Amino Acids In Hemolymph Research Articles

Improving the mechanical property of silk by feeding silkworm with chiral carbon dots

The influence of chiral materials on organisms is crucial. However, there is little research on the impact of chiral carbon dots (CDs), a kind of typical chiral materials, on biology. Herein, chiral CDs (L-/D-CDs) were synthesized using the thermal polymerization method from citric acid and chiral cysteine. The effect of chiral CDs on silkworms was explored through feeding silkworms with chiral CDs. The breaking strength of silk fibers (667.9 MPa) in D-CDs group exhibit a 71.4 % increase compared with control-silk (389.5 MPa), while the breaking strength of silk fibers in L-CDs group increases by 51.6 %. In addition, Fourier transform infrared spectra display CDs can prevent the transformation from random coil/α-helix structures to β-sheet structures. Furthermore, D-CDs group exhibit the highest percentage of four primary amino acids (glycine, alanine, serine, and tyrosine) relative to the total amino acids in silkworm hemolymph. This percentage is elevated by 70.5 % compared to the control group, thereby furnishing an ample supply of raw materials for the synthesis of silk proteins. In contrast, L-CDs group exhibit increase by 39.3 %. Our work provides new ideas and approaches for studying the effects of chiral materials on living organisms.

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.

Effect of amino acid enriched diets on hemolymph amino acid composition in honey bees.

Amino acids (AAs) are an abundant class of nectar solutes, and they are involved in the nectar attractiveness to flower visitors. Among the various AAs, proline is the most abundant proteogenic AA, and γ-amino butyric acid (GABA) and β-alanine are the two most abundant non-proteogenic AAs. These three AAs are known to affect insect physiology, being involved in flight metabolism and neurotransmission. The aim of this study was to investigate the effects of artificial diets enriched with either β-alanine, GABA, or proline on consumption, survival, and hemolymph composition in honey bees belonging to two different ages and with different metabolism (i.e., newly emerged and foragers). Differences in feed intake among diets were not observed, while a diet enriched with β-alanine improved the survival rate of newly emerged honey bees compared to the control group. Variations in the hemolymph AA concentrations occurred only in newly emerged honey bees, according to the diet and the time of hemolymph sampling. A greater susceptibility of young honey bees to enriched diets than older honey bees was observed. The variations in the concentrations of hemolymph AAs reflect either the accumulation of dietary AAs or the existence of metabolic pathways that may lead to the conversion of dietary AAs into different ones. This investigation could be an initial contribution to studying the complex dynamics that regulate hemolymph AA composition and its effect on honey bee physiology.

Utilization of Cottonseed Meal Protein Hydrolysate by Crustaceans: Insights on Growth Performance, Protein Turnover, and Metabolism in Chinese Mitten Crab Eriocheir sinensis

Plant protein hydrolysates could enhance the growth performance and diet utilization of aquaculture species. The mechanisms underlying their beneficial effects, however, remain unclear. The purpose of this study was to appraise the effects of cottonseed meal protein hydrolysate (CPH) supplementation on the growth performance, amino acid profiles, and protein turnover and metabolism of Eriocheir sinensis. A total of 240 crabs (initial weight: 37.32 ± 0.38 g) were randomly assigned to six groups, and fed six iso-protein feeds supplemented with varying levels of 0% (the control group), 0.2%, 0.4%, 0.8%, 1.6% and 3.2% of CPH. These diets were continuously fed to the crabs for 12 weeks. The findings demonstrated that, compared with the control group, adding 0.4–0.8% CPH to the diet significantly increased the specific growth rate, nitrogen retention efficiency, hepatopancreas index, body crude protein content, hepatopancreas alanine aminotransferase and glutamine synthetase activities, hemolymph total protein content, the hepatopancreas transcription of S6 kinase-poly-peptide 1, and the hepatopancreas protein levels of insulin-like growth factor-1 (IGF-1), protein kinase B (Akt), and target of rapamycin (TOR) of crabs. In contrast, when the dose of dietary CPH reached 3.2%, the forkhead box O1 (FoxO1) protein expression showed a significant decrease compared with the control group. In addition, CPH supplementation also increased the amount of amino acids and free amino acids in hepatopancreas and hemolymph, respectively. Together, these findings demonstrated that dietary supplementation of 0.4–0.8% CPH could activate the IGF-1/Akt/TOR pathway of E. sinensis, thereby improving growth performance, protein synthesis, and utilization. For cost considerations, the recommended dietary dose of CPH for E. sinensis is 0.8%. Due to the above benefits, CPH has the potential to be used as a growth promoter for other aquatic animals, especially crustaceans.

Effects of proline on survival, locomotion and amino acid haemolymph composition of Osmia cornuta (Latreille, 1805)

AbstractFlower nectar, a sugar‐rich solution containing amino acids as major secondary solutes, is the primary energy source for bees. Proline is one of the most abundant protein nectar amino acids, showing several effects on bee physiology. Mason bees are outstanding pollinators, often preferred to Apis mellifera for pollination of fruit trees. Among them, Osmia cornuta (Latreille) is one of the most successfully managed species on a commercial scale. In this work, the effects of 10 mM proline‐enriched diet administration on O. cornuta feed consumption, survival, behaviour and haemolymph amino acid composition were investigated. Feed intake was higher for the proline diet, while survival rate was not affected. Behaviour was affected only in the senescence of the mason bees with a reduction in locomotor activity induced by proline intake. Proline diet also affected the haemolymph amino acid composition, decreasing concentration of tyrosine, methionine, leucine and phenylalanine after 10 days of feeding. These results were discussed in relation to the potential involvement of proline in oxidative stress in insects and proline's ability to be converted to other amino acids. Further investigations are necessary to better understand the molecular mechanisms of proline effects on locomotion and oxidative stress.

The Phosphoserine Phosphatase Alters the Free Amino Acid Compositions and Fecundity in Cyrtorhinus lividipennis Reuter

The mirid bug Cyrtorhinus lividipennis (Reuter) is an important predator that consumes eggs and young nymphs of the brown planthopper Nilaparvata lugens as a primary food source and thus becomes an important member of the rice ecosystem. We identified and characterized the ClPSP gene in C. lividipennis encoding the phosphoserine phosphatase enzyme. The ClPSP has an open reading frame (ORF) of 957 bp encoding a protein with a length of 294bp and it possesses a haloacid dehalogenase-like (HAD) hydrolase, phosphoserine phosphatase, eukaryotic-like (HAD_PSP_eu) conserved domain. Furthermore, the in silico analysis of the ClPSP gene unveiled its distinct characteristics and it serves as a key player in the modulation of amino acids. The ClPSP showed expression in all developmental stages, with higher expression observed in the ovary and fat body. Silencing the ClPSP by RNA interference (RNAi) significantly decreased PSP enzyme activity and expression compared to dsGFP at two days after emergence (2DAE). The dsPSP treatment altered free hemolymph amino acid compositions, resulting in a significant reduction of serine (Ser) and Arginine (Arg) proportions and a significant increase of Threonine (Thr), Cystine (Cys), and Tyrosine (Tyr) in the C. lividipennis female at 2 DAE. Additionally, a hindered total protein concentration in the ovary and fat body, and reduced vitellogenin (Vg) expression, body weight, and number of laid eggs, were also observed. The same treatment also prolonged the preoviposition period and hindered ovarian development. Our data, for the first time, demonstrated the influential role of the PSP gene in modulating the fecundity of C. lividipennis and provide a platform for future insect pest control programs using the PSP gene in modulating fecundity.

Combined analysis of silk synthesis and hemolymph amino acid metabolism reveal key roles for glycine in increasing silkworm silk yields

Rearing silkworms (Bombyx mori) using formula feed has revolutionized traditional mulberry feed strategies. However, low silk production efficiencies persist and have caused bottlenecks, hindering the industrial application of formula feed sericulture. Here, we investigated the effects of formula feed amino acid composition on silk yields. We showed that imbalanced amino acids reduced DNA proliferation, decreased Fib-H, Fib-L, and P25 gene expression, and caused mild autophagy in the posterior silk gland, reducing cocoon shell weight and ratio. When compared with mulberry leaves, Gly, Ala, Ser, and Tyr percentages of total amino acids in formula feed were decreased by 5.26%, while Glu and Arg percentages increased by 9.56%. These changes increased uric acid and several amino acids levels in the hemolymph of silkworms on formula feed. Further analyses showed that Gly and Thr (important synthetic Gly sources) increased silk yields, with Gly increasing amino acid conversion efficiencies to silk protein, and reducing urea levels in hemolymph. Also, Gly promoted endomitotic DNA synthesis in silk gland cells via phosphoinositide 3-kinase (PI3K)/Akt/target of rapamycin (TOR) signaling. In this study, we highlighted the important role of Gly in regulating silk yields in silkworms.

Bacillus symbiont drives alterations in intestinal microbiota and circulating metabolites of lepidopteran host.

The symbiotic association between bacterial symbionts and insect hosts is a complicated process that is not completely understood. Herein, we used a silkworm model to study the association between symbiotic Bacillus and lepidopteran insect by investigating the changes in intestinal microbiota and hemolymph circulating metabolites of silkworm after symbiotic Bacillus subtilis treatment. Results showed that B. subtilis can generate a variety of primary and secondary metabolites, such as B vitamins and antimicrobial compounds, to provide micronutrients and enhance the pathogen resistance of their insect host. Shifts in the relative abundance of Enterococcus, Brevibacterium, Buttiauxella, Pseudomonas, Brevundimonas and Limnobacter had significant correlations with the concentrations of differential metabolites (e.g. phospholipids and certain amino acids) in insect hemolymph. The antimicrobial compounds secreted by B. subtilis were the primary driving force for the reconstruction of intestinal microbiota. Meanwhile, the altered levels of circulating metabolites in multiple metabolic pathways were potential adaptive mechanism of insect hosts in response to the shifts of intestinal microbiota. Our findings provided concrete evidence that bacterial intestinal symbiont can alter the physiological state of insects and highlighted the importance of the compositional alterations of intestinal microbiota as a source of variation in circulating metabolites of insect hosts.

Effects of dietary arginine on growth, anti-oxidative enzymes, biomarkers of immunity, amino acid metabolism and resistance to Vibrio parahaemolyticus challenge in abalone Haliotis discus hannai

A 110-day feeding trial was conducted to determine the effects of dietary arginine levels on growth performance, amino acid metabolism, immune response, and disease resistance of abalone Haliotis discus hannai (initial weight: 15.70 ± 0.03 g). Seven isonitrogenous (about 32% of crude protein) and isolipidic (about 3% of crude lipid) diets with 1.17% (the control), 1.36%, 1.68%, 2.19%, 2.47%, 2.82% and 3.43% of arginine were formulated. Each diet was randomly assigned to triplicate cages (5 layers, each layer was 40 cm × 35 cm × 14 cm) with 80 abalones per cage. At the end of the feeding trial, abalones were fasted for 72 h and anesthetized with 5% alcohol. Results showed that final body weight and biomass gain (BG) increased first and thereafter decreased, where the break-points (34.11 g and 934.41 g, respectively) were found in the 1.68% dietary arginine group. Based on the broken-line regression analysis of BG, the optimal dietary arginine level for abalone was estimated to be 1.64% of dry diet. Abalone fed with 2.47% of dietary arginine showed higher crude protein content in the soft body (17.59% wet weight) than other groups. The crude lipid content had a decreasing trend (from 2.02 to 1.28% wet weight) with increasing dietary arginine. The concentration of total free essential amino acids in hemolymph was significantly higher in 1.68% (207.30 μg/mL), 2.19% (199.17 μg/mL) and 2.47% (216.30 μg/mL) dietary arginine groups than that in the control group (P < 0.05). Activities of alanine aminotransferase, total nitric oxide synthase, total anti-oxidative capacity, lysozyme, and the contents of nitric oxide, total protein and albumin were significantly increased as dietary arginine levels increasing from 1.17% to 1.68% and then decreased (P < 0.05). In the digestive gland, the genes expression of nuclear factor erythroid 2-related factor 2, catalase, glutathione peroxidase and Cu/Zn superoxide dismutase were significantly increased in 1.68% dietary arginine group compared with that in the control group (P < 0.05), while the gene expression of kelch-like ECH-associated protein 1 showed an opposite trend. Meanwhile, the gene expression of tumor necrosis factor-α was decreased in 1.36% to 2.82% dietary arginine levels, and the gene expression of Arginase-I was increased in the 1.68% dietary arginine group. The cumulative mortality of abalone challenged with Vibrio parahaemolyticus (1.0 × 107 cfu/mL) for seven days was significantly decreased in the 1.68% dietary arginine group (36.67%) compared with those in the other groups except for the group with 2.19% of dietary arginine. In conclusion, dietary arginine level (1.68%) improved the growth, anti-oxidative capacity, immunity and disease resistance of abalone.

Silencing of a putative alanine aminotransferase (ALT) gene influences free amino acid composition in hemolymph and fecundity of the predatory bug, Cyrtorhinus lividipennis Reuter.

In Asian rice systems, Cyrtorhinus lividipennis Reuter is an important predator that preys on rice planthopper eggs and young nymphs, as a primary food source. Alanine aminotransferase (ALT) acts in many physiological and biochemical processes in insects. We cloned the full-length complementary DNA of C. lividipennis ClALT. Expression analysis showed higher expression in the fat body and midgut compared to other tissues. It is expressed in all C. lividipennis developmental stages and at least four organs. Silencing of ClALT by RNA interference significantly decreased the ClALT enzyme activity and ClALT expression compared to dsGFP-treated controls at 2 days after emergence (DAE). Silencing of ClALT influenced free hemolymph amino acid compositions, resulting in a reduction of Aspartic acid (Asp) and Alanine (Ala) proportions, and increased Cysteine (Cys) and Valine (Val) proportions in females at 2 DAE. dsClALT treatments led to decreased soluble total protein concentrations in ovary and fat body, and to lower reduced vitellogenin (Vg) expression, body weight, and the numbers of laid eggs. The double-stranded RNA viruse treatments also led to prolonged preoviposition periods and hindered ovarian development. Western blot analysis indicated that silencing ClALT also led to reduced fat body Vg protein abundance at 2 DAE. These data support our hypothesis that ClALT influences amino acid metabolism and fecundity in C. lividipennis.

Toxic effects of ammonia and thermal stress on the intestinal microbiota and transcriptomic and metabolomic responses of Litopenaeus vannamei

Ammonia and thermal stress frequently have harmful effects on aquatic animals. The intestine is an important barrier allowing the body to defend against stress. In this study, we investigated the intestinal microbiota and transcriptomic and metabolomic responses of Litopenaeus vannamei subjected to individual and combined ammonia and thermal stress. The results showed that obvious variation in the intestinal microbiota was observed after stress exposure, with increased levels of Firmicutes and decreased levels of Bacteroidetes and Planctomycetes. Several genera of putatively beneficial bacteria (Demequina, Weissella and Bacteroides) were abundant, while Formosa, Kriegella, Ruegeria, Rhodopirellula and Lutimonas were decreased; pathogenic bacteria of the genus Vibrio were increased under individual stress but decreased under combined stress. The intestinal transcriptome revealed several immune-related differentially expressed genes associated with the peritrophic membrane and antimicrobial processes in contrasting accessions. Haemolymph metabolomic analysis showed that stress exposure disturbed the metabolic processes of the shrimp, especially amino acid metabolism. This study provides insight into the underlying mechanisms associated with the intestinal microbiota, immunity and metabolism of L.vannamei in response to ammonia and thermal stress; ten stress-related metabolite markers were identified, including L-lactic acid, gulonic acid, docosahexaenoic acid, l-lysine, gamma-aminobutyric acid, methylmalonic acid, trans-cinnamate, N-acetylserotonin, adenine, and dihydrouracil.

Silencing of RpATG8 impairs the biogenesis of maternal autophagosomes in vitellogenic oocytes, but does not interrupt follicular atresia in the insect vector Rhodnius prolixus.

Follicular atresia is the mechanism by which the oocyte contents are degraded during oogenesis in response to stress conditions, allowing the energetic resources stored in the developing oocytes to be reallocated to optimize female fitness. Autophagy is a conserved intracellular degradation pathway where double-membrane vesicles are formed around target organelles leading to their degradation after lysosome fusion. The autophagy-related protein 8 (ATG8) is conjugated to the autophagic membrane and has a key role in the elongation and closure of the autophagosome. Here we identified one single isoform of ATG8 in the genome of the insect vector of Chagas Disease Rhodnius prolixus (RpATG8) and found that it is highly expressed in the ovary during vitellogenesis. Accordingly, autophagosomes were detected in the vitellogenic oocytes, as seen by immunoblotting and electron microscopy. To test if autophagosomes were important for follicular atresia, we silenced RpATG8 and elicited atresia in vitellogenic females by Zymosan-A injections. We found that silenced females were still able to trigger the same levels of follicle atresia, and that their atretic oocytes presented a characteristic morphology, with accumulated brown aggregates. Regardless of the difference in morphology, RpATG8-silenced atretic oocytes presented the same levels of protein, TAG and PolyP, as detected in control atretic oocytes, as well as the same levels of acidification of the yolk organelles. Because follicular atresia has the ultimate goal of restoring female fitness, we tested if RpATG8-silenced atresia would result in female physiology and behavior changes. Under insectarium conditions, we found that atresia-induced control and RpATG8-silenced females present no changes in blood meal digestion, survival, oviposition, TAG content in the fat body, haemolymph amino acid levels and overall locomotor activity. Altogether, we found that autophagosomes are formed during oogenesis and that the silencing of RpATG8 impairs autophagosome biogenesis in the oocytes. Nevertheless, regarding major macromolecule degradation and adaptations to the fitness costs imposed by triggering an immune response, we found that autophagic organelles are not essential for follicle atresia in R. prolixus.

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.

Combined effects of waterborne copper exposure and salinity on enzymes related to osmoregulation and ammonia excretion by blue crab Callinectes sapidus.

Copper is essential, but can be toxic to aquatic organisms when present in high concentrations. In freshwater crustaceans, copper inhibits enzymes related to ionic and osmoregulation and to the ammonia efflux, that leads to Na+ imbalance and inhibition of ammonia excretion. In the animals inhabiting estuarine or seawater, mechanisms of copper toxicity is not clear, but had been described as disruption of ionregulation and metabolism. To clarify the mechanism of copper toxicity in crustaceans inhabiting variable salinity, this work investigated whether copper affects ammonia excretion and enzymes used for ammonia balance and osmoregulation in the blue crab Callintectes sapidus acclimated to salinity 2 and 30 ppt. To achieve this, juveniles of the blue crab were exposed to 63.5 µg/L of copper at both salinities for 96 h. This is an environmentally realistic copper concentration. Results of ammonia efflux, free amino acids and Na+ concentrations in hemolymph, Na+/K+-ATPase, H+-ATPase and, carbonic anhydrase (CA) activities in gills were consistent with the osmoregulatory pattern adopted by the blue crab, which hyperosmoregulates at salinity 2 ppt and osmoconforms at 30 ppt. At 30 ppt copper reduced free amino acid in hemolymph of crabs, suggesting an effect of the metal on osmotic performance. At 2 ppt, copper significantly increased the H+-ATPase activity involved in ammonia excretion. This may be a compensatory response of crabs to maintain low levels of ammonia in their hemolymph; which can be increased by copper exposure. Results presented here are useful for the improvement of the Biotic Ligand Model (BLM) to predict copper toxicity for saltwater environments.

Comparative study on the utilization of crystalline methionine and methionine hydroxy analogue calcium by Pacific white shrimp (Litopenaeus vannamei Boone)

The objective of this study was to compare the supplemental effects of crystalline DL-methionine (DL-Met) and methionine hydroxy analogue calcium (MHA-Ca) on growth performance of Pacific white shrimp. Eight isoproteinic (355.3 ± 2.0 g/kg diet) and isolipidic (70.0 ± 2.2 g/kg diet) diets were prepared as positive diet (20% fish meal), negative diet (15% fish meal) and DL-Met, MHA-Ca-supplemented diets with the supplementation of 0.03%, 0.06%, 0.09% DL-Met and 0.04%, 0.07%, 0.1% MHA-Ca in negative diet respectively. Pacific white shrimp (0.92 ± 0.03 g) were fed one of the eight diets for 49 days. The results showed that dietary DL-Met did not affect weight gain (WG) and feed conversion ratio (FCR) (p > 0.05), but the supplementation of 0.1% MHA-Ca significantly increased WG, protein and lipid retention, and reduced FCR (p < 0.05) when compared to the negative group, and reached the similar levels as the positive control. The total free amino acids (TFAA) in haemolymph of MHA-Ca groups and PC, NC group peaked at the 3rd hr after feeding, but the peaking time of DL-Met groups was advanced to the 2nd hr. Activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in haemolymph of all MHA-Ca groups peaked at the 2nd hr, but DL-Met groups peaked at the 1st hr after feeding. The above results indicated that the supplementation of 0.1% MHA-Ca in a low fish meal diet could improve the growth performance and feed utilization of Pacific white shrimp, but dietary DL-Met did not significantly affect the growth.

Impact of Sampling and Cellular Separation on Amino Acid Determinations in Drosophila Hemolymph.

The fruit fly is a frequently used model system with a high degree of human disease-related genetic homology. The quantitative chemical analysis of fruit fly tissues and hemolymph uniquely brings chemical signaling and compositional information to fly experimentation. The work here explores the impact of measured chemical content of hemolymph with three aspects of sample collection and preparation. Cellular content of hemolymph was quantitated and removed to determine hemolymph composition changes for seven primary amine analytes. Hemolymph sampling methods were adapted to determine differences in primary amine composition of hemolymph collected from the head, antenna, and abdomen. Also, three types of anesthesia were employed with hemolymph collection to quantitate effects on measured amino acid content. Cell content was found to be 45.4 ± 22.1 cells/nL of hemolymph collected from both adult and larvae flies. Cell-concentrated fractions of adult, but not larvae, hemolymph were found to have higher and more variable amine content. There were amino acid content differences found between all three areas indicating a robust method to characterize chemical markers from specific regions of a fly, and these appear related to physiological activity. Methods of anesthesia have an impact on hemolymph amino acid composition related to overall physiological impact to fly including higher amino acid content variability and oxygen deprivation effects. Together, these analyses identify potential complications with Drosophila hemolymph analysis and opportunities for future studies to relate hemolymph content with model physiological activity.

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.

THE SECRETION OF DIGESTIVE ENZYMES AND CAECAL SIZE ARE DETERMINED BY DIETARY PROTEIN IN THE CRICKET Gryllus bimaculatus

In Gryllus bimaculatus, the size of the caecum decreases in the latter half of each instar to a stable minimal size with a steady minimal rate of digestive enzyme secretion until feeding resumes after ecdysis. The higher the percent protein in the newly ingested food, the faster and larger the caecum grows, and as a consequent the higher the secretion rate of trypsin and amylase. When hard boiled eggs (40% protein) are eaten the caecum is 2× larger, the trypsin secretion is almost 3× greater, and amylase 2.5× greater then when fed the same amount of apples (1.5% protein). Only dietary protein increases amylase secretion, whereas dietary carbohydrates have no effect on amylase secretion. The minimal caecal size and secretion rate must be supported by utilization of hemolymph amino acids, but the growth of the caecum and increasing enzymes secretions after the molt depend upon an amino acid source in the lumen. This simple regulation of digestive enzyme secretion is ideal for animals that must stop feeding in order to molt. This basic control system does not preclude additional regulation mechanisms, such as prandal, which is also indicated for G. bimaculatus, or even paramonal regulation.

Developmental Changes for the Hemolymph Metabolome of Silkworm (Bombyx mori L.).

Silkworm (Bombyx mori) is a lepidopteran-holometabolic model organism. To understand its developmental biochemistry, we characterized the larval hemolymph metabonome from the third instar to prepupa stage using (1)H NMR spectroscopy whilst hemolymph fatty acid composition using GC-FID/MS. We unambiguously assigned more than 60 metabolites, among which tyrosine-o-β-glucuronide, mesaconate, homocarnosine, and picolinate were reported for the first time from the silkworm hemolymph. Phosphorylcholine was the most abundant metabolite in all developmental stages with exception for the periods before the third and fourth molting. We also found obvious developmental dependence for the hemolymph metabonome involving multiple pathways including protein biosyntheses, glycolysis, TCA cycle, the metabolisms of choline amino acids, fatty acids, purines, and pyrimidines. Most hemolymph amino acids had two elevations during the feeding period of the fourth instar and prepupa stage. Trehalose was the major blood sugar before day 8 of the fifth instar, whereas glucose became the major blood sugar after spinning. C16:0, C18:0 and its unsaturated forms were dominant fatty acids in hemolymph. The developmental changes of hemolymph metabonome were associated with dietary nutrient intakes, biosyntheses of cell membrane, pigments, proteins, and energy metabolism. These findings offered essential biochemistry information in terms of the dynamic metabolic changes during silkworm development.

Effects of Titanium Dioxide Nanoparticles on the Synthesis of Fibroin in Silkworm (Bombyx mori).

Silkworm (Bombyx mori) is an economically important insect, and its silk production capacity largely depends on its ability to synthesize fibroin. While breeding of B. mori varieties has been a key strategy to improve silk production, little improvement of B. mori silk production has been achieved to date. As a result, the development of sericulture economy has not progressed well, pointing to the need of new ways for improvement of B. mori silk production. Titanium dioxide nanoparticles (TiO2 NPs), a food additive widely used for livestock, have been shown to promote animal growth and increase the protein synthesis in animals. However, no studies on effect of TiO2 NPs on fibroin synthesis in B. mori have been available. In this study, the differential expression profiles of genes and proteins in the silk gland of B. mori fed without or with TiO2 NPs (5 μg ml(-1)) were analyzed and compared using digital gene expression (DGE), reverse transcription quantitative polymerase chain reaction (RT-qPCR), semi-qPCR, and Western blot analysis. The effects of TiO2 NPs feeding on the activity of proteases in the midgut and the synthesis and transportation of amino acids in hemolymph were also investigated. DGE analyses showed that among a total of 4,741 genes detected, 306 genes were differentially expressed after the TiO2 NPs feeding, of which 137 genes were upregulated whereas 169 genes were downregulated. 106 genes were shown to be involved in fibroin synthesis, of which 97 genes, including those encoding cuticular protein glycine-rich 10, serine protease inhibitor 28, aspartate aminotransferase, lysyl-tRNA synthetase, and splicing factor arginine/serine-rich 6, and silk gland factor-1 (SGF-1), were upregulated with the maximum induction of 8.52-folds, whereas nine genes, including those encoding aspartylglucosaminidase, the cathepsin L in Tribolium castaneum, and similar to SPRY domain-containing SOCS box protein 3, were downregulated with the maximum reduction of 8.11-folds. Transcription levels of nine genes were further verified by RT-qPCR, and the results were consistent with those with DGE. Transcription and expression levels of fibroin light chain (Fib-L) gene were increased after TiO2 NPs feeding, indicating that TiO2 NPs improves fibroin synthesis. Compared with that of control, the mean protease activity was increased by 56.67% in the B. mori fed with TiO2 NPs, and the transport of four key amino acids used for fibroin synthesis in hemolymph was also increased. These findings indicated that TiO2 NPs feeding can improve the absorption and utilization of amino acids from the feed and could be a new way to increase the fibroin synthesis in B. mori.