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Chromosome banding in salmonid fishes: nucleolar organizer regions in Oncorhynchus.

Chromosome banding patterns obtained by silver staining and chromomycin a3 (CMA3) staining were analyzed in six species of Oncorhynchus: O. tshawytscha, O. kisutch, O. keta, O. nerka, and O. gorbuscha from North America and O. masou from Japan. Four different chromosomal locations of the nucleolar organizer regions (NORs) were found in different species. In O. tshawytscha, O. kisutch, and O. masou the NORs comprised the entire short arms of one medium-sized acrocentric chromosome pair. In O. nerka the NORs were found in an interstitial band on the short arms of one submetacentric chromosome pair and in O. gorbuscha proximal to the centromere on one metacentric chromosome pair. In O. keta the NORs were found on the telomeres of one small submetacentric chromosome pair. As in the related genera Salmo and Salvelinus chromomycin A3 positive bands were found at the same sites as the AgNORs in all species. Salmonid fish are assumed to be ancestral tetraploids and the considerable differences in chromosome number between different species are thought to be the result of chromosomal fusions after tetraploidization. In all members of the genus Oncorhynchus the rearrangements have resulted in the consolidation of the NORs on a single chromosome pair. The possible significance of intra- and inter-species NOR polymorphisms is discussed.

Animals↗

Mortality and kidney histopathology of chinook salmon Oncorhynchus tshawytscha exposed to virulent and attenuated Renibacterium salmoninarum strains.

An isolate of Renibacterium salmoninarum (strain MT 239) exhibiting reduced virulence in rainbow trout Oncorhynchus mykiss was tested for its ability to cause bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha, a salmonid species more susceptible to BKD. Juvenile chinook salmon were exposed to either 33209, the American Type Culture Collection type strain of R. salmoninarum, or to MT 239, by an intraperitoneal injection of 1 x 10(3) or 1 x 10(6) bacteria fish(-1), or by a 24 h immersion in 1 x 10(5) or 1 x 10(7) bacteria ml(-1). For 22 wk fish were held in 12 degrees C water and monitored for mortality. Fish were sampled periodically for histological examination of kidney tissues. In contrast to fish exposed to the high dose of strain 33209 by either injection or immersion, none of the fish exposed to strain MT 239 by either route exhibited gross clinical signs or histopathological changes indicative of BKD. However, the MT 239 strain was detected by the direct fluorescent antibody technique in 4 fish that died up to 11 wk after the injection challenge and in 5 fish that died up to 20 wk after the immersion challenge. Viable MT 239 was isolated in culture from 3 fish that died up to 13 wk after the immersion challenge. Total mortality in groups injected with the high dose of strain MT 239 (12%) was also significantly lower (p < 0.05) than mortality in groups injected with strain 33209 (73 %). These data indicate that the attenuated virulence observed with MT 239 in rainbow trout also occurs in a salmonid species highly susceptible to BKD. The reasons for the attenuated virulence of MT 239 were not determined but may be related to the reduced levels of the putative virulence protein p57 associated with this strain.

Animals↗

Evidence for a carrier state of infectious hematopoietic necrosis virus in chinook salmon Oncorhynchus tshawytscha.

In British Columbia, Canada, infectious hematopoietic necrosis virus (IHNV) is prevalent in wild sockeye salmon Oncorhynchus nerka and has caused disease in seawater net-pen reared Atlantic salmon Salmo salar. In this study, chinook salmon Oncorhynchus tshawytscha experimentally exposed to an isolate of IHNV found in British Columbia became carriers of the virus. When Atlantic salmon were cohabited with these virus-exposed chinook salmon, IHNV was isolated from the Atlantic salmon. Identification of chinook salmon populations that have been exposed to IHNV may be difficult, as virus isolation was successful only in fish that were concurrently infected with either Renibacterium salmoninarum or Piscirickettisia salmonis. Also, IHNV-specific antibodies were detected in only 2 of the 70 fish experimentally exposed to the virus. Two samples collected from chinook salmon exposed to IHNV while at a salt water net-pen site had a seroprevalence of 19 and 22%; however, the inconsistencies between our laboratory and field data suggest that further research is required before we can rely on serological analysis for identifying potential carrier populations. Because of the difficulty in determining the exposure status of populations of chinook salmon, especially if there is no concurrent disease, it may be prudent not to cohabit Atlantic salmon with chinook salmon on a farm if there is any possibility that the latter have been exposed to the virus.

Animals↗

Detection of viral DNA polymerase activity in salmon tumour tissue induced by herpes virus, Oncorhynchus masou virus.

DNA polymerase activities were surveyed in tumour tissue and normal tissue of cherry salmon (Oncorhynchus masou). High activity of DNA polymerase alpha was detected in the tumour tissue but not in the normal tissue. This indicates that the tumour cells replicate prosperously. Viral DNA polymerase activity was detected only in the tumour tissue, indicating that Oncorhynchus masou virus (OMV) DNA should replicate there. DNA polymerase beta activity was of same level in both tissues. This is the first evidence that herpesvirus DNA polymerase was detected in tumour tissue in association with herpesvirus.

Animals↗

A retrovirus in chinook salmon (Oncorhynchus tshawytscha) with plasmacytoid leukemia and evidence for the etiology of the disease.

A plasmacytoid leukemia (PL) has caused mortalities in chinook salmon (Oncorhynchus tshawytscha) reared in seawater netpens in western British Columbia, Canada, since 1988. Kidney or eye tissues from 11 of 13 fish from netpens with clinical PL had reverse transcriptase (RT) activity. This RT activity was associated with virus particles of retrovirus morphology and buoyant density. In a transmission experiment, PL-positive donor fish tissues also had RT activity and virus particles of retrovirus morphology and buoyant density, as did recipient fish tissues following development of the disease 6 weeks postinjection with a tissue homogenate from the donor fish. Kidney and spleen tissues from fish that developed PL following injection with an inoculum that was passed through a 0.22-micron filter, in a separate experiment (M. L. Kent and S. C. Dawe. Further evidence for a viral etiology in the plasmacytoid leukemia of chinook salmon Oncorhynchus tshawytscha. Dis. Aquat. Org., in press, 1992), also exhibited RT activity. The virus particles observed by electron-microscopic examination of tissues or sucrose fractions from PL-positive fish were enveloped and were about 110-nm diameter with a central electron-dense core. Polypeptides of about M(r) 120,000, 80,000, 42,000, 27,000, 25,000, 22,000, and 19,000 were observed when purified virus particles were examined by polyacrylamide gel electrophoresis analysis. Many infectious neoplasms of animals, including fishes, are caused by retroviruses. The evidence in this study shows the presence of a retrovirus in chinook salmon with PL and further suggests a retroviral etiology of the disease. We are tentatively calling this virus salmon leukemia virus.

Animals↗

The epithelial neoplasm observed in the cultured coho salmon, Oncorhynchus kisutch.

The epithelial neoplasms were observed on the mouth of the cultured coho salmon, Oncorhynchus kisutch. Histopathologically, the tumors were formed to be proliferative epithelial cells, but no change was observed in other organs. The virus from this tumor was isolated in RTG-2 and CHSE-214 cells and developed the cytopathic effect which characterized to be the formation of syncytia and the migration of chromatin. This virus was neutralized with anti-Oncorhynchus masou virus (OMV) rabbit serum.

Animals↗

MISET: an immunological technique for the serodiagnosis of Cryptobia salmositica (Sarcomastigophora: Kinetoplastida) infection in Oncorhynchus mykiss.

A serodiagnostic technique, microscopic immuno-substrate-enzyme technique (MISET), is described using the Cryptobia-Oncorhynchus mykiss system. The reactions of specific antibodies, phosphatase-labeled antibody, and substrate with subsequent color development on the parasite (cell membrane, flagella, kinetoplast, and nucleus) are observed using light microscopy. Using this technique, humoral response to Cryptobia salmositica was detected in 2 of 10 O. mykiss 7 days after infection. Subsequently, antibodies were detected in all 10 infected fish. Parasites cultured in minimum essential medium or from experimentally infected pink salmon, Oncorhynchus gobuscha, worked equally well. Antibodies eluted from antisera or whole blood dried on filter paper (stored at -20 C) gave similar reactions as those from antisera stored in containers. MISET is at least as sensitive as the indirect fluorescent antibody technique. When MISET is modified and adapted for other parasitic diseases (e.g., for those of medical and or of economic importance), it may be useful especially in smaller centers or in developing countries where equipment cost, maintenance of equipment, operating costs, and well trained personnel are important considerations. Because MISET worked with dried whole blood or serum on filter paper it may also be a useful field technique for large scale seroepidemiological surveys.

Animals↗

Isolation of apolipoproteins from carotenoid-carrying lipoprotein in the serum of chum salmon, Oncorhynchus keta.

Carotenoid-carrying lipoprotein (CCL) was rapidly isolated from the high density lipoprotein (HDL) fraction of the upstream migrating male chum salmon (Oncorhynchus keta) by a single-step density gradient ultracentrifugation. The two apolipoproteins (Mr = 24,000 and 12,000; designated apo-I and apo-II, respectively) were readily dissociated and separated in 0.1% SDS by gel filtration chromatography. Prominent features of the amino acid composition in the CCL included the relative high levels of glutamic acid, alanine, leucine, and lysine, and the low cysteine content. Apo-I, as well as the CCL, was rich in glutamic acid, alanine, leucine, and lysine. Compared to the amino acid composition of apo-I, apo-II included relatively high levels of glycine and tyrosine, and low threonine, serine, and arginine contents. When the intact CCL particle was treated with trypsin, apo-I was rapidly proteolyzed, while apo-II was resistant. However, both apo-I and apo-II isolated from the CCL particle were readily digested with trypsin. This suggested that a different structural arrangement rather than the amino acid compositions of the apolipoproteins was associated with the limited trypsin digestion of the CCL particle. Apo-II may be sheltered from the aqueous environment and lie partly within the CCL particle. The properties of both the HDL fraction and apolipoproteins from pink salmon (Oncorhynchus gorbuscha) were similar to those of the CCL from chum salmon.

Amino Acids↗

Immunocytochemical study of the somatolactin cells in the pituitary of Pacific salmon, Oncorhynchus nerka, and O keta at some stages of the reproductive cycle.

Cells of the intermediate lobe of salmonids, homologous to the PAS-positive cells of other teleost species, cannot be differentiated by normal staining techniques, but can be immunostained with an antiserum against cod somatolactin (SL). Immunocytochemical techniques were applied to pituitary sections of two Pacific salmon, Oncorhynchus nerka and O. keta. Samples of immature or early maturing fish were collected in the Pacific Ocean and from mature spawning fish from hatcheries near Seattle and Willard (Washington). SL cells were rather small and moderately immunoreactive in immature fish. They were slightly enlarged in the early stages of gonadal development and more often contacted the basal lamina through processes with terminal swellings, suggesting granule release into perivascular spaces. In spawning fish, cells were enlarged and frequently more granulated, showing a wide contact with the basal lamina and a proximodistal transport of granules. In addition, large and more or less degranulated cells were noted, also indicating an active release of SL granules. Spawning females tended to have more SL cells than equivalent males. The gradual stimulation of SL synthesis and release during sexual maturation suggests that SL may be involved in the control of some steps of reproduction as previously shown by the increase in SL plasma levels in maturing coho salmon.

Animals↗

Stimulation of non-specific immune functions in seawater-acclimated rainbow trout, Oncorhynchus mykiss, with reference to the role of growth hormone.

The influence of acclimation to seawater (SW) and growth hormone (GH) administration on immune functions was examined in the rainbow trout (Oncorhynchus mykiss). After 3 days acclimation to dilute SW (12 parts per thousand, ppt), an increase in plasma lysozyme activity was observed compared to the fish kept in fresh water (FW). No change was seen in plasma immunoglobulin M (IgM) levels. When they were transferred from dilute SW to full-strength SW (29 ppt) after a single intra-peritoneal injection of ovine or salmon GH, plasma sodium levels of GH-treated fish were significantly lower than those of the control fish injected with Ringer's solution 24 h after the transfer. The plasma level of IgM was not influenced by GH injection in the fish kept in FW nor in those transferred to SW. The administration of GH increased plasma lysozyme activity in the fish in FW, but no further increase was seen after SW transfer. The production of superoxide anions in peripheral blood leucocytes was stimulated by GH in both FW and SW. These results suggest that GH is involved in the stimulation of the non-specific immune functions in SW-acclimated salmonids.

Adaptation, Physiological↗

Genetic characterization of hybridization and introgression between anadromous rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki).

Interspecific hybridization represents a dynamic evolutionary phenomenon and major conservation problem in salmonid fishes. In this study we used amplified fragment length polymorphisms (AFLP) and mitochondrial DNA (mtDNA) markers to describe the extent and characterize the pattern of hybridization and introgression between coastal rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki). Hybrid individuals were initially identified using principle coordinate analysis of 133 polymorphic AFLP markers. Subsequent analysis using 23 diagnostic AFLP markers revealed the presence of F1, rainbow trout backcross, cutthroat trout backcross and later-generation hybrids. mtDNA analysis demonstrated equal numbers of F1 hybrids with rainbow and cutthroat trout mtDNA indicating reciprocal mating of the parental types. In contrast, rainbow and cutthroat trout backcross hybrids always exhibited the mtDNA from the recurrent parent, indicating a male hybrid mating with a pure female. This study illustrates the usefulness of the AFLP technique for generating large numbers of species diagnostic markers. The pattern of hybridization raises many questions concerning the existence and action of reproductive isolating mechanisms between these two species. Our findings are consistent with the hypothesis that introgression between anadromous populations of coastal rainbow and coastal cutthroat trout is limited by an environment-dependent reduction in hybrid fitness.

Animals↗

Validation of radioimmunoassays for two salmon gonadotropins (GTH I and GTH II) and their plasma concentrations throughout the reproductivecycle in male and female rainbow trout (Oncorhynchus mykiss).

RIAs were developed for the two salmon gonadotropins (GTH I and GTH II) and used to measure plasma GTH throughout the life of the rainbow trout, Oncorhynchus mykiss. The RIA for GTH II was specific and sensitive (< 0.001% cross-reaction with GTH I, mean sensitivity = 0.26 +/- 0.02 ng/ml). The RIA for GTH I was less specific and less sensitive than the GTH II RIA (9.7% cross-reaction with GTH II, mean sensitivity = 2.34 +/- 0.23 ng/ml). In both males and females, the levels of GTH II remained undetectable (< 0.3 ng/ml) throughout most of the reproductive cycle, until shortly preceding spermiation/ovulation, when they began to rise. Concentrations of plasma GTH II were maximal at spermiation/ovulation. In both sexes, plasma profiles of GTH I differed from those of GTH II. The plasma GTH I concentration in females was elevated during early vitellogenesis. It then fell to a basal level shortly before ovulation and finally was elevated again at ovulation. In males, increases in plasma GTH I were seen a year before spermiation and again later, during the final stages of testicular growth. These results support the contention that GTH I mediates gonadal growth, whereas GTH II regulates the final stages of maturation and ovulation/spermiation. In rainbow trout, plasma profiles of GTH I and GTH II mimic the cycles of plasma FSH and LH, respectively, in the ovulatory cycle of higher vertebrates.

Animals↗

Detection of humoral antibodies to Renibacterium salmoninarum in rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar challenged by immersion and in naturally infected populations.

Humoral antibodies to heat-stable antigens of Renibacterium salmoninarum (Rs) were detected by enzyme-linked immunosorbent assay (ELISA) in rainbow trout Oncorhynchus mykiss and in Atlantic salmon Salmo salar challenged by immersion. A slow antibody response was found: 3% (1/30) was positive 4 wk after immersion and 72% (26/36) was positive after 8 wk. All 30 fish sampled after 4 wk were found to be infected, as determined by bacterial culture and/or the presence of soluble antigens in the kidney. At 6, 8 and 12 wk after immersion the proportion of positives indicated by ELISA was 58%. The Rs infection was detected by cultivation in 36% of sampled fish collected on the same occasion. Elevated antibody titres to Rs were detected in samples from both Atlantic salmon (59% in 1 farm) and from rainbow trout (20% in 1 of 5 sampled farms) in naturally exposed populations all of which classified positive for bacterial kidney disease (BKD). Elevated antibody titres were detected among sampled fish from populations of rainbow trout and salmon with clinical BKD. Samples collected from farm populations of rainbow trout, salmon and brown trout Salmo trutta, exposed to Rs but without clinical BKD, were negative in the ELISA, although Rs bacteria or soluble antigens were detected at the same sampling. The antibody ELISA method cannot be recommended for general fish health monitoring purposes, but may be a valuable tool for monitoring the disease progression during controlled experiments.

Actinobacteria↗

Characterization of growth hormone nycthemeral plasma profiles in catheterized rainbow trout (Oncorhynchus mykiss).

This study was conducted in order to characterize the nycthemeral plasma profiles of growth hormone (GH) in 41 prepubertal (2+) rainbow trout (Oncorhynchus mykiss). The possible influences of day-night alternation and of the food factor (quantity ingested, timing of the feed) on these plasma profiles were also analysed. Blood samples were taken every hour during a 24-hour period through a catheter inserted in the dorsal aorta. An assay of cortisol levels showed that the fish were in satisfactory physiological condition. The plasma concentrations of GH fluctuated significantly during the course of a nycthemere. The basal level was very low (0.32 +/- 0.01 ng/ml), and was interrupted by peaks that occurred at a rate of 2.1 +/- 0.1 peaks/24 hr, an amplitude of 2.0 +/- 0.3 ng/ml, and a duration of 3.5 +/- 0.2 hr. As a result, mean GH levels over a 24-hr period were low (0.7 +/- 0.1 ng/ml). Peaks occurred irregularly and asynchronously in individual fish and displayed no rhythmicity. Our study shows that there is a sexual difference in GH profiles in rainbow trout. No significant difference was observed between daily and nightly levels of GH (0.6 +/- 0.07 vs. 0.8 +/- 0.1 ng/ml). No influence of the diet on the plasma profiles of GH was observed. Average levels of GH over a 24 hr period are not significantly correlated with the growth rate of the fish. This study shows that circulating levels of GH in rainbow trout are markedly lower than in other vertebrates.

Animals↗

Early embryonic expression of the growth hormone family protein genes in the developing rainbow trout, Oncorhynchus mykiss.

In fish, growth hormone (GH), prolactin (PRL) and somatolactin (SL) are three major peptide hormones produced in the pituitary gland. Using reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis of the amplified products, the expression of GH, PRL, and SL genes were determined during the embryonic development in rainbow trout (Oncorhynchus mykiss). The mRNA for GH, PRL and SL were detected not only in embryos prior to or after organogenesis of the pituitary gland but also in mature oocytes as maternal messages. After hatching, all of these three mRNA species were detected at very high levels. Since the ontogenesis of the pituitary gland takes place on embryonic day (ED) 14, these observations suggest that the GH family protein genes are expressed in the developing embryos prior to the formation of the pituitary gland. Using the same RT-PCR assay, PRL mRNA was detected only in the head part of the fry whereas GH and SL mRNA were detected in both the head and trunk. In adult animals, though high levels of GH mRNA were primarily detected in the pituitary gland, brain, gill, and heart, low levels of GH mRNA were also detected in the kidney, liver, pyloric ceaca, and ovary. Results of the current study and those reported by Yang et al. (1997. Gen Comp Endocrinol 106:271-281) suggest that GH and SL genes are also expressed in extrapituitary tissues even after the organogenesis of the pituitary gland. Furthermore, these results suggest that these hormones may play important roles yet to be identified during embryonic development in fish.

Animals↗

Involvement of estrogens in the process of sex differentiation in two fish species: the rainbow trout (Oncorhynchus mykiss) and a tilapia (Oreochromis niloticus).

In order to study the physiological implication of sex steroid hormones in gonadal sex differentiation in fish, we first investigated the potential role of estrogens using two fish models: the rainbow trout (Oncorhynchus mykiss) and a tilapia species (Oreochromis niloticus). All experiments were carried out on genetically all-male (XY) and all-female (XX) populations. In vivo treatments with an aromatase inhibitor (ATD, 1,4,6- androstatriene-3-17-dione) result in 100% masculinization of an all-female population in rainbow trout (dosage 50 mg/kg of food) and 75.3% in tilapia (dosage 150 mg/kg of food). In tilapia, the effectiveness of the aromatase inhibition by ATD is demonstrated by the marked decrease of the gonadal aromatase activity in treated animals versus control. No masculinization is obtained following treatment with an estrogen receptor antagonist (tamoxifen) in both species. Aromatase and estrogen receptor gene expression was studied in rainbow trout by semi-quantitative RT-PCR in gonads sampled before, during and after sex-differentiation. Aromatase mRNA is specifically detected in female gonads, 3 weeks before the first sign of histological sex-differentiation, i.e., first female meiosis. Aromatase expression in male gonads is at least a few hundred times less than in female gonads. Estrogen receptor gene is expressed in both male and female gonads at all stages with no dimorphic expression between sexes. Specific aromatase gene expression before ovarian differentiation was also demonstrated using virtual Northern blot, with no expression detected in male differentiating gonads. From these results it can be concluded that estrogen synthesis is crucial for ovarian differentiation, and transcription of the aromatase gene can be proposed as a key step in that process in fish.

Animals↗

Proteome analysis of Oncorhynchus species during embryogenesis.

To understand the molecular mechanisms underlying normal and abnormal development of two salmonids, masu salmon (Oncorhynchus masou) and rainbow trout (O. mykiss), we used two-dimensional (2-D) electrophoresis to construct a series of 2-D maps during the embryonic period. We identified all visible protein spots on the 2-D map by assigning numbers for masu salmon and rainbow trout, and we determined N-terminal sequences of proteins for one hundred of the spots, that appear at very high concentrations in the whole embryos of masu salmon and rainbow trout. We also characterized embryonic stages according to the periods of appearance of spots. Most of the N-terminal sequences were identical or at least highly similar to partial sequences reported for vitellogenin (Vtg) of O. mykiss. A potential proteolytic processing of Vtg for rainbow trout is discussed in relation to the time of appearance and relative position of Vtg fragments within the complete protein sequence.

Amino Acid Sequence↗

Na(+) and Cl(-) transport by the urinary bladder of the freshwater rainbow trout (Oncorhynchus mykiss).

Freshwater (FW) rainbow trout (Oncorhynchus mykiss) urinary bladders mounted in vitro under symmetrical saline conditions displayed electroneutral active absorption of Na(+) and Cl(-) from the mucosal side; the transepithelial potential (V(t)) was 0.1 mV, and the short-circuit current was less than 1 microA cm(-2). Removal of Na(+) from mucosal saline decreased Cl(-) absorption by 56% and removal of Cl(-) decreased Na(+) absorption by 69%. However, active net absorption of both Na(+) and Cl(-) was not abolished when Cl(-) or Na(+) was replaced with an impermeant ion (gluconate or choline, respectively). Under physiological conditions with artificial urine (¿Na(+) = 2.12 mM, ¿Cl(-) = 3.51 mM) bathing the mucosal surface and saline bathing the serosal surface, transepithelial potential (V(t)) increased to a serosal positive approximately +7.6 mV. Unidirectional influx rates of both Na(+) and Cl(-) were 10-20-fold lower but active absorption of both ions still occurred according to the Ussing flux ratio criterion. Replacement of Na(+) with choline, or Cl(-) with gluconate, in the mucosal artificial urine yielded no change in unidirectional influx of Cl(-) or Na(+), respectively. However, kinetic analyses indicated a decrease in maximum Na(+) transport rate (J(max)) of 66% with no change in affinity (K(m)) in the low Cl(-) mucosal solution relative to the control solution. Similarly, there was a 79% decrease in J(max) values for Cl(-), again with no change in K(m), in the low-Na(+) mucosal bathing. The mucosal addition of DIDS, amiloride or bumetanide (10(-4) M) had no effect on either Na(+) or Cl(-) transport, under either symmetrical saline or artificial urine/saline conditions. Addition of the three drugs simultaneously (10(-4) M), or chlorothiazide (10(-3) M), under symmetrical saline conditions also had no effect on Na(+) or Cl(-) transport rates. Cyanide (10(-3) M) addition to mucosal artificial urine caused a slowly developing decrease of Na(+) influx to 59% and Cl(-) influx to 50% in the period after drug addition. Na(+) and Cl(-) reabsorption appears to be a partially coupled process in the urinary bladder of O. mykiss; transport mechanisms are both dependent upon and independent of the other ion.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗