Intercross population study reveals that co-mutation of mitfa genes in two subgenomes induces red skin color in common carp(Cyprinus carpio wuyuanensis)

Common carp are among the oldest domesticated fish in the world.As such,there are many food and ornamental carp strains with abundant phenotypic variations due to natural and artificial selection.Hebao red carp(HB,Cyprinus carpio wuyuanensis),an indigenous strain in China,is renowned for its unique body morphology and reddish skin.To reveal the genetic basis underlying the distinct skin color of HB,we constructed an improved highfidelity(HiFi) HB genome with good contiguity,completeness,and correctness.Genome structure comparison was conducted between HB and a representative wild strain,Yellow River carp(YR,C.carpio haematopterus),to identify structural variants and genes under positive selection.Signatures of artificial selection during domestication were identified in HB and YR populations,while phenotype mapping was performed in a segregating population generated by HB×YR crosses.Body color in HB was associated with regions with fixed mutations.The simultaneous mutation and superposition of a pair of homologous genes(mitfa) in chromosomes A06 and B06 conferred the reddish color in domesticated HB.Transcriptome analysis of common carp with different alleles of the mitfa mutation confirmed that gene duplication can buffer the deleterious effects of mutation in allotetraploids.This study provides new insights into genotype-phenotype associations in allotetraploid species and lays a foundation for future breeding of common carp.

Pharmacokinetics of Enrofloxacin and Its Metabolite in Carp (Cyprinus carpio) After a Single Oral Administration in Medicated Feed

A precise and reliable analytical method of high performance liquid chromatography-tandem mass spectrometry(HPLCMS/MS)was developed to measure trace levels of enrofloxacin(ENR)and its major metabolite ciprofloxacin(CIP)in carp tissues.Optimized chromatographic separation was obtained on a Waters Xterra MS C_(18) reversed-phase column using gradient elution with methanol and 0.1%formic acid aqueous solution including 5mmolL^(-1) of ammonium acetate.The established method was applied to study the pharmacokinetics and distribution of ENR and CIP in tissues of carp following a single oral administration in feed at a dosage of 40mgkg^(-1) bw(body weight).Data were analyzed using DAS 2.0 dynamics software,and the experimental results suggest that ENR was rapidly absorbed and extensively distributed in carp tissues through systemic circulation,and the pharmacokinetic characteristics can be described with a two-compartment model.The elimination half-lives(t_(1/2β))from muscle,liver,gill,plasma and skin were 131,160,104,132 and 310 h,respectively.The areas under the drug concentration-time curves(AUC)for these tissues were 491,972,750,249 and 706hmgkg^(-1),respectively.The maximum concentration(C_(max))values were 13,29,37,9 and 5mgkg^(-1) with peak times(t_(max))of 8,4,4,2 and 4 h,respectively.Ciprofloxacin,the active metabolite of ENR,was also detected in carp tissues,indicating that only 1.54%of de-ethylation of ENR occurs in carp.At a water temperature of 18℃,the drug withdrawal time was determined to be no less than 24 d while the carp was fed at a single dosage of 40mgkg^(-1).

Ontogeny of Immune Related Organs during Early Development of Carp (Cyprinus Carpio L.)

In this paper, ontogeny of immune-related organs during theearly development of carp was studied by histochemical technique-lcineBlue staining under acid conditions and PAS reaction(AB-PAS staining). Thekidney appeared one day before hatching, spleen, liver and pancreas emergedon the same day of hatching, and thymus was not found until the third day afterhatching. Ontogeny of these immune related organs of the carp in thisresearch is earlier than that reported by Botham.

Preliminarily study on the maximum handling size, prey size and species selectivity of growth hormone transgenic and non-transgenic common carp Cyprinus carpio when foraging on gastropods

The present study preliminarily examined the differences in maximum handling size, prey size and species selectivity of growth hormone transgenic and non-transgenic common carp Cyprinus carpio when foraging on four gastropods species( Bellamya aeruginosa, Radix auricularia, Parafossarulus sinensis and Alocinma longicornis) under laboratory conditions. In the maximum handling size trial, five fish from each age group(1-year-old and 2-year-old) and each genotype(transgenic and non-transgenic) of common carp were individually allowed to feed on B. aeruginosa with wide shell height range. The results showed that maximum handling size increased linearly with fish length, and there was no significant difference in maximum handling size between the two genotypes. In the size selection trial, three pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on three size groups of B. aeruginosa. The results show that the two genotypes of C. carpio favored the small-sized group over the large-sized group. In the species selection trial, three pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on thin-shelled B. aeruginosa and thick-shelled R. auricularia, and five pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on two gastropods species( P. sinensis and A. longicornis) with similar size and shell strength. The results showed that both genotypes preferred thin-shelled Radix auricularia rather than thick-shelled B. aeruginosa, but there were no significant difference in selectivity between the two genotypes when fed on P. sinensis and A. longicornis. The present study indicates that transgenic and non-transgenic C. carpio show similar selectivity of predation on the size-and species-limited gastropods. While this information may be useful for assessing the environmental risk of transgenic carp, it does not necessarily demonstrate that transgenic common carp might have lesser environmental impacts than non-transgenic carp.

Quantitative structure-activity relationships for the Toxicity of Substituted Benzenes to Cyprinus carpio

To measure the 96h-LC50 values of 32 substituted benzenes to the carp and to study the relationship between quantitative structure-activity and structural parameters of chemicals. Methods The acute toxicity values of 32 substituted benzenes to the carp were determined in a semistatic test. The energy of the lowest unoccupied molecular orbital, and the highest occupied molecular orbital, the dipole moment and the molecular weight of substituted benzenes were calculated by the quantum chemical method MOPAC6.0. Results The range of the toxicity of studied compounds was broad, and the most toxic compound was pentachlorophenol, while the least toxic compound was 4-methylaniline. By the stepwise regression analyses, a series of Quantitative structure-activity relationships (QSAR) equations were derived from all compounds and subclasses. The equation log1/LC50=0.759logP +2.222 (R2 (adj)=0.818) was found to fit well and the average predicted percentage error was 6.16%. Conclusion The toxicity of anilines and phenols to the carp could be modeled well by logP alone, whereas the toxicity of the halogenated benzenes and nitrobenznes not containing hydroxyl or amino group can be controlled by hydrophobic and electronic factors.

Biological Significance of [^(14)C]Phenol Accumulation in Different Organs of a Murrel, Channa punctatus, and the Common Carp, Cyprinus carpio

Phenol, a ubiquitous component of industrial effluents, is a common pollutant of water resources and a serious threat to fish.The present work demonstrates that a significant amount of phenol is retained by various tissues of the common carp.Cyprinus carpio.and the snake-headed murrel.Channa punclatus.The rate of [^(14)C] phenol accumulation was higher in the carp than in the murrel.It is suggested that retention of phenol in the brain and ovary may seriously afiect the reproductive potential of the fish. 1990 Academic Press.Inc.

Karyotype Analysis of Cyprinus carpio in Dongping Lake

To investigate the genetic composition of the carp （Cyprinus carpio） in Dongping Lake and to provide eytogenetie data for exploration, protection and appropriate utilization of the germplasm resources, karyotype of the carp was analyzed in this study. In detail, colehieine was given to the fish an intraperitoneal route 24 h after the intraperitoneal injection of phytohemagglutinin （PHA） solution to arrest cells in metaphase. The kidney cells were collected for the preparation of chromosome specimens, which were observed under a microscope. Ten cells having correct number of chromosomes and clear morphology were photographed and zoomed to measure the relative length, arm ratio, ann number （NF） and other parameters of chromosomes. The results showed that the chromosome number of C. earpio in Dongping Lake was （2n = 100） ; the karyotype formula was （2n =20m ＋38sm ＋20st ＋22t）. The total number of chromosome arms （NF） was 158. By comparing the karyotype of Dongping Lake carp with that of other Cyprinus species, we concluded that Dongping Lake carp is more highly evolved among Cyprinus species.

Effect of Certain Toxicants on Gonadotropin-Induced Ovarian Non-Esterified Cholesterol Depletion and Steroidogenic Enzyme Stimulation of the Common Carp Cyprinus carpio in vitro

Isolated ovarian tissues from the common carp, Cyprinus carpio were incubated in vitro to obtain a discrete effect of four common toxicants of industrial origin, namely phenol, sulfide, mercuric chloride and cadmium chloride, on gonadotropin-induced alteration of nonesterified and esterified cholesterol and steroidogenic enzymes, △5-3β-HSD and 17β-HSD activity. Stage II ovarian tissue containing 30-40% mature oocytes were shown to be most responsive to gonadotropins in depleting only nonesterified cholesterol moiety and stimulating the activity of both. Safe doses of above mentioned toxicants when added separately to stage II ovarian tissue with oLH (1 μg/incubation) gonadotropin-induced depletion of nonesterified cholesterol and gonadotropin-induced stimulation of the activity of both enzymes was significantly inhibited. Esterified cholesterol remained almost unaltered. Findings clearly indicate the impairment of gonadotropin induced fish ovarian steroidogenesis by the four toxicants separately.

Exploring the sublethal genotoxic effects of class II organophosphorus insecticide quinalphos on freshwater fish Cyprinus carpio

The genotoxic effects of quinalphos(QP)were evaluated in the erythrocytes of Cyprinus carpio using the comet,micronucleus,and nuclear abnormality assays.The 96 h LC_(50) value of quinalphos,estimated by static renewal system,was 10.9µL/L.On the basis of the LC_(50) value,two sublethal concentrations(1.09 and 2.18µL/L)of the compound were used together with cyclophosphamide(5 mg/L)as the positive control.The fish were exposed for 35 d,and sampling was done at the end of 7,14,21,28,and 35 d.Our results reveal a significant dose-dependent increase in the frequencies of DNA strand breaks,micronucleus,and nuclear abnormalities.Similar responses were also observed in fish exposed to the positive genotoxin cyclophosphamide.The collective findings demonstrated that quinalphos exerts a genotoxic effect on C.carpio.The results further confirmed that the comet,micronucleus,and nuclear abnormality assays are useful tools in determining the potential genotoxicity of pesticides towards aquatic organisms.

Effects of Biological Flocs on Growth Performance of Cyprinus carpio var.Furui No.2 and Aquaculture Water Quality

[Objectives]To study the effects of biological flocs on growth performance of Cyprinus carpio var.Furui No.2 and aquaculture water quality.[Methods]The comparative experiment method was adopted.An experimental group and a control group were set up,with 3 replicates in each group.The experimental group used beet molasses as the carbon source which was added once a week.The breed,quantity,and specifications of stocked fish in each group were exactly the same.The experiment lasted for 60 d.Fish growth indicators were measured every 7 d.After one week of the experiment,ammonia nitrogen and nitrite were monitored once a week,and dissolved oxygen was monitored once a day.[Results]The survival rate of fish,the average weight of,the weight gain rate,and the specific growth rate of the experimental group were significantly higher than those of the control group(P<0.05),and the feed conversion rate was significantly lower than that of the control group(P<0.05).One week after adding the carbon source in the experimental group,the ammonia nitrogen was lower than the control group,and after three weeks it was significantly lower than the control group(P<0.05);after adding the carbon source,the nitrite in the experimental group was lower than the control group,there was no significant difference in the first three weeks(P>0.05),and there was a significant difference after three weeks(P<0.05).The feed conversion rate of the experimental group was significantly lower than that of the control group(P<0.05).[Conclusions]Adding a carbon source can significantly reduce ammonia nitrogen,nitrite and other toxic and harmful substances in the aquaculture water,promote the growth of aquaculture fish and increase the feed utilization rate.

Effect of Mercuric Chloride and Cadmium Chloride on Gonadal Function and Its Regulation in SexuallyMature Common Carp Cyprinus carpio

Gonadal function in fish, CJprinus carpio was significantly affected by sublethal doses of mercuric chloride (HgCl2 ) and cadmium chloride (CdCl2 ) in chronic (45 days ) exposure.Parameters investigated were nonesterified (NE) and esterified (E) cholestcrol of ovary,liver and serum and ovarian 3β-Hydroxysteroid and 17β-Hydroxysteroid dehyrogenaseenzyme activity and serum and pituitary gonadotropin (GtH) levels. Both the pollutantswere able to reduce the hypothalamic extract (HE) or gonadotropin releasing hormone(GnRH) induced pituitary GtH release in vitro. Short term (96h) exposure of the fish tothe pollutants had no significant effect on the gonadal function. ln addition to thedeleterious effect of pollutants on the gonadal steroidogenesis and pituitary gonadotropin release, using [4-14C] cholesterol as a tracer it was found that for 45 days expeure, HgCl2'had an adverse effect on the transport of cholesterol from circulation to ovary.

The Use of Cyanobacteria Arthrospira platensis and Cladoceran Daphnia magna as Complementary Protein and Lipid Sources in Transitional Diet for Common Carp (Cyprinus carpio L.) Nursery

This study was conducted to evaluate the use of cyanobacteria Arthrospira platensis and cladoceran Daphnia magna biomass as complementary protein and lipid sources in diet supplementation for common carp (Cyprinus carpio L.) nursery. Three experimental diets containing A. platensis and D. magna meal at different concentrations were compared to the commercial (control) diet. Each experimental diet (ED) was set to contain D. magna and A. platensis meal at a specific combination: 2% and 5%, 2% and 7%, and 4% and 5% for ED1, ED2 and ED3, respectively. The protein and lipid content of the experimental diets ranged from 43.20% to 44.60% dry weight (DW) and 10.64% to 13.42% DW, respectively;while the protein and lipid content of the control diet were 43.00% DW and 6.72% DW, respectively. After 20 days of feeding period, ED3 group obtained the highest final body weight (BW) (58.18 ± 35.24 mg), total biomass (1936 ± 1625 mg), food conversion rate (1.34 ± 0.04), and specific growth rate (12.86 ± 0.03% BW day–1) among all treatment groups (P > 0.05);while ED1 group obtained the highest survival (75.5% ± 7.47%) among all treatment groups (P > 0.05). The total bacterial count and total pathogenic Aeromonas sp. in the culture water of the treatment groups (ranged from 2.00 to 2.65 × 105 CFU mL–1 and 3.50 to 4.12 × 103 CFU mL–1, respectively) were lower compared to the water of the control group fed (3.73 × 105 and 4.70 × 103 CFU mL–1, respectively). No significant differences in physicochemical water quality parameters were observed among treatments (P > 0.05). The current study suggests that the combination of A. platensis and D. magna biomass can be used as complementary protein and lipid sources in diet supplementation for common carp larvae and can result in a comparable fish growth, survival and feed utilization in common carp culture at the nursery phase.

Effects of BDE-209 exposure on growth performance, intestinal digestive enzymes, and intestinal microbiome in common carp (Cyprinus carpio L.)

Polybrominated diphenyl ether has become a widespread environmental pollutant in aquatic environments.BDE-209,a prominent PBDEs found in the environment and wildlife,has been proven to cause oxidative stress,immune damage and endocrine system disorders in fish.However,the effects of BDE-209 on the intestinal health of fish remain unclear.This research aimed to determine the effects of BDE-209 on the growth performance,digestive enzymes activities and intestinal microbiota of common carp.After 60-day exposure,the growth performance of common carp were remarkably decreased(P<0.05).Moreover,the levels of protease,amylase and lipase activities remarkably decreased by BDE-209 exposure(P<0.05).In addition,metagenomics analysis revealed that the alpha-diversity values of intestinal microbiota were significantly decreased in BDE-209 exposure groups(P<0.05).The results also indicated that BDE-209 exposure could alter the intestinal microbial compositions.At the phylum level,the proportion of Fusobacteria was markedly reduced(P<0.05),while the proportions of Bacteroidetes and Firmicutes were significantly increased(P<0.05)along with the increasing concentrations of BDE-209.At the genus level,the proportion of Shewanella was higher(P<0.05)in BDE-209 exposure groups than in control group,while the abundance of Cetobacterium showed an opposite trend.Furthermore,the proportions of some aquatic probiotics(such as Bacillus,Rhodopseudomonas and Lactobacillus)were significantly enriched(P<0.05)in control group,and the content of short-chain fatty acid producing bacterium(such as Faecalibacterium,Bacteroides and Clostridium)were decreased along with the increasing BDE-209 concentrations,but no significant differences were found among the four groups(P>0.05).Overall,exposure to BDE-209 could alter the intestinal microbiota of common carp,which may affect their growth and threaten their health.

Immunomolecular response of CD4^(+),CD8^(+),TNF-α and IFN-γ in Myxobolus-infected koi(Cyprinus carpio)treated with probiotics

A poor environment increases fish’s susceptibility to myxosporean infection that can cause the death of larval fish,especially for koi fish(Cyprinus carpio).This study aimed to determine the effect of probiotics,local anti-parasitic drugs(kutuklin),and the chemical compound diflubenzuron treatments on the koi immune response.This study used PCR with specific primer 18S SSU rDNA and DNA sequencing to detect Myxobulus phylogenetic.The treatments were divided into 5 groups:Treatment(A)(healthy koi without treatment),(B)(infected koi without treatment),(C)(infected koi with 0.55 mL/30 L probiotics),(D)(infected koi with 1μL/g of feed kutuklin),and(E)(infected koi with 0.02 mg/5 L dimilin).Myxospore has observed with Scanning Electron Microscopy(SEM)and 4′,6-diamidino-2-phenylindole(DAPI)fluorescence staining.The histological analysis using semi-quantitative scoring methods,and flow cytometry was conducted to analyse the immune response of Cluster of differentiation 4(CD4^(+)),Cluster of differentiation 8(CD8^(+)),Tumor Necrosis Factor-alpha(TNF-α),Interferon gamma(IFN-γ)cells in the gills.Results show that the histological analysis indicated edema,hyperplasia,lamella fusion,congestion,and hypertrophy lesions in infected koi.Treatment with probiotics shows the lowest damage(30.6%).The immune responses of CD4^(+)and CD8^(+)cells to dimilin treatment were 10.54%and 16.86%,respectively.The largest TNF-αand IFN-γresponse were for the kutuklin treatment(29.26%)and probiotics treatment(8.23%).

Cloning and expression analysis in mature individuals of two chicken type-Ⅱ GnRH (cGnRH-Ⅱ) genes in common carp (Cyprinus carpio)

Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide fam-ily, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II pre-cursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplica-tion hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the ex-pression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.

Hepatoprotective and antioxidant effects of dietary Glycyrrhiza polysaccharide against TCDD-induced hepatic injury and RT-PCR quantification of AHR2,ARNT2,CYPIA mRNA in Jian Carp(Cyprinus carpio var.Jian)

To evaluate the protective effects of Glycyrrhiza polysaccharide（GPS） against 2,3,7,8-tetrachlorodibenzo-p-dioxin（TCDD）-induced hepatotoxicity in Jian carp,the fish were fed diets containing GPS at doses of 0.1,0.5 and 1.0 g/kg for 60 days before an intraperitoneal injection of 0.6 μg/kg TCDD at a volume of 0.05 mL/10 g body weight.At 72 hr post-injection,blood and liver samples were taken for biochemical analysis and the fish liver samples were used for the preparation of pathological slices.The results showed that increases in alanine aminotransferase（GPT）,aspartate aminotransferase（GOT）,lactate dehydrogenase（LDH）,and alkaline phosphatase（AKP） in serum induced by TCDD were significantly inhibited by pre-treatment with 1.0 g/kg GPS.Following the 1.0 g/kg GPS pre-treatment,total protein（TP）,albumin（Alb）,catalase（CAT）,glutathione peroxidase（GPx）,total antioxidant capacity（T-AOC） and superoxide dismutase（SOD） activities in liver tissue increased significantly,malondialdehyde（MDA） formation（P ＆lt; 0.05 or P ＆lt; 0.01） was significantly inhibited,and the expression of cytochrome P4501A（CYP1A）,aryl hydrocarbon receptor 2（AHR2） and aryl hydrocarbon receptor nuclear translocator 2（ARNT2） mRNA（P ＆lt; 0.05） was significantly enhanced.Histological observations on fish liver were obtained by preparing paraffin tissue sections via HE staining,and the results showed that histological changes were obviously reduced by 0.5 and 1.0 g/kg GPS.GPS significantly reduced liver tissue damage caused by TCDD.Overall,these results proved the hepatoprotective effect of GPS in protecting against fish liver injury induced by TCDD,and supported the use of GPS（1.0 g/kg） as a hepatoprotective and antioxidant agent in fish.

Quantitative genetic estimates of growth-related traits in the common carp(Cyprinus carpio L.):A review

The common carp(Cyrpinus carpio L.)is the oldest cultured and the most domesticated fish species,as well as one of the most important freshwater fishes in the world.However,scientific studies on evaluating the growth-related quantitative traits in this fish are limited.Heritability,the most important parameter in selective breeding programs,was extensively studied for the growth-related traits.The values varied widely among the experiments and methods used because of the existence of common environmental,dominance and maternal effects.However,correlations in phenotypic and genetic levels first evaluated several years ago were limited.On the other hand,heterosis was widely reported and easily obtained for growth-related traits in the common carp.Meanwhile,genotype environment interaction and prediction of breeding values have been studied recently,and are very important in conducting selective breeding programs.The developmental quantitative genetics of growth-related traits was first analyzed in the common carp for reasonable selection during ontogeny.It is expected that genetic improvement will be achieved by carrying out direct selective breeding in the common carp.

Acute toxicity test of pesticide abamectin on common carp(Cyprinus carpio)

Objective:To determine acute toxicity of abamectin(abamectin used for agricultural fields and also is a common acaridae used in farms)to common carp(Cyprinus carpio).Methods:In this research,common carps were exposed to abamectin for 96 h.LC_(50) values of 24 h,48 h,72 h and 96 h were attained by probit analysis software SPSS Version 16.Fish were exposed to different concentrations(1,2,3,6,12 and 15 mg/L)of abamectin for 96 h and physicochemical properties of water used for these experiments were stable and every mortality was recorded daily.Results:The 96 h LC_(50)of abamectin for Cyprinus carpio was 1.243 mg/L.Conclusions:Eventually toxicity values indicated that abamectin has same toxicity in studied other specie and we can state lower value of LC_(50)for studied specie in compare with most species.

Assessment of toxicity of two nitroaromatic compounds in the freshwater fish Cyprinus carpio

This study was conducted to evaluate the toxicological response ofp-nitrotoluene and p-nitroaniline to the key fish species, Cyprinus carpio. A freshwater fish bioassay based on the 96 h LC50 was used to estimate the single and joint toxicity of the two chemicals. The toxicity of p-nitrotoluene was greater than that of pnitroaniline based on 96 h LCso values of 40.74 mg.L^-1 and 48.99mg.L^-1, respectively. Both compounds had moderate toxicity toward Cyprinus carpio, and this toxicity increased with the exposure duration and con- centration. Binary mixtures of the compounds were more toxic than the individual compounds at 96 h, and they acted upon partial addition. When the exposure time was longer, the toxicity increased for mixtures of compounds with the same concentration or toxicity. The results of this study suggest that exposure to a combination of these chemicals would result in a higher environmental risk in aquatic systems than exposure to either compound alone. Further research is needed to investigate the combined effects and sublethal toxicity of p-nitrotoluene and p- nitroaniline, since they are both still used in China.