evolution. Excess Mn, however, can be toxic. Some Extracts of the gray-brown callus show that most photosynthetic pigments (both Chl and carotenoids) are missing or drastically reduced, as monitored by absorption at 445 nm of their elution profile (Fig. Ia). Monitoring the A at 400 nm, however, reveals other pigments, one major and several minor peaks. We identified the major peak as protoporphyrin IX, as its absorption spectrum and elution profile are identical to those of purified standard. A comparison of their absorption spectra from the HPLC is shown in Figure 2. Both have a single main peak at about 400 nm, with very minor peaks at about 500, 535, and 575 nm. In addition, both the purified standard and the main (Fig. 3) pigment in the medium around them. Growth inhibition and accumulation of a reddish-brown pigment in tobacco callus exposed to 10 and 100 mm Mn have recently been reported also by Petolino and Collins (6).The amount of protoporphyrin IX-like pigment present in callus was determined using a standard curves from HPLC chromatography and fluorimetry. Mn treated callus contained 0.2 to 0.4 nmol (0.1-0.2 ug)/g wet weight, compared to 0.01 to 0.02 nmol (0.005-0.01 fig) for control callus.The accumulation of more protoporphyrin IX than Mg protoporphyrin IX monomethyl ester could be explained by two factors. First, the Mg may be lost in the tissue; Mg protoporphyrin in plastids is reported to be photolabile (2). Second, with decreasing cell growth, less protoporphyrin IX would be drawn off for heme synthesis.Our results support the hypothesis that Mn toxicity is mediated, at least in part, by inhibition of Chl synthesis. Chl synthesis is more sensitive to Mn inhibition than growth, since growth proceeds at Mn levels which completely inhibit Chl synthesis. However, carotenoid accumulation is also apparently inhibited by Mn, either directly or by the absence of a Chl precursor.Using absorption at 400 nm but not 445 nm and the elution profile (2.7 min) as a method of detection, the protoporphyrin IX-like pigment was also found in callus growing on normal medium (Fig. lb)

The photosynthetic rates of intact sporophytes or gametophytes of the fern Todea barbara grown in sterile culture were measured using an infrared gas analyzer. We have overcome some of the attendant difficulties of comparing sporophyte and gametophyte photosynthesis by using cultured sporophytes and gametophytes in sterile tube culture where conditions can be controlled and the two life cycle stages compared under identical conditions. The entire plant is used in both gametophyte and sporophyte gas exchange measurements, thereby making the comparison of the two more physiologically meaningful in the sense that the root system and stem are included as an integral part of the sporophyte plant being measured. MATERIALS AND METHODSCultures of Todea barbara L., originally obtained from R. H. Wetmore, were grown on a medium (7) consisting of 6.25 mm NH4NO3, 1.47 mm KH2PO4, 0.81 mM MgSO4, and 0.51 mm CaCl2, supplemented with 0.033 mm ferric citrate, 1 ml/l trace element solution (8), and 0.8% (w/v) agar (pH 5.5). Sporophytes and gametophytes were separated from mixed cultures, grown for several weeks, and transferred to slightly slanted tubes of fresh medium where they were allowed to grow for at least 2 weeks before measurements were taken. Gametophytes in sterile culture are similar in size and morphology to those in nature. Cultured sporophytes are much smaller than naturally occurring plants and their leaves tend to be juvenile in form. However, they have roots, stems, and leaves in roughly the same proportion as naturally occurring sporophytes. Sporophyte tubes contained one complete plant with roots and six to eight leaves, the largest leaf being 3-4 cm in length. Gametophyte tubes contained several gametophytes which covered the surface of the agar. Cultures were maintained at 22 C and a combination of cool-white fluorescent and incandescent lamps at an intensity of 160 ,uE m-2 s-' was used for illumination on a 12-h on, 12-h off cycle. Cultures were grown in 25-mm tubes in which the agar surface was slanted so that they could be illuminated at the same angle during growth and during the measurement of photosynthesis.Photosynthetic rates were determined by measuring the change in CO2 concentration in a tank air mixture (0.033% CO2, 21% 02,

The light-induced transient states of chlorophyll-protein 668 (Cp668) and its photoconverted form Cp743 were investigated using flash photolysis. Short lived transient species induced by a short flash were detected in both Cp668 and Cp743. The Cp668 transient had a half decay time of 2.0 milliseconds and showed a broad absorption band at 460 nanometers. The Cp743 transient had a half decay time of only 0.6 millisecond and had a major absorption peak at 410 nanometers in addition, to a broad absorption band around 530 nanometers. Both transient signals were quenched by oxygen. Cp668 had a temperature-dependent delayed fluorescence at room temperature with a half-life of 2.0 milliseconds, the same as the life-time of the absorption transient. This suggests that the transient species observed was a triplet state of chlorophyll.The light-induced transients of both Cp668 and Cp743 were formed with high efficiency. A very low quantum efficiency was found for the photoconversion of Cp668 to Cp743 suggesting another intermediate in the conversion sequence. The photoconversion reaction requires oxygen suggesting that an intermediate in the reaction sequence might be superoxide or singlet oxygen.A Chl-protein complex absorbing light at 668 nm was first discovered by Yakushiji and co-workers in the leaf extract of Chenopodium album (15). It was identified as Chl-protein 668 or Cp668 because of its absorption maximum at 668 nm (15). Later the protein was found to be widely distributed among other plants, particularly in the family of Chenopodeaceae including Atriplex and in related families (12).The protein has several unique properties. In the presence of oxygen, Cp668 is photoconverted to another form called Cp743 with the major A peak shifted to 743 nm from the original at 668 nm. The fully converted form, Cp743, still has some A remaining at 668 nm. The protein is water-soluble, extractable from macerated plants in dilute buffer without any detergents. The weight of the protein is about 78,000 daltons and it contains both Chl a and Chl b (13). The protein is remarkably stable in the dark, and is also particularly resistant to heat treatment (10).Most of the interest in Cp668 has centered on its photoconvertibility. Takamiya and co-workers (13) Cytf (8). The cellular location of Cp668 was found to be in the chloroplasts (1).In order to understand more fully the photochemistry of Cp668 we have examined the light-activated transient state of both Cp668 and Cp743 using flash photolysis and delayed fluorescence measurements. Measurement of the quantum yield of photoconversion of Cp668 was also made. MATERIALS AND METHODSCp668 was prepared from the buffer extract of macerated stems of young plants of Atriplex rosea. The extract was precipitated between 0.3 and 0.7 ammonium sulfate saturation. The precipitate was resuspended in 0.1 M phosphate buffer (pH 7.2) and dialyzed against the same buffer. The dialysate was then heated at 60 C for 5 min. Centrifugation of the sample at 144,000g for 40 min removed denatured pr...

1. In a study of five ponds sensitive to add precipitation, we document seasonal , acidification profiles and assess the impact of short-term acid pulses on the reproductive success of resident sunfish (Lqyomis spp.). 2. Three years of water sampling at 2-3 week intervals showed substantial seasonal variation in pH and alkalinity consistent with a carbonate buffering system. Though all ponds shared a common seasonal pattern in pH, ponds with relatively low pH and alkalinity showed the greatest variation in these parameters. Spring minima may dispose some of the ponds towards episodes of extreme acidity during heavy spring rains.3. Otolith analysis of young-of-the-year sunfish revealed recruitment failures for eggs laid early in spring in ponds with relatively low alkalinity and pH, and, in the most extreme case, missing day classes at subsequent irregular intervals even though average pH and alkalinity were well above those demonstrated to affect centrarchid fishes. 4. Age-class distributions of sunfishes revealed gaps in adult age distributions which could be traced through 3 years of the study, but there was no clear-cut relationship between pond acidification and tbe age structure of adult fish. 5. Seasonal profiles of acidity may enable researchers to predict the time during which a pond or lake may be highly sensitive to add inputs. Comparative otolith analysis of young-of-the-year fishes and short-term continuous monitoring of water chemistry may provide an early warning of biological effects of addification in sensitive bodies of water.