Starvation on changes in growth and fatty acid composition of juvenile red swamp crawfish, Procambarus clarkii

The effect of starvation on physiological and biochemical indicators of body, muscle and hepatopancreas were investigated for juvenile red swamp crawfish (Procambarus clarkii), and the results showed the effect was obvious. In a 70-day starvation experiment, mean body weight, exuvial rate, ab- dominal muscle ratio and hepatosomatic index decreased significantly. The percentage of protein, fat, ash and moisture were also significantly affected. The protein content decreased sharply at Day 30 onward and the percentage of fat decreased rapidly from Day 50 and on. The percentages of ash and moisture started to increase significantly at Day 40 and 30 respectively. After the starvation experiment, the hepa- topancreatic total and neutral lipid content decreased slightly from 23.90% to 20.39% and 73.75% to 70.35%, while the hepatopancreatic polar lipid content increased from 23.81% to 27.32%. On the other hand, all the muscular total, neutral and polar lipid contents, and the muscular fatty acid compositions did not change significantly except for the hepatopancreatic fatty acid composition. The relative percentages of 14:0, 16:0, 16:1n-7 and 18:1n-9 were decreased by 55.17%, 11.11%, 8.5% and 6.70% from the initial values, and the relative percentages of 18:2n-6, 18:3n-3, 20:4n-6, 20:5n-3 and 22:6n-3 increased obvi- ously from 5.2%, 3.9%, 2.4%, 10.6%, and 13.7% to 7.1%, 5.0%, 2.8%, 11.5%, and 14.4% respectively. Preference of utilization of fatty acids from hepatopancreas during starvation was as follows: 14:0>16:0>16:1n-7>18:1n-9, and the conservation of highly unsaturated fatty acids was in the order of: 18:2n-6 >18:3n-3>20:4n-6>20:5n-3>22:6n-3.     

Seasonal variation of biochemical components in clam (<Emphasis Type="Italic">Saxidomus purpuratus</Emphasis> Sowerby 1852) in relation to its reproductive cycle and the environmental condition of Sanggou Bay,China

Seasonal variation of biochemical components in clam (Saxidomus purpuratus Sowerby 1852) was investigated from March 2012 to February 2013 in relation to environmental condition of Sanggou Bay and the reproductive cycle of clam. According to the histological analysis, the reproductive cycle of S. purpuratus includes two distinctive phases: a total spent and inactive stage from November to January, and a gametogenesis stage, including ripeness and spawning, during the rest of the year. Gametes were generated at a low temperature (2.1°C) in February. Spawning took place once a year from June to October. The massive spawning occurred in August when the highest water temperature and chlorophyll a level could be observed. The key biochemical components (glycogen, protein and lipid) in five tissues (gonad, foot, mantle, siphon and adductor muscle) were analyzed. The glycogen content was high before gametogenesis, and decreased significantly during the gonad development in the gonad, mantle and foot of both females and males, suggesting that glycogen was an important energy source for gonad development. The protein and lipid contents increased in the ovary during the gonad development, demonstrating that they are the major organic components of oocytes. The lipid and protein contents decreased in the testis, implying that they can provide energy and material for spermatogenesis. The results also showed that protein stored in the mantle and foot could support the reproduction after the glycogen was depleted.     

Development of fatty acid biomarkers for the identification of wild and aquacultured sea cucumber (Apostichopus japonicus)

In this study,the fatty acids(FAs) of the organs and tissues of sea cucumber(Apostichopus japonicus) were profiled in order to compare the FA composition of sea cucumber collected from natural habitat(wild) and cages(cultured).The differences in FA contents in dermomuscular tube,peripharyngeal annulus,gonad and intestine(with or without content) between the wild and the cultured were determined.The main fatty acids in all organs and tissues were 20:5n-3,16:1n-7,20:4n-6,22:6n-3,18:0,and 18:1n-7.The basically different FAs of body wall and digestive tube were 16:1n-7,18:1n-9 and 20:1n-11.The ratio of saturated to mono-and polyunsaturated FAs in digestive tube was independent on inside content while there was a redistribution of the total amount of n-3 and n-6 fatty acids.The comparison of FA composition of the wild and the cultured sea cucumber showed that 20:5n-3,16:1n-7 and 18:1n-7 predominated the wild while 20:4n-6 predominated the cultured.The content of branched-chain fatty acids in the wild was 3%–4% and about 9% in the cultured.The possible FAs for identifying the wild and the cultured sea cucumbers were selected.It was suggested that the indexes such as the ratio of either(n-3:n-6) to(n-7:n-6) or(n-3) +(n-7) to(n-6) may serve as the biomarkers distinguishing the wild and the cultured sea cucumber.     

Lipid and fatty acid compositions of cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>), haddock (<Emphasis Type="Italic">Melanogrammus aeglefinus</Emphasis>) and halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis>)

This study was conducted to compare lipid and fatty acid composition of cod, haddock and halibut. Three groups of cod (276 g ± 61 g), haddock (538 g ± 83 g) and halibut (3704 g ± 221 g) were maintained with commercial feeds mainly based on fish meal and marine fish oil for 12 weeks prior to sampling. The fatty acid compositions of muscle and liver were determined by GC/FID after derivatization of extracted lipids into fatty acid methyl esters (FAME). Lipids were also fractionated into neutral and polar lipids using Waters silica Sep-Pak?. The phospholipid fraction was further separated by high-performance thin-layer chromatography (HPTLC) and the FAME profile was obtained. Results of the present study showed that cod and haddock were lean fish and their total muscle lipid contents were 0.8% and 0.7%, respectively, with phospholipid constituting 83.6% and 87.5% of the total muscle lipid, respectively. Halibut was a medium-fat fish and its muscle lipid content was 8%, with 84% of the total muscle lipid being neutral lipid. Total liver lipid contents of cod, haddock and halibut were 36.9%, 67.2% and 30.7%, respectively, of which the neutral lipids accounted for the major fraction (88.1%–97.1%). Polyunsaturated fatty acids were the most abundant in cod and haddock muscle neutral lipid. Monounsaturated fatty acid level was the highest in halibut muscle neutral lipid. Fatty acid compositions of phospholipid were relatively constant. In summary, the liver of cod and haddock as lean fish was the main lipid reserve organ, and structural phospholipid is the major lipid form in flesh. However, as a medium-fat fish, halibut stored lipid in both their liver and muscle.     

Lipid and Fatty Acid Compositions of Cod(Gadus morhua),Haddock(Melanogrammus aeglefinus)and Halibut(Hippogiossus hippoglossus)

This study was conducted to compare lipid and fatty acid composition of cod,haddock and halibut.Three groups of cod(276 g±61 g),haddock(538 g±83 g)and halibut(3704 g±221 g)were maintained with commercial feeds mainly based on fish meal and marine fish oil for 12 weeks prior to sampling.The fatty acid compositions of muscle and liver were determined by GC/FID after derivatization of extracted lipids into fatty acid methyl esters(FAME).Lipids were also fractionated into neutral and polar lipids using Waters silica Sep-Pak(R).The phospholipid fraction was further separated by high-performance thin-layer chromatography(HPTLC)and the FAME profile was obtained.Results of the present study showed that cod and haddock were lean fish and their total muscle lipid contents were 0.8% and 0.7%,respectively,with phospholipid constituting 83.6% and 87.5% of the total muscle lipid,respectively.Halibut was a medium-fat fish and its muscle lipid content was 8%,with 84% of the total muscle lipid being neutral lipid.Total liver lipid contents of cod,haddock and halibut were 36.9%,67.2% and 30.7%,respectively,of which the neutral lipids accounted for the major fraction(88.1%-97.1%).Polyunsaturated fatty acids were the most abundant in cod and haddock muscle neutral lipid.Monounsaturated fatty acid level was the highest in halibut muscle neutral lipid.Fatty acid compositions of phospholipid were relatively constant.In summary,the liver of cod and haddock as lean fish was the main lipid reserve organ,and structural phospholipid is the major lipid form in flesh.However,as a medium-fat fish,halibut stored lipid in both their liver and muscle.     

Lipids and fatty acids in Calanus sinicus during over-summering in the southern Yellow Sea

Over-summering is a crucial period for Calanus sinicus in the southern Yellow Sea,where it is a key member of the zooplankton community.Lipids play an important role in copepod diapause,which is part of their over-summering strategy.We investigated how different fatty acids and lipid classes,including wax esters,changed during over-summering of C.sinicus during three cruises in June and August 2011 and November 2010,corresponding to the pre-,during and post-diapause periods,respectively.Large amounts of lipids were accumulated,mainly wax esters as previously found in C.finmarchicus during its diapause,and most of the storage lipids were used during over-summering.Wax ester polyunsaturated fatty acids(PUFAs) showed the most variation of the fatty acids(FAs),while the percentage composition of FAs in polar lipids was relatively stable.Selective use of wax ester PUFAs has already been shown to play important roles in the winter diapause of Calanus species in other regions,and our FA results show that this is the case for the Yellow Sea Cold Bottom Water(YSCBW) population that diapauses in summer.     

Changes of Protein and Lipid Contents,Amino Acid and Fatty Acid Compositions in Eggs and Yolk-Sac Larvae of American Shad(Alosa sapidissima)

To investigate the changes of the biochemical composition of American shad(Alosa sapidissima) eggs and larvae at embryonic and early larval stages, samples were collected at different development stages from artificial fertilization to the end of yolk absorption including 2 h, 12 h and 30 h after fertilization and newly hatched larvae including 1 and 3 days after hatching. The composition of lipid, fatty acids, protein and amino acids were analyzed. The content of total protein exhibited a decreasing trend during embryogenesis and larval development, and a significant reduction was detected after hatching(P 0.05). The total lipid content remained relative stable. A significant reduction was detected in almost all amino acids after hatching except for glycine(P 0.05), while a significant decrease was found in the content of cysteine, proline, tyrosine, valine, isoleucine, leucine and phenylalanine during the yolk-sac phase(P 0.05). On the other hand, all the groups of fatty acids remained stable during the period of embryogenesis. But after hatching, a significant decrease was found in the content of C18:2n-6, C18:3n-6, SFA and ratio of EPA/ARA(P 0.05), while a significant increase was found in the content of C18:3n-3, C20:4n-6, C22:6n-3 and ratio of n-3/n-6(P 0.05). In conclusion, the combined data suggested that American shad utilizes the protein content as preferential energy substrates during embryonic and early larval developments with some specificity in the consumption of different amino acids.     

Lipid oxidation and fatty acid composition in salt-dried yellow croaker (<Emphasis Type="Italic">Pseudosciaena polyactis</Emphasis>) during processing

Lipid oxidation in salt-dried yellow croaker (Pseudosciaena polyactis) was evaluated during processing with commonly used analytical indices, such as the peroxide value (POV), the thiobarbituric acid reactive substances (TBARS) value, and oxidative-relative lipoxygenase (LOX) activity. Additionally, fatty acids were analyzed using gas chromatography-mass spectrometry. Both POV and TBARS increased significantly (P < 0.05) at the rinsing stage. POV reached its peak value of 3.63 meq O₂ per kg sample at the drying stage, whereas TBARS constantly increased from 0.05 to 0.20 mg MDA per kg sample. Processing of salt-dried yellow croaker had an extremely significant (P < 0.01) effect on LOX activity. Twenty-six fatty acids were identified. Combined eicosapentaenoic acid (EPA; C20:5n3) and docosahexaenoic acid (DHA; C22:6n3) content varied between (19.20 ± 0.37) mg g^?1 and (23.45 ± 1.05) mg g^?1. The polyunsaturated fatty acid/saturated fatty acid (PUFA/SFA) ratio in yellow croaker was 0.73–1.10, and the n-6/n-3 PUFA ratio was approximately 0.13–0.20. The contents of most fatty acids varied significantly (P < 0.05) during the different processing stages, and these differences were caused by lipid oxidation. C18:0, C16:1n7, C19:0, and C22:6n3 showed clear changes in principle component one of a principle components analysis. These fatty acids are potential markers for evaluating lipid oxidation in fish muscle because there was a significant correlation between these markers and TBARS and LOX activity (P < 0.05) with Pearson’s coefficients > 0.931.     

EFFECT OF LIGHT INTENSITY ON THE TOTAL LIPID AND FATTY ACID COMPOSITION OF SIX STRAINS OF MARINE DIATOMS

The effect of light intensity ( 1500 Ix and 5000 Ix) on the total lipid and fatty acid composition of six strains of marine diatoms Cylindrotheca fusiformis (B211 ), Phaeodactylum tricornutum (Bl14, Bl18 and B221) Nitzschia closterium (B222) and Chaetoceros gracilis (B13) was investigated.The total lipids of B13, Bl14, and B211 grown at 5000 Ix were lower than those grown at 1500 Ix. No evident changes were observed in Bl18, B221 and B222. Fatty acid composition changed considerably at different light intensity although no consistent correlation between the relative proportion of a single FA and light intensity. The major fatty acids of the 6 strains were 14:0, 16:0, 16:l(n- 7) and 20:5(n-3) . Cylindrothecafusiformis had high percentage of 20:4n - 6 (9.2 - 10.9% ) . The total polyunsaturated fatty acid /rl all 6 strains decreased with increasing light intensity. The percentage of the highly unsaturated fatty acid eicosapentaenoic acid (EPA) decreased with increasing light intensity in all strains except Chaetoceros gracilis.     

Fatty acid composition of <Emphasis Type="Italic">Euphausia superba</Emphasis>, <Emphasis Type="Italic">Thysanoessa macrura</Emphasis> and <Emphasis Type="Italic">Euphausia crystallorophias</Emphasis> collected from Prydz Bay,Antarctica

The information of trophic relationship is important for studying the Southern Ocean ecosystems. In this study, three dominant krill species, Euphausia superba, Thysanoessa macrura and Euphausia crystallorophias, were collected from Prydz Bay, Antarctica, during austral summer of 2009/2010. The composition of fatty acids in these species was studied. E. superba and T. macrura showed a similar fatty acid composition which was dominated by C14:0, C16:0, EPA (eicosapentenoic acid) and DHA (decosahexenoic acid) while E. crystallorophias showed higher contents of C18:1(n-9), C18:1(n-7), DHA and EPA than the former two. Higher fatty acid ratios of C18:1(n-9)/18:1(n-7), PUFA (polyunsaturated fatty acid)/SFA (saturated fatty acid), and 18PUFA/16PUFA indicated that E. crystallorophias should be classified as a typical omnivore with a higher trophic position compared with E. superba and T. macrura.     

Comparison of lipids in organs of the starfish Asterias amurensis associated with different treatments

Lipids were extracted from organs of the starfish Asterias amurensis associated with different treatments(raw-control,boiling and heating),and then analyzed for lipid content,lipid oxidation index,lipid classes and fatty acid composition.Results showed that boiling softened the hard starfish shells,thus facilitating the collection of starfish organs.As compared with raw organs,the boiled organs had lower water content and higher lipid content,possibly due to the loss of water-holding capacity caused by protein denaturation.Both boiling and heating increased the peroxide value(PV),thiobarbituric acid(TBA) value and carbon value(CV) of lipids.Despite slight increases in the content of complex lipids,associated lipid composition had no substantial variations upon boiling and heating.For simple lipids,the content of 1,2-diglyceride decreased in boiled and heated organs,with free fatty acids observed on thin layer chromatography(TLC).However,neither boiling nor heating significantly changed the fatty acid compositions of simple or complex lipids in starfish organs,suggesting that these two treatments had no significant effects on complex lipids in starfish organs.Together,our results indicated that boiling of starfish soon after capture facilitated the handling and extraction of useful complex lipids consisting of abundant glucosylceramide and eicosapentaenoic acid(EPA)-bounded phospholipids.     

Seasonal changes of reproductive activity and biochemical composition of pen shell <Emphasis Type="Italic">Atrina pectinata</Emphasis> Linnaeus, 1767 in Bohai Sea,China

Seasonal variations in the biochemical composition and the reproductive cycle of pen shell Atrina pectinata in Bohai Sea were investigated from May 2013 to April 2014. Histological analysis indicated that the reproductive cycle of A. pectinata can be divided into two phases, inactive stage and gametogenesis, which were equally and significantly influenced by seawater temperature and food availability. Gametogenesis began in late autumn (October), and completed in June and July. Spawning took place in August, coinciding with the highest water temperature and the richest phytoplankton. The significantly high glycogen content in adductor muscles sustained throughout the late active and ripeness stages, but plummeted during spawning and inactive stages. The protein content in female gonads exhibited a synchronous increase along with oocyte diameter and lipid content, suggesting that the female gonads could accumulate protein and lipid for vitelline in A. pectinata. Furthermore, the RNA/DNA ratio was found to be a useful index to indicate the level of gonad maturation in both males and females. The findings of the present study provided a foundation for the fishery resource administration and the aquaculture development of this species.     

Distribution characteristics of lipids in hadal sediment in the Yap Trench

In this study, cored sediment samples collected by the Jiaolong Submersible at 6 779 m depth from the hadal zone of the Yap Trench in May 2016 were sliced in 1-cm interval from top to bottom, and lipids in each sediment layer were quantitatively and qualitatively analyzed. The vertical distribution profiles of the lipids in the sediment sample, their main existing forms, and their possible sources were investigated. The results show that the concentration of lipid in the surface sediment was the highest with the carbon number from 12 to 27, dominated by medium and short-chain lipids. The total concentration of fatty acids in surface sediment was much higher than those in the offshore and deep-sea areas, being up to 325.77 μg/g due to the funnel effect caused by the “V” terrain of the trench. Fatty acid 18.0 was the most abundant lipids in the sediment sample. Abnormal high concentrations of fatty acid 18.ω7 and alkanes indicated the existence of hydrothermal fluids in the study area. In addition, saturated fatty acids and monounsaturated fatty acids existed mainly in free form, and polyunsaturated fatty acids existed mainly in bound form. Most of the alkanes were in bound form, and their major source was autochthounous input. The carbon number of alcohols in the sediment sample ranged from 12 to 20, mainly existed in bound form. The source of fatty acids was mainly autochthonous input, and the neutral lipids had both marine and terrestrial origin. This is the first study of lipids in hadal sediment of the Yap Trench. The results will promote deeper understanding of organic carbon cycle in marine environment.     

Fatty Acid Composition of Euphausia superba,Thysanoessa macrura and Euphausia crystallorophias Collected from Prydz Bay,Antarctica

The information of trophic relationship is important for studying the Southern Ocean ecosystems.In this study,three dominant krill species,Euphausia superba,Thysanoessa macrura and Euphausia crystallorophias,were collected from Prydz Bay,Antarctica,during austral summer of 2009/2010.The composition of fatty acids in these species was studied.E.superba and T.macrura showed a similar fatty acid composition which was dominated by C14:0,C16:0,EPA(eicosapentenoic acid) and DHA(decosahexenoic acid) while E.crystallorophias showed higher contents of C18:1(n-9),C18:1(n-7),DHA and EPA than the former two.Higher fatty acid ratios of C18:1(n-9)/18:1(n-7),PUFA(polyunsaturated fatty acid)/SFA(saturated fatty acid),and 18PUFA/16 PUFA indicated that E.crystallorophias should be classified as a typical omnivore with a higher trophic position compared with E.superba and T.macrura.     

The Response of Bio-Component Production of Nannochloris oculata to the Combinations of Monochromatic Light

Illuminating conditions are crucial factors affecting the microalgal growth and biosynthesis.The effects of illuminating spectral quality on the growth and bio-component production of Nannochloris oculata were investigated.The results indicated that a high proportion of Red-light enhanced the pigments and carbohydrate production but reduced those of the biomass and lipid.Mono-chromatic Blue-light has advantageous effects on lipid production compared with Red-and White-light.The optimal light spectrum for the protein production was the combination of Red-and Blue-light at a ratio of 4:3 or 5:2.Among the seven fatty acids identified from N.oculata,the contents of C16:0,C18:0,and C18:3(n-3)in the lipid were inhibited by the increased proportion of Red-light while those of C18:2(n-9),C16:2(n-6),and C20:0 were inhibited by Blue-light.Monochromatic Red-and Blue-light and their com-binations were proposed as a promising illuminating strategy for the large-scale cultivation aiming for various bio-components.     

Seasonal variation of the glycogen enzyme activity in diploid and triploid Pacific oyster gonad during sexual maturation

The glycogen content and the activities of two key enzymes in glycogen metabolism, glycogen phosphorylase and gly- cogen synthetase, in the gonad of diploid and triploid Pacific oysters (Crassostrea gigas) were compared during maturation. The glycogen content in the gonad of diploids decreased with gametogenesis (by 85.7%), but the glycogen content in the gonad of trip- loids did not vary significantly. Activity of glycogen phosphorylase (GP) in the gonad of diploids decreased with gametogenesis (by 55.5%), while GP activity of triploids did not vary significantly during maturation. Activity of glycogen synthetase (GS) in the gonad of diploids increased slightly with gametogenesis, reaching a peak in June. Activity of GS declined sharply from June to July, which might be due to gonad spawning. GS activity of triploid oysters in spawning time (July and August) was significantly higher than that in other months, which might be explained with a ‘compensating’ mechanism for the higher glycogen content in triploids.     

Responses in growth, lipid accumulation, and fatty acid composition of four oleaginous microalgae to different nitrogen sources and concentrations

Nitrogen deficiency is an effective strategy for enhancing lipid production in microalgae. Close relationships exist among lipid production, microalgal species, and nitrogen sources. We report growth, lipid accumulation, and fatty acid composition in four microalgae （Chloroeoccum ellipsoideum UTEX972, Chlorococcum nivale LB2225, Chlorococcum tatrense UTEX2227, and Scenedesmus deserticola JNU19） under nitrate- and urea-nitrogen deficiencies. We found three patterns of response to nitrogen deficiency： Type-A （decrease in biomass and increase in lipid content）, Type-B （reduction in both biomass and lipid content）, and Type-C （enhancement of both biomass and lipid content）. Type-C microalgae are potential candidates for large-scale oil production. Chlorococcum ellipsoideum, for example, exhibited a neutral lipid production of up to 239.6 mg/（L＇d） under urea-nitrogen deficiency. In addition, nitrogen deficiency showed only a slight influence on lipid fractions and fatty acid composition. Our study provides useful information for further screening hyper-lipid microalgal strains for biofuel production.     

Effect of growth phase on the fatty acid compositions of four species of marine diatoms

The fatty acid compositions of four species of marine diatoms (Chaetoceros gracilis MACC/B13, Cylindrotheca fusiformis MACC/B211, Phaeodactylum tricornutum MACC/B221 and Nitzschia closterium MACC/B222), cultivated at 22 ℃±1 ℃ with the salinity of 28 in f/2 medium and harvested in the exponential growth phase, the early stationary phase and the late stationary phase, were determined. The results showed that growth phase has significant effect on most fatty acid contents in the four species of marine diatoms. The proportions of 16:0 and 16: 1n-7 fatty acids increased while those of 16 :3n-4 and eicosapentaenoic acid (EPA) decreased with increasing culture age in all species studied. The subtotal of saturated fatty acids (SFA) increased with the increasing culture age in all species with the exception of B13. The subtotal of monounsaturated fatty acids (MUFA) increased while that of polyunsaturated fatty acids (PUFA) decreased with culture age in the four species of marine diatoms. MUFA reached their lowest value in the exponential growth phase, whereas PUFA reached their highest value in the same phase.     

Effect of Long-Term Low-Salinity Culture on the Survival,Growth, and Nutrient Composition of Mud Crab Scylla paramamosain

This study investigated the effects of long-term low-salinity on the growth performance,proximate composition,amino acid and fatty acid profiles of Scylla paramamosain.The salinity of the low-salinity-culture group(LC)and maintained-salinity-cul-ture group(MC)were set at 5 and 25 respectively.After rearing for 30 d,the survival rate(SR)of the LC group was significantly lower,whereas the average daily molting frequency(ADMF)was significantly higher than those in MC group(P<0.05).There were no significant difference for weight gain rate(WGR)and specific growth rate(SGR)between the two groups.The moisture content of the LC group was slightly higher than the MC group without significant difference.The contents of ash,crude lipid and crude pro-tein in the LC group were lower than those in the MC group,but no significant differences were found except for ash content.A total of 17 free amino acids and 23 fatty acids were identified from the muscle of S.paramamosain.The total amino acids(TAA),essen-tial amino acids(EAA)and essential amino acid index(EAAI)had no significant difference between the two groups.The crabs from LC group had a significantly higher HUFA,including EPA,DHA,ARA.The percentage of n-3 PUFA and the ratio of n-3/n-6 PUFA were significantly higher in the LC group.These results indicated that S.paramamosain rearing at low salinity had significantly de-creased SR;however,the low-salinity environment significantly promoted the accumulation of unsaturated fatty acids(UFAs),espe-cially the n-3 PUFA,which might be related to the osmoregulation mechanism.