A hepatic metabolomics-based diagnostic approach to assess lethal toxicity of dithiocarbamate fungicide polycarbamate in three marine fish species.

The present study was performed to evaluate the toxic effect of the dithiocarbamate fungicide polycarbamate (PC) on the hepatic metabolic profiles of three marine fish species, red sea bream (Pagrus major), spotted halibut (Verasper variegatus), and marbled flounder (Pleuronectes yokohamae). First, juvenile fish were exposed to graded concentrations of PC for 96h; the 96-h LC50 values obtained were 22-29, 239-553, and 301-364µgL-1 for red sea bream, spotted halibut, and marbled flounder, respectively, indicating that red sea bream possessed higher sensitivity to PC than the two benthic species. Second, the fish were exposed to lethal-equivalent concentration (H group) or sub-lethal (one-tenth of the H group concentrations; L group) for 24 and 96h and gas-chromatography based metabolomics approach was employed to explore the crucial biomarker metabolite associated with lethal toxicity. Of the 53 metabolites identified, only reduced glutathione (GSH) was consistently elevated in the H group for the three fish species at 96h. The calculated cut-off value of GSH (mM) based on receiver operating curve analysis between H group and the other treatment groups (control, solvent control, and L group) was obtained at 0.56mM, which allowed to distinguish between the groups with high confidence for the three fish species. These results are the first to demonstrate the potential of using GSH as a possible biomarker metabolite and its usefulness of threshold cut-off value for diagnosing life-threatening health conditions of fish.

Dietary taurine supplementation ameliorates the lethal effect of phenanthrene but not the bioaccumulation in a marine teleost, red sea bream, Pagrus major.

The present study was performed to evaluate the effect of dietary taurine on the hepatic metabolic profiles of red sea bream (Pagrus major) and on phenanthrene (a polyaromatic hydrocarbon) toxicity and bioaccumulation. The fish were fed a diet supplemented with 0% (TAU0%), 0.5% (TAU0.5%), or 5% (TAU5%) taurine for 40-55d and subjected to phenanthrene acute toxicity and bioaccumulation tests. Taurine deficiency in feed severely affected the hepatic metabolic profiles of fish, which indicated a complementary physiological response to taurine deficiency. For the acute toxicity test, fish were fed the test diets for 55d and were then exposed to 0-893µg/L phenanthrene for 96h. Tolerance to phenanthrene was significantly improved by 0.5% of taurine inclusion in feed relative to TAU0%, but not by 5.0% inclusion. Reduced glutathione in the liver, which acts as an oxygen-free radical scavenger, was associated with a reduction in the toxicity of phenanthrene. For the bioaccumulation test, fish were fed the test diets for 40d and were thereafter chronically exposed to 20µg/L phenanthrene for 13d followed by depuration for 3d. The activity of hepatic biomarker, ethoxyresorufin-O-deethylase, was increased by phenanthrene exposure in the taurine inclusion groups. However, phenanthrene concentrations in the liver and muscle of fish fed TAU5.0% tended to be higher than those of fish fed TAU0% and TAU0.5% during the exposure period. These results indicate that 0.5% of taurine inclusion in feed plays an important role in the alleviation of phenanthrene toxicity but not bioaccumulation. Furthermore, larger amount of taurine inclusion (TAU5%) did not show marked beneficial effects against phenanthrene exposure. This study provides insight about a major concern of environmental contaminants into aquatic environment and can be effectively used for improvement of aquaculture.

Development of a method to assess the ichthyotoxicity of the harmful marine microalgae Karenia spp. using gill cell cultures from red sea bream (Pagrus major ).

The present study reports the development of a method to investigate ichthyotoxicity of harmful marine microalgae using cultured red sea bream (Pagrus major) gill cells. The cultured gill cells formed adherent 1-2 layers on the bottom of the culture plate and could tolerate seawater exposure for 4 h without significant alteration in cell survival. The microalgae Karenia mikimotoi, Karenia papilionacea, K. papilionacea phylotype-I, and Heterosigma akashiwo were cultured, then directly exposed to gill cells. After K. mikimotoi and K. papilionacea phylotype-I exposure, live cell coverage was significantly lower than in the cells exposed to a seawater-based medium (control cells; P < 0.05). Toxicity of K. mikimotoi cells was weakened when cells were ruptured, and was almost inexistent when the algal cells were removed from the culture by filtration. Significant cytotoxicity was detected in the concentrated ruptured cells, and in the concentrated of ruptured cells after freezing and thawing though cytotoxicity was weakened; whereas, cytotoxicity almost disappeared after heat treatment. In addition, examination of the distribution of toxic substances from the ruptured cells showed that cytotoxicity mainly occurred in the fraction with the resuspended pellet after centrifugation at 3000×g.

Effect of taurine supplementation on hepatic metabolism and alleviation of cadmium toxicity and bioaccumulation in a marine teleost, red sea bream, Pagrus major.

This study was performed to unravel the mechanism of the beneficial action of taurine on marine teleost fish, red sea bream (Pagrus major), by analyzing the hepatic metabolism. Moreover, the ameliorative effects of the nutrient against cadmium toxicity and bioaccumulation were further evaluated. The fish were fed a diet containing 0 % (TAU0 %), 0.5 % (TAU0.5 %), or 5.0 % (TAU5.0 %) taurine for 40-55 days (d) and subjected to cadmium acute toxicity and bioaccumulation tests. Taurine deficiency in feed severely affected growth and the hepatic metabolic profiles of the fish, including a remarkable increase in myo-inositol, aspartate, and ß-alanine in the TAU0 % group, which indicates a complementary physiological response to taurine deficiency. For the acute toxicity test, fish were fed the test diets for 55 d and were then exposed to different dose of cadmium ranging from 0 to 5.6 mg/L for 96 h. Fish fed taurine had a higher tolerance to cadmium than those not fed taurine. For the bioaccumulation test, fish were fed the test diets for 40 d and then were chronically exposed to 0.2 mg/L of cadmium for 28 d followed by depuration for 21 d. Cadmium concentrations in the liver and muscle of fish fed TAU5.0 % were significantly lower than those of fish fed TAU0 % for the first 7 d of exposure and the first 7 d of elimination. Our findings suggest a possible mechanism for the beneficial role played by taurine and that the inclusion of taurine in fish aquaculture feed may reduce cadmium contamination of fish intended for human consumption.

Bioconcentration and Elimination of the Dithiocarbamate Fungicide Polycarbamate in Marine Teleost Fish and Polychaete.

We evaluated the bioconcentration and elimination of polycarbamate, a popular antifoulant classified as a dithiocarbamate fungicide, in a marine teleost fish, mummichog (Fundulus heteroclitus), and a polychaete (Perinereis nuntia). Following polycarbamate exposure and depuration, the calculated bioconcentration factor (BCF) and elimination rate constant (k2) were 3.1 and 0.17 day(-1) in fish and 1.5-7.8 and 0.13-0.18 day(-1) in the polychaete, indicating that the fungicide has low bioconcentration potential in both organisms. Given the BCF of 3.1, the permissible environmental concentration level of polycarbamate in water was calculated to be 6.1 µg L(-1) to satisfy the threshold pesticide residue levels in fish permitted by law, which is far below the permissible levels in Hiroshima Bay, Japan. Therefore, we consider that the current polycarbamate contamination level in terms of bioconcentration is not likely to be an alarming issue in coastal environments.

[Uterine cervical cancer screening in kitakyushu city: present and future].

Uterine cervical cancer is the most common primary gynecologic malignant tumor in Japan. Conventional cervical screening Papanicolaou (Pap) test has been shown to be extremely effective in reducing cervical cancer incidence and mortality, but the consultation rate for cancer screening in Japan is markedly low, at 20% of prescribed subjects, in comparison with other developed countries. In 2001, 15,501 women (6.8%) received a Pap test in Kitakyushu City, and that was less than half of national average. From 2009, free coupons for uterine cervical cancer screening were distributed to Japanese woman who were 20, 25, 30, 35 or 40 years of age as part of the project for women-specific cancer screening. The rate of participation in Pap testing was 22.3% in 2012, with 31,970 women receiving cervical tests. It was almost as high as the national level. It's obvious that high risk human papillomavirus (HPV) is responsible for cervical cancer incidence and HPV mainly infects through sexual practice. The rate of early cervical neoplasms and invasive cancers is currently increasing in young women. Abnormal Pap tests were detected in 2.3% of the women in 2008. To increase the population's participation in this screening process, a cost-effective and efficient system should be established. National and local governments, medical institutions, companies, and educational institutions must have an accurate understanding of the current situation, and take an assertive approach in order to decrease the mortality rate of uterine cervical cancer.

Relationship closeness of tolerance to two neonicotinoids with their internal body burden in two estuarine resident marine crustaceans.

The estuarine resident crustacean sand shrimp, Crangon uritai, has a higher tolerance to neonicotinoid insecticides than that of the kuruma prawns, Penaeus japonicus. However, the reason for the differential sensitivities between the two marine crustaceans remains to be understood. This study explored the mechanism underlying differential sensitivities based on insecticide body residues after exposing both said crustaceans to two insecticides (acetamiprid and clothianidin) with or without oxygenase inhibitor piperonyl butoxide (PBO) for 96 h. Two graded-concentration groups were formed; group H (1/15-1 times the 96-h LC50 values) and L (one-tenth the concentration of group H). Results showed that the internal concentration in survived specimens tended to be lower in sand shrimp than in kuruma prawns. Co-treatment of PBO with two neonicotinoids not only increased sand shrimp mortality in the H group, but also altered metabolism of acetamiprid into its metabolite, N-desmethyl acetamiprid. Furthermore, molting during the exposure period enhanced bioconcentration of insecticides, but not affects survival. Collectively, the higher tolerance of sand shrimp than that of kuruma prawns to the two neonicotinoids can be explained by lower bioconcentration potential and more involvement of oxygenase in their alleviating lethal toxicity.

Desorption of polycyclic aromatic hydrocarbons from polyethylene microplastics in two morphologically different digestive tracts of marine teleosts: Gastric red seabream (Pagrus major) and agastric mummichog (Fundulus heteroclitus).

In this study, we elucidated the desorption potency of polycyclic aromatic hydrocarbons (PAHs) sorbed on microplastics (MP; polyethylene) in the digestive tract of two fish species: gastric red seabream and agastric mummichog. In our in vitro assay system using the real gut sample of unexposed fish, the digestive tract was firstly removed from the fish and divided into three parts. Then, MP that had previously been sorbed with 16 PAHs were incubated with extracts of the gut contents or tissue with buffer or only a buffer. The desorption potency of PAHs was individually assessed for gut contents and tissue, which revealed that PAH desorption from MP was elevated in extracts of the gut contents compared with that in the buffer alone for both fish species. PAH desorption potency was the highest in the midgut for gastric red seabream and in the foregut for agastric mummichog, which indicates that PAH desorption from MP varies among different parts of the digestive tract and among fish with distinct gut morphology. In the midgut contents of red seabream and foregut contents of mummichog, the desorption fraction was 5.6% and 8.1% of the total PAHs sorbed on MP, respectively. The desorption fraction enhancement achieved by adding gut contents extracts tended to be greater with an increase in the n-octanol/water partition ratio, suggesting that enhancement of the desorption fraction in the digestive tract depends on the physicochemical properties of PAHs. Thus, morphological differences in digestive tracts and PAH properties should be considered when evaluating the effect of MP vector on pollutant exposure in fish.

Microplastic uptake and gut retention time in Japanese anchovy (Engraulis japonicus) under laboratory conditions.

To explore the impact of microplastic (MP) pollution on planktivorous fish, we examined the uptake and retention of MPs by Japanese anchovy (Engraulis japonicus) under laboratory conditions. MP uptake was size selective in adult anchovy-0.3-mm MPs were taken up in significantly larger amounts than 0.85-mm MPs-but not in juveniles. There were no significant differences in the uptake of MPs of three different colors, suggesting that anchovy do not select for MP coloration. More than 90% of the MPs were excreted within 20 h of ingestion, indicating that MP retention time is similar to the processing time of food items. Our findings suggest that Japanese anchovy tend to take up MPs that are equivalent in size to prey items, but that the impacts of MP ingestion are likely to be limited under the current state of oceanic MP contamination.

Molting enhances internal concentrations of fipronil and thereby decreases survival of two estuarine resident marine crustaceans.

In aquatic arthropods, molting is a pivotal physiological process for normal development, but it may also expose them to higher risks from xenobiotics, because the organism may take up additional water during that time. This study aimed to assess the effects of molting on bioconcentration and survival after 96-h exposure to insecticide fipronil with or without oxygenase (CYP450s) inhibitor piperonyl butoxide (PBO) of two estuarine resident marine crustacean species: the sand shrimp Crangon uritai and the kuruma prawn Penaeus japonicus, with 96-h LC50 value of fipronil = 2.0 µg/L and 0.2 µg/L, respectively. Two graded concentrations included group high (H) (equivalent to the 96-h LC50 values) and low (L) (one-tenth of the H group concentration). Molting and survival were individually checked, and internal concentrations of fipronil and its metabolites (fipronil desulfinyl, fipronil sulfide, fipronil sulfone) were measured. The results showed that, only fipronil and fipronil sulfone were detected from organism, and that internal concentrations of these insecticides in molted specimens were higher than those of unmolted ones but comparable with those of dead ones. Accordingly, mortality was more frequent in molted specimens than those that were unmolted. Furthermore, involvement of oxygenase and higher lethal body burden threshold may confer higher tolerance to fipronil in sand shrimp than in the kuruma prawn. This study is the first to demonstrate that the body-residue-based approach is useful for deciphering the causal factors underlying fipronil toxicity, but highlights the need to consider physiological factors in arthropods, which influence and lie beyond body burden, molting and drug metabolism.

Integrated transcriptomic and metabolomic analyses reveal mechanism underlying higher resistance of the marine oligochaete Thalassodrilides cf. briani (Clitellata: Naididae) to heavy contamination of sediments with polycyclic aromatic hydrocarbons.

In some coastal areas, sediments are contaminated with various chemical compounds, causing significant threats to marine organisms. Therefore, the development of remediation techniques is important. Here, we focused on bioremediation using marine benthic animals such as aquatic oligochaetes. The oligochaete Thalassodrilides cf. briani is highly resistant to contamination of sediments with toxic chemicals. We examined whether T. cf. briani could decompose high-concentration polycyclic aromatic hydrocarbons (PAHs) in sediments. Furthermore, relevant genes expressed in T. cf. briani exposed to contaminated sediment were comprehensively examined using next-generation sequencing, and its metabolites were identified by metabolomic analysis using gas chromatography-mass spectrometry. T. cf. briani reduced the concentration of 16 PAHs in the sediment from 55,900 to 45,200 ng/g dry weight in 50 days, thereby reducing total PAH concentrations by approximately 20%. The results of transcriptomic analysis suggest that activation of a drug-metabolizing enzyme system may promote the metabolism of harmful chemical substances during excretion of chemicals from the body. According to the results of principal component analysis based on the values of 43 types of metabolomes identified by metabolomic analysis, groups were divided according to the difference in the number of exposure days. In addition, levels of glutamine, which is important for maintaining digestive tract functions, increased. This suggests that the digestive tract function promotes the metabolism and detoxification of foreign substances. Furthermore, transcriptome analysis revealed that glutamate dehydrogenase increased 1.3-fold and glutamine synthetase increased 1.7-fold, confirming the increase in glutamine. Thus, we conclude that T. cf. briani adapted to the polluted sediment by regulating its metabolism.

Estimation of the uptake and gut retention of microplastics in juvenile marine fish: Mummichogs (Fundulus heteroclitus) and red seabreams (Pagrus major).

We investigated the impact of microplastics (MPs) on marine fish by estimating the uptake and retention by mummichogs (Fundulus heteroclitus) and red seabreams (Pagrus major) of MPs similar in size (≥0.25 mm) and composition (polyethylene) to MPs detected in fish intestines. Results revealed a correlation between MP concentrations in aquarium water and the content of MPs in the gastrointestinal tracts of exposed fish. More than 95% were excreted from both species within 25 h; this retention time is similar to the processing time of food items. The rate of excretion showed little dependence on MP size, but there was some dependence on fish species and MP shape. These results suggest that MPs similar to those we studied have little direct adverse impact on these two marine fish species.

Ecological risk assessment of an antifouling biocide triphenyl (octadecylamine) boron in the Seto Inland Sea, Japan.

In this study, we derived the predicted no-effect concentrations (PNEC) for triphenyl (octadecylamine) boron (TPB-18) and investigated the occurrence of triphenylboranes (TPBs), including TPB-18, for ecological risk assessment in the Seto Inland Sea, Japan. We tested algal growth inhibition, crustacean immobilization, and reproductive toxicity and performed toxicity tests in fish to assess acute and chronic toxicity and generate the PNEC for TPB-18. The minimum toxicity value was 0.30 µg/L, as determined by the 72-h no-observed-effect concentration (NOEC) for the alga Chaetoceros gracilis. The 5th-percentile of hazardous concentration (HC5), derived from NOECs using the species sensitivity distributions approach, was 0.059 µg/L, which indicated the PNEC of 0.0059 µg/L. In comparison, the highest concentration in seawater sampled from the Seto Inland Sea was 0.00034 µg/L, suggesting that the ecological risks posed by TPB-18 are currently low.

Occurrence of neonicotinoids and fipronil in estuaries and their potential risks to aquatic invertebrates.

This study aimed to evaluate and qualify field-based potential risks of seven neonicotinoid and phenylpyrazole (fipronil) insecticides on aquatic invertebrates, including estuary-resident marine crustaceans. One hundred and ninety-three estuarine water samples, with salinity ranging from 0.5 to 32.7, were collected from four estuarine sites in the Seto Inland Sea of Japan, in 2015-2018 and the insecticide levels were measured. Five neonicotinoid and fipronil insecticides were successfully identified, and their occurrence varied temporally. Marine crustaceans were simultaneously harvested every month from one of the estuarine water sampling sites in 2015-2017. Three predominant crustacean species, kuruma prawn (Penaeus japonicus), sand shrimp (Crangon uritai), and mysid (Neomysis awatschensis), were captured and their seasonal presence was species independent. A 96-h laboratory toxicity study with the insecticides using kuruma prawn, sand shrimp, and a surrogate mysid species (Americamysis bahia) indicated that fipronil exerted the highest toxicity to the three crustaceans. Using both toxicity data and insecticide occurrence in estuarine water (salinity ≥10, n = 169), the potential risks on the three marine crustaceans were quantified by calculating the proportion of mixture toxicity effects (Pmix). The Pmix of seven neonicotinoids on the crustaceans was less than 0.8%, which is likely to be too low to indicate adverse effects caused by the insecticides. However, short temporal detection of fipronil (exclusively in June and July) significantly affected the Pmix, which presented the maximal Pmix values of 21%, 3.4%, and 72% for kuruma prawn, sand shrimp, and mysid, respectively, indicating a significant effect on the organisms. As for estuarine water (salinity <10), some water samples contained imidacloprid and fipronil exceeding the freshwater benchmarks for aquatic invertebrates. The present study provides novel insights into the seasonally varying risks of insecticides to estuarine crustaceans and highlights the importance of considering whether ecological risk periods coincide with crustacean presence.

Toxicity of the biocide polycarbamate, used for aquaculture nets, to some marine fish species.

We investigated toxic effects of the antifouling biocide polycarbamate (PC) on marine fish by conducting acute, early-life stage toxicity (ELS), and embryo toxicity tests. Mummichog (Fundulus heteroclitus) 96-h LC50 values for hatched larvae (body weight about 2.0 mg) and juveniles (660 ±â€¯36 mg) were about 12 and 630 µg/L, respectively. The ELS test using mummichog embryos yielded a lowest-observed-effect concentration of 3.9 µg/L and a no-observed-effect concentration of 2.1 µg/L with growth as the most sensitive endpoint. The embryo toxicity test for spotted halibut (Verasper variegatus) revealed a 10-d EC50 of 8.1 µg/L with abnormality as an endpoint. During the ELS and embryo toxicity tests, morphological abnormalities (notochord undulation) were induced in the embryos. Biochemical and gene-expression analysis suggest that PC-induced morphological abnormalities involve disruption of lysyl oxidase-mediated collagen fiber organization, essential for notochord formation, and inhibition of gene expression related to notochord formation.

Detection and assessment of androgenic potency of endocrine-disrupting chemicals using three-spined stickleback, Gasterosteus aculeatus.

The male three-spined stickleback (Gasterosteus aculeatus) produces a glue protein named "spiggin" that is used as a cementing substance for building its nest. The synthesis of spiggin is under the control of androgenic stimulation. Therefore, spiggin is an effective biomarker of any androgenic activity displayed by environmental chemicals, similarly to the use of vitellogenin as an estrogenic biomarker. The aim of this study was to establish a quantification system for spiggin mRNA to develop a highly sensitive system for evaluating environmental androgens. In this process, two different types of cDNA encoding spiggin (SPG-1 and SPG-2) were isolated. They closely resemble each other in primary structure and features. In addition, the transcriptions of both spiggin gene were induced by only androgenic stimulation in a receptor-mediated manner. These findings suggest the multiplicity albeit specificity of spiggin in the stickleback. The quantification system for spiggin mRNA was established using a real-time RT-PCR technique. This system enables accurate quantification within a wide range of spiggin mRNA from 10(1) to 10(6) copies.

Identification of two forms of vitellogenin-derived phosvitin and elucidation of their fate and roles during oocyte maturation in the barfin flounder, Verasper moseri.

A new method for visualizing small and multiple phosvitins (Pvs) in oocytes from a marine teleost was developed by a combination of gel filtration, alkaline phosphatase treatment, and SDS-PAGE followed by silver staining. Three distinct Pv polypeptides having molecular masses of 15 kDa, 8 kDa, and 7 kDa were visualized in vitellogenic follicle extract of barfin flounder, Verasper moseri. N-terminal amino acid sequencing identified two different N-termini that fell into the PvA (7 kDa) and PvB (15 kDa and 8 kDa) groups, which were derived from two forms of vitellogenin (Vg), VgA and VgB, respectively. Analysis of time-course change in phosphorus-rich peaks of gel chromatography fractions of follicle extracts from different maturational stages demonstrated a rapid degradation of Pvs during mid-phase of oocyte maturation. Quantitative analysis of free amino acids in maturing follicles revealed an increment of serine content but not of phosphoserine, indicating the occurrence of dephosphorylation concomitant with Pv degradation. Measurement of phosphatase activity in follicles and eggs at different maturational stages demonstrated a significant activation of phosphatase especially under acidic conditions. This suggested that Pv degradation and dephosphorylation are regulated by changes in ooplasm pH during oocyte maturation. Our results also suggested that the Pvs in barfin flounder vitellogenic oocytes bind to much lower amounts of calcium and magnesium than those of masu salmon, Oncorhynchus masou. This indicates that the Pvs in the barfin flounder, a marine teleost spawning its eggs in seawater, do not play a role in the transport and deposition of calcium and magnesium into oocytes.

Differences in the ability of two marine annelid species, Thalassodrilides sp. and Perinereis nuntia, to detoxify 1-nitronaphthalene.

Bioremediation is a promising method for remediating environmentally polluted water. We investigated the abilities of two benthic annelid species to biotransform 1-nitronaphthalene, a nitrated polycyclic aromatic hydrocarbon. We used an oligochaete, Thalassodrilides sp. (Naididae), collected from the sediment beneath a fish farm and a polychaete, Perinereis nuntia, which was obtained from a commercial source. Populations of both organisms were exposed to 1400 µg L(-1) of 1-nitronaphthalene in seawater for 3 days in the dark at 20 °C. The concentration of the pollutant decreased to 12 µg L(-1) in the seawater containing the Thalassodrilides sp. and to 560 µg L(-1) in the seawater containing P. nuntia. The 1-nitronaphthalene concentration in the bodies of the animals increased from 12 to 94 µg kg(-1) in Thalassodrilides sp. and from 0.90 µg kg(-1) to 38,000 µg kg(-1) in P. nuntia. After 3 days, 99% and 40% of the 1-nitronaphthalene had been biotransformed in the Thalassodrilides sp. and P. nuntia experimental groups, respectively. We then tested the acute toxicity of residual 1-nitronaphthalene from the same water using mummichog (fish) larvae. After the larvae had been exposed for 96 h, the percentage of apparently unaffected larvae remaining was 83.3% in Thalassodrilides sp. group but only 16.7% in the P. nuntia group. Clearly, of the two species we studied, Thalassodrilides sp. had a superior ability to convert 1-nitronaphthalene into substances that were nontoxic to mummichog larvae. Therefore, we recommend the use of this species for bioremediation of chemically polluted sediments.

Incorporation and utilization of multiple forms of vitellogenin and their derivative yolk proteins during vitellogenesis and embryonic development in the mosquitofish, Gambusia affinis.

We previously demonstrated the presence of three forms of vitellogenin (Vg), two 600 kDa Vgs (600Vg; VgA and VgB) and a 400 kDa Vg (400Vg; phosvitinless Vg) in plasma from maturing female viviparous mosquitofish, Gambusia affinis. For further quantitative elucidation of the accumulation and utilization of the multiple Vg-derived yolk proteins, two sandwich enzyme-linked immunosorbent assays (ELISA) were developed using antisera against 600Vgs and a 400 kDa yolk protein (400Yp; derived from 400Vg), respectively. Contents of 560 kDa yolk protein (560Yp; lipovitellins derived from 600Vg) and 400Yp measured by the ELISAs increased in accordance with the growth of vitellogenic oocytes, keeping their proportional ratio (mol/mol) at about 4:1. A similar ratio obtained for plasma Vgs suggests that the proportional accumulation of the multiple Vg-derived yolk proteins is regulated by the hepatic synthesis and secretion of their precursor Vgs. When egg homogenate was analyzed by gel chromatography, three peaks, consisting of 560Yp, 400Yp and 28 kDa native beta'-component, were observed. The elution profile showed no change until embryos reached the early neurula stage, however, the relative height of the 560Yp peak as compared to the 400Yp one decreased after retinal pigmentation. Results from measurements of 560Yp and 400Yp at each embryonic stage supported the occurrence of unequal utilization of the two yolk proteins. The proportional ratios (mol/mol) of 560Yp content versus 400Yp content gradually decreased from 4.1 fold in early neurula embryo to 1.4 fold in larva just before parturition. The present study thus demonstrated unequal utilization of the multiple Vg-derived yolk proteins in developing embryos of mosquitofish.