Free Amino Acids In Haemolymph Research Articles

The osmoregulatory mechanism in response to hypoosmotic stress and the key role of ABCC1 in osmoregulation in the mud crab, Scylla paramamosain

Scylla paramamosain is an important mariculture animal. In the present study, we examined the physiological and biochemical characteristics of juvenile S. paramamosain under hypoosmotic stress and further verified the function of ABCC1, a key transport protein in gill tissue. The results showed that Na+/K+-ATPase, Ca2+-ATPase, and Na+/K+/2Cl- cotransporter in gill tissues increased first and then decreased. In addition, SOD and CAT activities also began to gradually increase, protecting gill tissues from oxidative damage. The hemolymph osmolarity decreased rapidly from 792 mOsm/kg to 560 mOsm/kg within 6 h, and then rose to 626.25 mOsm/kg at 48 h, and then remained stable. At the same time, Na+, Cl-, K+, and Ca2+ concentrations were decreased to varying degrees. There were significant changes in free amino acids such as aspartic acid, glutamic acid, and asparagine in hemolymph in response to acute salt depletion. By injection of ABCC1 inhibitor/activator, it was found that while ABCC1 transporter activity in the MK-571 group showed a decreasing trend at 9 h, Na+/K+ -ATPase activity began to increase and reached a peak at 48 h. Compared with the control group, the levels of aspartic acid, glutamic acid, asparagine, glycine, arginine, alanine, lysine, and proline in the MK-571 group showed significant differences at 6 h; Significant differences in glutamate, asparagine, arginine, alanine, lysine, and proline appeared at 12 h in the Thiethylperazine group. These results suggested that the dynamic changes of gill ion transport enzymes, antioxidant enzymes, inorganic ions, and free amino acids in hemolymph occurred in S. paramamosain under hypoosmotic stress, and the ABCC1 transporter played an important role in this regulatory process.

Physiological response of the razor clam Sinonovacula constricta exposed to hyposalinity stress

The razor clam Sinonovacula constricta is an economically important bivalve species in China, which inhabits the mudflat of intertidal and estuaries areas and frequently suffers the low salinity stress due to heavy rain events. The present study investigated the effects of hyposalinity stress (salinity 10, 5, 3, and 1) on osmoregulation and haemolymph microbiota in S. constricta after 7 days of exposure. The results showed that the survival rate of razor clams was only significantly reduced in the 1 ppt group compared to the control group (salinity 20) (P < 0.05). Haemolymph osmolality in razor clams reduced significantly with decreasing salinities (P < 0.05). Taurine, arginine, alanine and ornithine were the dominant free amino acids (FAAs) with higher contents in the haemolymph. Exposure to low salinities altered the composition of haemolymph FAAs, with alanine being the highest in the control group and taurine being the highest in the low salinity (LS) groups. Hyposalinity stress caused an increase in Na+/K+-ATPase (NKA) activity in gills, and histological analysis revealed severe hyperplasia in gills and a significant reduction in the length of gill filaments (P < 0.05). The microbial community variation in the haemolymph assessed by 16S rRNA sequencing explains that bacterial communities was shifted under low salinity stress. At the genus level, the relative abundance of Marinobacterium, Marinomonas and Pseudomonas changed significantly under hyposalinity stress (P < 0.05). These findings give further insight into the physiological responses and potential adaptation strategies of razor clams to hyposalinity stress and provide information for the development of inland low salinity culture.

Feed intake, gastro-intestinal transit and haemolymph free amino acids in the shrimp Penaeus monodon are influenced by marine meal supplementation

Aquaculture production can be improved by understanding how ingredients can affect feed intake and digestion. This is an area poorly understood for many aquaculture species, including for the shrimp Penaeus monodon. To investigate these processes, we measured feed intake, the rate at which digesta moves through the gastro-intestinal tract, the change of free amino acids (FAA) circulating in the haemolymph following feeding, and how different test ingredients affect these processes in this species. Diets supplemented with microbial Novacq™ (NQ), krill meal (KM) or whole squid meal (SQ) and a control diet were used across two separate experiments. In the first, feed intake was measured in communally housed shrimp for 60, 120 and 360 min to examine the effects of diet on ingestion. Feed intake after 360 min was greater in shrimp offered the SQ diet while no effect was observed at earlier time points. In the second experiment, individually housed shrimp were used to measure feed intake, the rate at which digesta passed through the gut and the change of individual FAA in the haemolymph over a 30 min period. For this experiment, addition of SQ and NQ increased both the rate at which digesta travels through the gut and post-prandial concentration of FAA in haemolymph at 5 min. Krill meal had the highest haemolymph concentration of amino acids at 10 min and all dietary treatments showed a convergence of haemolymph FAA by 30 min. The different ingredients appear to affect the appearance of FAA in haemolymph through different mechanisms. For SQ, the FAA content of the diet was high, being 2.8 times that of the others (27.6 vs. 10.0 g kg−1), and it is hypothesized that the availability of these FAA resulted in the rapid increase in haemolymph observed for this treatment. In contrast, two other ingredients also led to rapid increases in haemolymph FAA. However, for these diets the amount of FAA present was similar to the control indicating that other characteristics of the meals may be causal. Our data indicate that specific ingredients can increase both the rate at which digesta travels through the gut and the rate of absorption of amino acids and that these mechanisms could explain increases in both feed duration and intake.

POTENTIAL OF PYRETHRUM EXTRACT AGAINST LARVAL BIOCHEMISTRY OF RICE-MOTH, CORCYRA CEPHALONICA STAINT. (LEPIDOPTERA: PYRALIDAE)

Sub lethal doses of pyrethrum extract caused a significantly dose dependent reduction in the levels of total protein, DNA, RNA, RNA/DNA ratio and a significantly dose dependent enhancement in total free amino acids in haemolymph and fat body tissues of the larva of rice- moth, C. cephalonica. 0.0004% dose level of this extract caused maximum effect on these biochemical parameters.

Supercooling capacity along with up-regulation of glycerol content in an overwintering butterfly, Parnassius bremeri

Eggs of an endangered butterfly, Parnassius bremeri, hatch during December and young larvae grow during winter and early spring. Cold tolerance is required for survival and growth of the young larvae. Supercooling points (SCPs) were below −20°C for the young larvae compared to −9.8°C for the last fifth instar (L5) larvae, which developed during May. To investigate the depression of SCPs in young third instar (L3) larvae, polyols and free amino acids in hemolymph were quantified using a BioLC. Compared to L5 larvae, L3 larvae had higher polyols (glycerol and mannitol). However, L5 larvae had much higher levels of trehalose than L3 larvae in hemolymph. Increase of glycerol in L3 larvae was explained by enhanced expression of genes associated with glycerol metabolism. RNA-Seq analysis indicated that expression level of glycerol-3-phosphate dehydrogenase in L3 larvae was higher than that of L5 larvae and more number of glycerol kinase contigs was detected in L3 larvae. In contrast, trehalose-6-phosphate dehydrogenase was highly expressed in L5 larvae by more than 3.2 folds compared to L3 larvae. These results suggest that deep supercooling along with glycerol and other small cryoprotectants is associated with cold tolerance of young larvae of P. bremeri.

Effects of Gibberellic Acid on Hemolymph-Free Amino Acids of Galleria mellonella (Lepidoptera: Pyralidae) and Endoparasitoid Pimpla turionellae (Hymenoptera: Ichneumonidae)

Abstract The impacts of the plant growth regulator, gibberellic acid (GA3) were investigated on hemolymph free amino acids (FAAs) of Galleria mellonella L. (Lepidoptera: Pyralidae) larvae and its endoparasitoid Pimpla turionellae L. (Hymenoptera: Ichneumonidae) reared on host treated with different doses (50–5,000 ppm) of GA3 in diet. High-performance liquid chromatographic analysis of the hemolymph revealed that out of the 20 amino acids detected, 18 FAAs were found in the hemolymph of treated and untreated host larvae, whereas 19 were present in that of parasitoid. The total pool of FAAs in host larvae after treatment by GA3 fluctuated among doses and the ratio of essential amino acids in the pool increased to reach 9.54% at 1,000 ppm. Parasitoid larval total FAA concentration exhibited an increase at all treatments. However, the ratio of essential amino acids in the pool decreased &amp;gt;50% at all doses except for 50 ppm. Dose-dependent variations in individual FAAs in larval hemolymph of both species were also detected. Tyrosine, methionine, isoleucine, lysine, and tryptophan generally occurred in lowest amount both in control and treated G. mellonella larvae. The amount of serine, asparagine, glycine, isoleucine, leucine, and phenylalanine displayed differences only among GA3 doses in treatments. Great variations in the amount of all amino acids occurred at most of the treatment doses in wasp larvae hemolymph. Our study indicated that application of GA3 resulted in different effects on the quantity of FAAs associated with energy metabolisms of the pest species and its natural enemy.

Free amino acid pools as effectors of osmostic adjustment in different tissues of the freshwater shrimp macrobrachium olfersii (crustacea, decapoda) during long-term salinity acclimation

To examine osmotic regulation during long-term acclimation to a hyperosmotic medium, hemolymph osmolality, [Na+] and total protein, tissue hydration, and free amino acid (FAA) pools in abdominal muscle, gills, central nervous tissue and hemolymph were quantified in the diadromous freshwater (FW) shrimp, Macrobrachium olfersii, during direct exposure to 21‰S seawater over a 20-day period. Hemolymph osmolality and [Na+] reach stable maxima within 24 h while total protein is unchanged. Muscle and nerve tissues rapidly lose water while gills hydrate; all tissues attain maximum hydration (+5%) by 5 days, declining to FW values except for gills. Total FAA are highest in muscle, reach a maximum by 2 days (+64%), declining to FW values. Gill FAA increase by 110% after 24 h, diminishing to FW values. Nerve FAA increase 187% within 24 h, and remain elevated. Hemolymph FAA decrease (−75%) after 24 h, stabilizing well below the FW concentration. During acclimation, muscle glycine (+247%), gill taurine (+253%) and proline (+150%), and nerve proline (+426%), glycine (+415%) and alanine (+139%) increase, while hemolymph leucine (−70%) decreases. Total FAA pools contribute 10–20% to intracellular (22–70 mmol/kg) and 0.5–2.4% to hemolymph (3–7 mOsm/kg) osmolalities during direct acclimation from FW. These data emphasize the modest participation of FAA pools in intracellular osmotic regulation during physiological adaptation by M. olfersii to osmotic challenge, accentuating the role of anisosmotic extracellular regulation, suggesting that, during the invasion of freshwater by the Crustacea, dependence on intracellular adjustment employing FAA as osmotic effectors, has become progressively reduced.

Changes in free amino acids in the hemolymph of giant freshwater prawn Macrobrachium rosenbergii exposed to varying salinities: relationship to osmoregulatory ability

Changes in free amino acids (FAA) in the hemolymph of the giant freshwater prawn, Macrobrachium rosenbergii, were examined in individuals exposed to varying salinities for up to 1 week. In freshwater and under conditions of low salinity, total FAA concentrations were maintained between approximately 0.85 and 1 mM and did not exhibit changes in response to salinity exposure. Under high salinities, total FAA concentrations increased dramatically, reaching up to 2.1 mM depending on treatment. Examination of individual amino acid concentrations revealed that these increases were based on specific changes in glycine, arginine, alanine, proline and lysine. Among these, alanine showed the greatest increases, resulting in levels six-fold higher under high salinity than in freshwater and under low salinity. The other amino acid species showed increases of 2.5-fold compared to original values. These five FAAs in freshwater and under low salinity together occupied approximately 45% of total FAA contents and under high salinity comprised more than 70% of total FAA contents. These results suggest that specific hemolymph FAAs are involved in mediating response to salinity exposure in freshwater prawns.

Effect of pyriproxyfen and photoperiod on free amino acid concentrations and proteins in the hemolymph of the Colorado potato beetle, Leptinotarsa decemlineata (Say)

A North Dakota strain of the Colorado potato beetle, Leptinotarsa decemlineata (Say), was reared under both short- (8L:16D) and long-day (17L:7D) conditions. Age-related and pyriproxyfen- (JHA-) induced changes in hemolymph free amino acids and proteins were examined. Under a short-day photoperiod, the total free amino acid concentration in the hemolymph increased gradually up to 20 days of adult life, but the long-day beetles showed marked increases during the first 10 days and then decreased afterwards. Proline, glutamine and valine were the most abundant free amino acids in both sexes of beetles held under either short- or long-day photoregims. JHA treatment of diapausing adults, held under either short- or long-day conditions after treatment, terminated diapause as indicated by re-emergence from the vermiculite, feeding, mating, changes in free amino acid levels, the disappearance of diapause protein 1 and appearance of vitellogenin in the hemolymph. Furthermore, most of the JHA-treated females held under long-day conditions also matured oocytes and oviposited, but those held under short-day conditions did not.

Quantitative determination of free amino acids in the hemolymph of autogenous and anautogenous strains of Culex tarsalis (Diptera: Culicidae).

Autogenous and anautogenous strains were selected from the egg rafts of Culex tarsalis Coquillett collected from Coachella Valley, California. In autogenous (21-25 generations) and anautogenous (0-4 generations) strains used for hemolymph free amino acid analysis, the autogenous oviposition rates were 73.6-83.6% and 2.7-3.9%, the proportions of females undergoing autogenous ovarian development 120 h postemergence were 75.6 and 8.9%, respectively. To study the presence and quantities of free amino acids and explore their relationship to autogenous ovarian development, the 6 legs of females from both autogenous and anautogenous strains were removed at the coxo-femoral joints, and hemolymph was collected by centrifuging the bodies of 50-100 females. Proteins in the hemolymph were precipitated with 10% sulphosalicylic acid. On days 1, 4, 7, and 10 postemergence, a total of 17 free amino acids was found in females from the autogenous strain, and a total of 16 was found in females from the anautogenous strain in which asparagine (Asn) was absent. Comparisons of free amino acid concentrations between these 2 strains indicated that there were significant differences in total free amino acids and 13 individual free amino acids except Glu, Gln, Pro, and Met on day 1, total free amino acids and 14 individual free amino acids except Gln, Pro, and on day 4, and total free amino acids and 12 individual free amino acids except Asp, Glu, Gln, Pro, and Met on day 7 postemergence. There were no significant differences in the concentrations of individual and total free amino acids on day 10 postemergence. Female age affected free amino acid content in the autogenous strain, where total free amino acids and 14 individual free amino acid content in the autogenous strain, where total free amino acids and 14 individual free amino acids increased from day 1, peaked on day 4, declined on day 7, and declined further on day 10 postemergence. In the anautogenous strain, only total free amino acids and Ser, Gly, Ala, and Leu exhibited age-dependent changes in concentration, and the magnitude of changes in concentration was less than that in autogenous strain. Conclusively, autogenous ovarian development was the major reason for the changes in hemolymph free amino acid concentration postemergence. The current and previous findings indicate that the free amino acids, Arg, Gly, Ile, Leu, Lys, Phe, Ser, Thr, and Val putatively may be essential for vitellogenesis in mosquitoes.

Neuroendocrine Control of Osmotic Regulation in the Freshwater Shrimp Macrobrachium olfersii (Wiegmann) (Crustacea, Decapoda): Free Amino Acid Concentrations in the Hemolymph

The participation of neuroendocrine factors present within the central nervous system in the regulation of hemolymph free amino acid (FAA) concentrations was examined in the freshwater shrimp Macrobrachium olfersii. Test shrimps were injected intramuscularly with homogenates prepared from the eyestalks (ES), ventral nerve cord (VNC), supraesophageal (SEG), or thoracic ganglia (TG) of donor shrimps previously exposed for 6 hr to a high-salinity medium (HSM, 21% salinity). After injection of the homogenate, the shrimps were maintained for up to 6 hr in either freshwater (FW) or HSM. Hemolymph was sampled by cardiac puncture and prepared for reverse phase HPLC, derivatizing the FAA with phenylisothiocyanate. An FAA profile was determined and the [FAA]:[Cl-] ratios for the four FAA present in highest concentration (Gly, Arg, Ala, and Pro for ES and VNC experiments; Glu, Leu, Ala, and Val for SEG and TG experiments) were obtained. Nonparametric analyses revealed specific, notable effects resulting from homogenate injection, e.g., ES homogenate increased [Pro]/[Cl-] ratios in FW-exposed shrimps; SEG homogenate increased [Glu]/[Cl-] and [Val]/[Cl-] ratios in HSM exposed shrimps; and TG homogenate increased [FAA]/[Cl-] ratios for Glu, Leu, Ala, and Val in HSM-exposed shrimps. Total FAA concentrations decreased after exposure of the shrimps to HSM but were increased by the injection of ES homogenate in FW exposed shrimps and by TG homogenate in HSM exposed shrimps. The total [FAA]/[Cl-] ratio was also increased by TG homogenate in HSM-exposed animals. There were no clear effects on [Cl-] alone. These data are consistent with the notion that FAA titers in the hemolymph of M. olfersii reflect anisosmotic extracellular regulation and its modulation by endocrine factors which accumulate in neurosecretory cells of the ES, SEG, and TG, particularly the latter. The role of such modulation in the osmoregulatory process in vivo is discussed.

Effect of squid extracts on time course appearance of glucose and free amino acids in haemolymph in Penaeus japonicus after feeding: Preliminary results

Squid protein and squid protein extracts have been shown to improve both growth rate and food conversion in Penaeus japonicus. In the present study the effects of squid protein and two extracts on haemolymph levels of glucose and free amino acids were measured at different times after feeding, in P. japonicus fed a semi-purified diet. Glucose and free amino acid levels increased faster in the group the squid extract-supplemented diet. These results suggest an effect of squid extract on the absorption of nutrients (intestinal transport or endocytosis) but an effect on the hydrolysis of protein and starch by digestive enzymes is also possible.

Correlation between osmoregulation and cell volume regulation.

The euryhaline crab, Callinectes sapidus, behaves both as an osmoregulator when equilibrated in salines in the range of 800 mosM and below and an osmoconformer when equilibrated in salines above 800 mosM. There exists a close correlation between osmoregulation seen in the whole animal in vivo and cell volume regulation studied in vitro. Hyperregulation of the hemolymph osmotic pressure and cell volume regulation both occurred in salines at approximately 800 mosM and below. During long-term equilibration of the crabs to a wide range of saline environments, the total concentration of hemolymph amino acids plus taurine remained below 3 mM. During the first 6 h after an acute osmotic stress to the whole animal, the hemolymph osmotic pressure and Na activity gradually decreased, whereas the free amino acids remained below 3 mM. As the hemolymph osmotic pressure decreased below approximately 850 mosM, the amino acid level began to increase to 17-25 mM. This change was primarily due to increases in glycine, proline, taurine, and alanine. The likely source of the increase in hemolymph free amino acids in vivo is the free amino acid loss from muscle cells observed during cell volume regulation in vitro.

Erratum to : Changes in the concentration of &alpha;-ketoglutaric acid and free amino acids in haemolymph of the silkworm Bomyx mori, infected with a nuclear-polyhedrosis virus

Erratum to : Changes in the concentration of &alpha;-ketoglutaric acid and free amino acids in haemolymph of the silkworm <i>Bomyx mori</i>, infected with a nuclear-polyhedrosis virus

Changes in the concentration of α-ketoglutaric acid and free amino acids in haemolymph of the silkworm Bomyx mori, infected with a nuclear-polyhedrosis virus

Changes in the concentration of α-ketoglutaric acid and free amino acids in haemolymph of the silkworm Bomyx mori, infected with a nuclear-polyhedrosis virus

Significance of periodic fluctuations in the haemolymph proteins and their catabolic products during starvation and repeated injury in Scylla serrata (Forskal)

AbstractThe fate of haemolymph proteins during the course of prolonged starvation was investigated. Two different experimental protocols were set up. In experiment 1, sampling of the haemolymph was done in a group of six crabs at 3‐day intervals up to 33 days (group 1). In experiment 2, separate groups of crabs were sampled at 3‐day intervals up to 33 days (group 2).There is no change in body weight. The haemolymph volume and water increase significantly indicating hydration of haemolymph. The haemolymph protein concentration and quantity decline significantly in both groups during the course of prolonged starvation. Concomitantly, the haemolymph free amino acids concentration and quantity increase in both the groups, indicating degradation of protein during the course of starvation.That degradation of protein cannot occur continuously but rather takes place on specific days in both groups is indicated by the increase in the products of protein catabolism—namely, free amino acids and ammonia. The results also provide evidence for the mobilization of proteins from storage sites. Nonprotein dry weight increases markedly during the course of starvation, the significance of which is discussed.

AMESON MICHAELIS(MICROSPORIDA) IN THE BLUE CRAB,CALLINECTES SAPIDUS:PARASITE-INDUCED ALTERATIONS IN THE BIOCHEMICAL COMPOSITION OF HOST TISSUES

Ameson michaelis' interaction with its blue crab host significantly altered the biochemical constituents of host tissues. Modifications in skeletal muscle protein and carbohydrate metabolism were reflected in substantial variations in hemolymph composition. Blood osmolality, Cl-, and Na+ ion levels decreased with heavy parasitic invasion, while K+ ion and ninhydrin positive substances (NPS) levels increased in both light and heavy infections. Microsporidiosis resulted in a general increase in all hemolymph free amino acids detected except glutamic acid.Effects of A. michaelis sporogenesis were observed by comparing the biochemical composition of thoracic and cheliped skeletal musculature. Protein and carbohydrate levels were lower in infected thoracic muscle. The opposite trend was observed for tissue free-amino-acids (ninhydrin-positive substances). The concentration of nine of the sixteen amino acids detected remained unchanged with infection. Skeletal muscle glutamate, proline, glycine, alanine, and ar...

Hydromineral regulation in the saline water corixid Trichocorixa reticulata (Hemiptera: Corixidae)

The aquatic corixid Trichocorixa reticulata (Guerin-Meneville) inhabits coastal marshes, brackish water ponds and salt ponds of high salinity, suggesting the presence of well developed mechanisms for hydromineral regulation. Groups of corixids acclimated in salinities ranging from fresh water to just above 300% sea water (100‰) were analyzed for total body water content, haemolymph ionic and osmotic levels, and haemolymph free amino acids. Results indicate an excellent ability to maintain haemolymph Na +, Cl −, Mg 2+ and K + hyperosmotic to the medium at low salinities and hyposmotic at high salinities. Calcium appears to conform closely to changes in external medium, becoming hyposmotic at very high salinities (80‰). Total haemolymph osmotic pressure was well regulated, the freezing point depression varying from 0.75°C in distilled water to 1.15°C in salinities of 100‰. Total body water was maintained at approx. 75% of the total animal wet weight at all salinities tested. Free amino acids were maintained between 40–60 mM in all tests and did not appear to change with salinity.

Hemolymph of Anopheles stephensi from Uninfected and Plasmodium berghei-Infected Mosquitoes. 2. Free Amino Acids

Determinations were made of free amino acids in hemolymph collected from adult female Anopheles stephensi mosquitoes. The hemolymph first was fractionated by extraction and precipitation procedures, after which qualitative determinations of free amino acids were made by high voltage thin layer electrophoresis, and thin layer chromatography. Subsequent quantitative determinations were made with an automatic amino acid analyzer. The concentration of total free amino acids in the hemolymph rose 60--70% after the mosquito took a blood meal, and remained relatively constant thereafter. When mosquitoes took a blood meal infected with the rodent malaria parasite Plasmodium berghei, the rise in total free amino acids was only 15--25%. The chief differences that occurred with individual free amino acids was that infected mosquitoes had greater increases in arginine, greater decreases in valine and histidine, and a total loss of detectable methionine.

Relationship between ammonia excretion rates and hemolymph nitrogenous compounds of a euryhaline bivalve during low salinity acclimation.

1. Free amino acid concentrations in the hemolymph increase rapidly in company with a decrease in the tissue free amino acid pool of propped-open M. demissus exposed to low salinity.2. As acclimation proceeds, blood amino acid concentrations level off and an increase in both hemolymph ammonia concentration and external ammonia excretion occurs.3. Finally, hemolymph amino acid concentrations decrease toward control levels, followed by hemolymph ammonia concentration and external ammonia excretion rates.4. Following complete acclimation of mussels to high or low salinity, ammonia excretion rates are the same. However, hemolymph free amino acid concentrations are lower in animals adapted to low salinity.5. These results show that during acclimation of mussels to low salinity, free amino acids are released intact from the cells into the hemolymph. The subsequent transitory increase in hemolymph ammonia concentrations and external ammonia excretion rates is probably due to degradation of the effluxed amino aci...