Hemolymph Proteins Research Articles

Influence of stocking density and exposure to white spot syndrome virus in biological performance, metabolic, immune, and bioenergetics response of whiteleg shrimp Litopenaeus vannamei

The risk of disease in shrimp farming has been suggested to increase with stocking density, although it is not clear if this is related to an increased susceptibility to pathogens based on poor physiological condition or a lower capacity of immune response. This study analyzed the influence of stocking density on the metabolic and immune response to exposure to white spot syndrome virus (WSSV) on whiteleg shrimp Litopenaeus vannamei. Juvenile shrimp were submitted to two stocking densities (20 and 100 shrimp m2) for eight weeks; water quality, growth, and survival were recorded. Then, some shrimp were sampled for determining biochemical composition, adenylic nucleotides, arginine phosphate (Arg-P), total hemocyte count (THC), and clotting time to determine baseline values. Other shrimp were challenged with WSSV for 24h and then analyzed for the same variables. Concentration of nitrogenous compounds and phosphorus were significantly higher in tanks with the higher shrimp density. Growth and survival were significantly lower by 42% and 18%, respectively, under high density conditions. No outstanding effect of stocking density on biochemical and immunological variables were observed, except for higher levels of hemocyanin in hemolymph and lactate in muscle in shrimp under low density conditions. In contrast, the WSSV challenge induced several metabolic disruptions, including a higher adenylic energy charge (AEC) in the hepatopancreas and higher content of Arg-P in muscle, higher content of triglycerides in hemolymph, hepatopancreas, and muscle, and higher content of glucose, protein, hemocyanin, and cholesterol in hemolymph. These responses correspond an increased energy demand caused by exposure to virus and/or a phase of metabolic depression. Conditions of high stocking apparently do not increase the susceptibility of shrimp to WSSV since no differences were observed in the severity of infection and THC. However clotting time, glucose, lactate and triglycerides were more importantly affected by WSSV in shrimp coming from the high density condition, suggesting a “dual stress” effect that could in turn affect susceptibility to pathogens.

Effect of Borrelial Infection on Haemolymph and Ovarian Protein Concentrations During Vitellogenesis in Ornithodoros erraticus, a vector of Relapsing Fever in Egypt

Abstract Changes in the total protein concentration of haemolymph and ovaries in Ornithodoros erraticus mated fed females uninfected and infected with Borrelia crocidurae were studied during vitellogenesis and at the end of oviposition. In the uninfected mated females, engorgement was followed by a decrease in the haemolymph total protein concentration immediately and for the following 2 days after feeding, then the level of haemolymph protein gradually increased on the following days to reach maximum levels on 5 – 9 d.a.f. The ovary total protein level increased gradually on the 1st d.a.f, then the increase continued evidently on the following days to reach a maximum on 9 d.a.f (day of oviposition).At the end of oviposition the level of haemolymph and ovaries total protein largely decreased. The increase in the level of haemolymph proteins was positively correlated with the increase in the ovary total protein, ovary weight and number of mature oocytes inside the ovary. Changes in the haemolymph and ovaries total protein concentration of mated females infected with Borrelia crocidurae followed the same pattern of the uninfected ones. However, borrelial infection reduced the level of haemolymph and ovaries total protein during most of the preoviposition period (3 – 9 d.a.f). Also the weight of ovary and number of mature oocytes were lower in the infected than uninfected on the 5th- 9th and 3rd-9thd.a.f, respectively. Freshly deposited eggs of the infected females had lower total protein concentration than those deposited by uninfected females.

Effect of Some Essential Oils and Natural Botanical Extracts on Food Consumption and Some Physiological Aspects of Mulberry Silkworm, Bombyx mori L.

The effects of different concentrations of marjoram oil, thyme oil, (1%, 0.5%, and 0.25%) and their botanical extracts (2%, 1%, and 0.5%) as a nutritional additives offered to silkworm larvae during the 4th and 5th larval instars on food consumption, Approximate weight of digested food (A.D), Approximate digestibility (A.D%), efficiency of conversion of food ingested to body substance (E.C.I%), and efficiency of conversion of food digested to body substance (E.C.D%) were studied. Also, to determine their effects on total carbohydrates and total protein in larval haemolymph. Results showed significant variation in most of the studied parameters of oils and botanical extracts different concentrations compared to control; E.C.D% was found maximum with 0.5% marjoram oil (74.995%) and thyme oil (71.465%), in the botanical extracts 2% of both thyme, and marjoram exhibited the highest ratio (63.170% and 60.975%), respectively compared with control which recorded the minimum (38.830%). The efficiency with which the ingested food is converted into body substances (E.C.I%) 0.5% of marjoram and thyme oils showed the highest efficiency of conversion (21.000% and 20.555%), respectively compared to (13.445%) for the control. 2% of both thyme (19.960%) and marjoram extract (18.845%) showed also the highest ECI% while the control recorded the least result. The highest AD% was shown by control group (43.185%) and AD of the control group exhibited the highest among them (3.400 gm). Results revealed a significant variation between the both larval instars in most parameters. The quantity of food consumed in both oils and extracts varied significantly between the both instars with no significant values among different concentrations of them. Total protein exhibited the highest value when feeding larvae on mulberry leaves supplemented with 0.5% of both marjoram and thyme oils (41.067 and 39.867 mg/ml haemolymph), respectively, and 2% of both thyme and marjoram extracts (36.9 and 36.567 mg/ml haemolymph), respectively while the control group showed the minimum value (22.633 mg/ml haemolymph). The same trend was observed for total carbohydrates. It can be concluded that mulberry leaves fortified with different concentrations of marjoram and thyme oils and extracts were proved to be more efficient in rearing mulberry silkworm as it improved food acquisition, high ingestion, high (E.C.I & E.C.D) and low (A.D),also it increased protein and carbohydrates metabolism.

Hemolymph proteins of Anopheles gambiae larvae infected by Escherichia coli

Anopheles gambiae is a major vector of human malaria and its immune system in part determines the fate of ingested parasites. Proteins, hemocytes and fat body in hemolymph are critical components of this system, mediating both humoral and cellular defenses. Here we assessed differences in the hemolymph proteomes of water- and E. coli-pricked mosquito larvae by a gel-LC-MS approach. Among the 1756 proteins identified, 603 contained a signal peptide but accounted for two-third of the total protein amount on the quantitative basis. The sequence homology search indicated that 233 of the 1756 may be related to defense. In general, we did not detect substantial differences between the control and induced plasma samples in terms of protein numbers or levels. Protein distributions in the gel slices suggested post-translational modifications (e.g. proteolysis) and formation of serpin-protease complexes and high Mr immune complexes. Based on the twenty-five most abundant proteins, we further suggest that major functions of the larval hemolymph are storage, transport, and immunity. In summary, this study provided first data on constitution, levels, and possible functions of hemolymph proteins in the mosquito larvae, reflecting complex changes occurring in the fight against E. coli infection.

Apolipophorin-III Acts as a Positive Regulator of Plasmodium Development in Anopheles stephensi.

Apolipophorin III (ApoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune responses of insects. Here we report the molecular and functional characterization of Anopheles stephensi Apolipophorin-III (AsApoLp-III) gene. This gene consists of 679 nucleotides arranged into two exons of 45 and 540 bp that give an ORF encoding 194 amino acid residues. Excluding a putative signal peptide of the first 19 amino acid residues, the 175-residues in mature AsApoLp-III protein has a calculated molecular mass of 22 kDa. Phylogenetic analysis revealed the divergence of mosquitoes (Order Diptera) ApoLp-III from their counterparts in moths (Order: Lepidoptera). Also, it revealed a close relatedness of AsApoLp-III to ApoLp-III of An. gambiae. AsApoLp-III mRNA expression is strongly induced in Plasmodium berghei infected mosquito midguts suggesting its crucial role in parasite development. AsApoLp-III silencing decreased P. berghei oocysts numbers by 7.7 fold against controls. These effects might be due to the interruption of AsApoLp-III mediated lipid delivery to the developing oocysts. In addition, nitric oxide synthase (NOS), an antiplasmodial gene, is also highly induced in AsApoLp-III silenced midguts suggesting that this gene acts like an agonist and protects Plasmodium against the mosquito immunity.

Influence of the Desert Plant,Lotus corniculatuson the Protein Quantity and Glutathione-S-Transferase Activity ofSchistocerca gregaria

ABSTRACT: The desert plant, Lotus corniculatus (Fabaceae), inhabits the Saudi Arabian Sahara Desert. In these areas the desert locust, Schistocerca gregaria (Orthoptera, Acrididae), feeds on L. corniculatus containing the plant allelochemical linamarin; a cyanogenic glucoside that is usually toxic to polyphagous insects. Under laboratory conditions this plant was fed to S. gregaria. Haemolymph proteins and allelochemicals detoxification in S. gregaria, fed on L. corniculatus were assessed. Female 5th instar nymphs were reared on L. corniculatus or on wheat seedlings, Triticum aestivum, as control. In insects, glutathione-S-transferase is one of the most important detoxifying enzymes. The activity of glutathione-S-transferase (in units /gram) in the mid-gut tissues of 5th instar nymph females fed on L. corniculatus was significantly higher than in those fed on T. aestivum. The quantity of haemolymph proteins (in mg 100µl1) in adult locust females resulting from nymphs fed on L. corniculatus was significant...

Evaluation of three non-genetically modified soybean cultivars as ingredients and a yeast-based additive as a supplement in practical diets for Pacific white shrimp Litopenaeus vannamei

Two growth trials and a physiology assessment were conducted to evaluate three non-genetically modified (GM) soybean cultivars as ingredients in practical diets for Pacific white shrimp, Litopenaeus vannamei. In addition, a commercially available fermented yeast product was evaluated as dietary supplement. For the growth trials (46 and 35 days, respectively, for trials 1 and 2), the basal diet was primarily composed of soybean meal (SBM), fishmeal (FM), whole wheat, corn protein concentrate, poultry meal (PM, pet food grade) and corn starch. Non-GM cultivars were processed with novel methodologies to produce Navita™ ingredients (N1, N2 and N3) which were incorporated at low (L) or high (H) levels into the experimental diets, in partial replacement of FM and full replacement of conventional SBM. The last two formulations incorporated the fermented yeast for a total of nine experimental diets (Table 1). Results from the growth trials indicate that shrimp fed diet 5 (HN2) exhibited significantly lower (p < .05) weight gain as compared to shrimp fed diets 1 and 2 (basal and LN1, respectively) in trial 1, as well as compared to animals fed diets 1, 2, 3 and 8 (basal, LN1, HN1 and basal + yeast) in trial 2. The feed conversion ratio significantly increased for shrimp fed diet 5, in contrast with shrimp fed diets 1, 2, 3 and 8 in trials 1 and 2, as well as compared to shrimp fed diets diet 6 (LN3) in trial 2. For the physiological assessment (stress and immune responses), only the effects of diets 1, 3, 8 and 9 (basal, HN1, basal + yeast and HN1 + yeast, respectively) were investigated. Granular cell counts were significantly higher for shrimp fed the yeast-containing diets. Haemolymph glucose and haemolymph packed cell volume were significantly reduced for shrimp fed diets 3, 8 and 9. No significant differences were observed in total haemocyte counts, hyaline cells counts, semi-granular cells counts, haemolymph protein, haemocyte phagocytic capacity and haemocyte respiratory burst activity. Results of this work indicate that selective soy breeding technology coupled with novel processing options has the potential to increase the nutritional value of conventional SBM for shrimp feeds. Trends on immune responses were more difficult to elucidate possibly due to the limited length of the feeding trial.

Anti-oxidative effects of silkworm storage protein 1 in HeLa cell

Abstract Silkworm hemolymph contains unique proteins that exhibit anti-apoptotic activity in mammalian cells. Among them, 30 K protein, which is one of the major anti-apoptotic molecules in silkworm hemolymph, has been well investigated. However, little is known about the biological functions of storage protein 1 (SP1), another main protein in silkworm hemolymph. In this study, the anti-apoptotic and anti-oxidative activities of SP1 were analyzed. A stable cell line expressing SP1 was constructed, which showed strong anti-apoptotic effect induced by staurosporine treatment. In addition, the cell line exhibited resistance to oxidative stress caused by hydrogen peroxide. For practical applications of SP1, recombinant SP1 was produced in Escherichia coli, and the supplementation of recombinant SP1 into culture medium exhibited anti-apoptotic and anti-oxidative activities. In addition, SP1 was found to be a cell-penetrating protein and localized in the cytosol as well as on the plasma membrane. The findings showed that SP1 itself is not an anti-oxidant; rather, it mediates intracellular anti-oxidative activity. In conclusion, the cellular resistance of SP1 to apoptosis and oxidative stress will provide a new strategy that could be utilized in the bio-industry for the production of biologics as well as for the development of anti-aging cosmetics.

A Venom Serpin Splicing Isoform of the Endoparasitoid Wasp Pteromalus puparum Suppresses Host Prophenoloxidase Cascade by Forming Complexes with Host Hemolymph Proteinases

To ensure successful parasitism, parasitoid wasps inject venom along with their eggs into their hosts. The venom serves to suppress host immune responses, including melanization. Venom from Pteromalus puparum, a pupal endoparasitoid, inhibits melanization of host hemolymph in vitro in a dose-dependent manner. Using assay-guided fractionation, a serpin splicing isoform with phenoloxidase inhibitory activity was identified as P puparum serpin-1, venom isoform (PpS1V). This serpin gene has 16 predicted splicing isoforms that differ only in the C-terminal region. RT-PCR results show that the specific serpin isoform is differentially expressed in the venom gland. Recombinant PpS1V (rPpS1V) suppresses host prophenoloxidase (PPO) activation rather than inhibiting the phenoloxidase directly. Pulldown assays show that PpS1V forms complexes with two host hemolymph proteins, here named Pieris rapae hemolymph proteinase 8 (PrHP8) and P. rapae prophenoloxidase-activating proteinase 1 (PrPAP1), based on gene sequence blasting and phylogenetic analysis. The role of rPrPAP1 in the PPO activation cascade and its interaction with rPpS1V were confirmed. The stoichiometry of inhibition of PrPAP1 by PpS1V is 2.3. PpS1V also inhibits PPO activation in a non-natural host, Ostrinia furnacalis, through forming a complex with O. furnacalis serine protease 13 (OfSP13), an ortholog to PrPAP1. Our results identify a venom-enriched serpin isoform in P. puparum that inhibits host PPO activation, probably by forming a complex with host hemolymph proteinase PrPAP1.

Gut symbiotic bacteria stimulate insect growth and egg production by modulating hexamerin and vitellogenin gene expression

Recent studies have suggested that gut symbionts modulate insect development and reproduction. However, the mechanisms by which gut symbionts modulate host physiologies and the molecules involved in these changes are unclear. To address these questions, we prepared three different groups of the insect Riptortus pedestris: Burkholderia gut symbiont-colonized (Sym) insects, Burkholderia-non-colonized (Apo) insects, and Burkholderia-depleted (SymBurk-) insects, which were fed tetracycline. When the hemolymph proteins of three insects were analyzed by SDS-PAGE, the hexamerin-α, hexamerin-β and vitellogenin-1 proteins of Sym-adults were highly expressed compared to those of Apo- and SymBurk--insects. To investigate the expression patterns of these three genes during insect development, we measured the transcriptional levels of these genes. The hexamerin-β gene was specifically expressed at all nymphal stages, and its expression was detected 4–5 days earlier in Sym-insect nymphs than that in Apo- and SymBurk--insects. However, the hexamerin-α and vitellogenin-1 genes were only expressed in adult females, and they were also detected 6–7 days earlier and were 2-fold higher in Sym-adult females than those in the other insects. Depletion of hexamerin-β by RNA interference in 2nd instar Sym-nymphs delayed adult emergence, whereas hexamerin-α and vitellogenin-1 RNA interference in 5th instar nymphs caused loss of color of the eggs of Sym-insects. These results demonstrate that the Burkholderia gut symbiont modulates host development and egg production by regulating production of these three hemolymph storage proteins.

APOL1-G1 in Nephrocytes Induces Hypertrophy and Accelerates Cell Death.

People of African ancestry carrying certain APOL1 mutant alleles are at elevated risk of developing renal diseases. However, the mechanisms underlying APOL1-associated renal diseases are unknown. Because the APOL1 gene is unique to humans and some primates, new animal models are needed to understand the function of APOL1 in vivo We generated transgenic Drosophila fly lines expressing the human APOL1 wild type allele (G0) or the predominant APOL1 risk allele (G1) in different tissues. Ubiquitous expression of APOL1 G0 or G1 in Drosophila induced lethal phenotypes, and G1 was more toxic than was G0. Selective expression of the APOL1 G0 or G1 transgene in nephrocytes, fly cells homologous to mammalian podocytes, induced increased endocytic activity and accumulation of hemolymph proteins, dextran particles, and silver nitrate. As transgenic flies with either allele aged, nephrocyte function declined, cell size increased, and nephrocytes died prematurely. Compared with G0-expressing cells, however, G1-expressing cells showed more dramatic phenotypes, resembling those observed in cultured mammalian podocytes overexpressing APOL1-G1. Expressing the G0 or G1 APOL1 transgene in nephrocytes also impaired the acidification of organelles. We conclude that expression of an APOL1 transgene initially enhances nephrocyte function, causing hypertrophy and subsequent cell death. This new Drosophila model uncovers a novel mechanism by which upregulated expression of APOL1-G1 could precipitate renal disease in humans. Furthermore, this model may facilitate the identification of APOL1-interacting molecules that could serve as new drug targets to treat APOL1-associated renal diseases.

A Mini Review of Functional Proteins in Silkworm &lt;i&gt;Bombyx mori L&lt;/i&gt; Haemolymph

This review focuses on the state of the art on haemolymph proteins in lepidopteron insect silkworm Bombyx mori L. Insects are most successful animals in the world. Their success is mostly attributed to their physiological well being and adaptability to most of the environmental conditions is an important reason to be considered evaluating their success. Proteins in insect especially the ones circulating in the blood are seen to be the major contributors for the insects successful survive, owing to their open type circulatory system the haemolymph in insects soaks all the major organs systems in the body and thus making it an important body fluid for regulation of homeostasis. This calls for an detailed study on proteomics of the haemolymph of Bombyx mori which along with being an economical insect, it is being considered as model organism for most frontier sciences like biomaterials, biomedical applications and pharmacokinetic studies, Research on lepidopteron insects has been instrumental in the discovery and the biochemical understanding of many haemolymph plasma proteins. In the present review we analyzed the haemolymph proteins based on their function and molecular characterization. We also could deduce the role of these proteins in insect physiology by comparative analysis with earlier reported proteins.

Biochemical and histopathological changes of Biomphalaria alexandrina snails exposed to bisphenol A

ABSTRACTThe effects of sub-lethal concentrations of bisphenol A on some biochemical parameters in Biomphalaria alexandrina snail's hemolymph and tissues as well as on histological changes of the hermaphrodite gland were evaluated. Adult snails were exposed to sub-lethal concentrations for four weeks and samples of snails were investigated after two and four weeks. Total protein and albumin levels in hemolymph, as well as lipid peroxidation and glutathione contents of snails’ tissues, were measured. After two weeks of exposure, total protein content and albumin levels decreased in all groups. Elevation of lipid peroxidation levels was correlated with decreasing glutathione content in the treated snails. Histological examination of the hermaphrodite gland revealed deformations in eggs and affected sperm production.

Characterization of Antibacterial Activities of Eastern Subterranean Termite, Reticulitermes flavipes, against Human Pathogens.

The emergence and dissemination of multidrug resistant bacterial pathogens necessitate research to find new antimicrobials against these organisms. We investigated antimicrobial production by eastern subterranean termites, Reticulitermes flavipes, against a panel of bacteria including three multidrug resistant (MDR) and four non-MDR human pathogens. We determined that the crude extract of naïve termites had a broad-spectrum activity against the non-MDR bacteria but it was ineffective against the three MDR pathogens Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), and Acinetobacter baumannii. Heat or trypsin treatment resulted in a complete loss of activity suggesting that antibacterial activity was proteinaceous in nature. The antimicrobial activity changed dramatically when the termites were fed with either heat-killed P. aeruginosa or MRSA. Heat-killed P. aeruginosa induced activity against P. aeruginosa and MRSA while maintaining or slightly increasing activity against non-MDR bacteria. Heat-killed MRSA induced activity specifically against MRSA, altered the activity against two other Gram-positive bacteria, and inhibited activity against three Gram-negative bacteria. Neither the naïve termites nor the termites challenged with heat-killed pathogens produced antibacterial activity against A. baumannii. Further investigation demonstrated that hemolymph, not the hindgut, was the primary source of antibiotic activity. This suggests that the termite produces these antibacterial activities and not the hindgut microbiota. Two-dimensional gel electrophoretic analyses of 493 hemolymph protein spots indicated that a total of 38 and 65 proteins were differentially expressed at least 2.5-fold upon being fed with P. aeruginosa and MRSA, respectively. Our results provide the first evidence of constitutive and inducible activities produced by R. flavipes against human bacterial pathogens.

Thiol oxidation of hemolymph proteins in oysters Crassostrea brasiliana as markers of oxidative damage induced by urban sewage exposure

Urban sewage is a concerning issue worldwide, threatening both wildlife and human health. The present study investigated protein oxidation in mangrove oysters (Crassostrea brasiliana) exposed to seawater from Balneário Camboriú, an important tourist destination in Brazil that is affected by urban sewage. Oysters were exposed for 24 h to seawater collected close to the Camboriú River (CAM1) or 1 km away (CAM2). Seawater from an aquaculture laboratory was used as a reference. Local sewage input was marked by higher levels of coliforms, nitrogen, and phosphorus in seawater, as well as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), linear alkylbenzenes (LABs), and fecal steroid in sediments at CAM1. Exposure of oysters to CAM1 caused marked bioaccumulation of LABs and decreased PAH and PCB concentrations after exposure to both CAM1 and CAM2. Protein thiol oxidation in gills, digestive gland, and hemolymph was evaluated. Lower levels of reduced protein thiols were detected in hemolymph from CAM1, and actin, segon, and dominin were identified as targets of protein thiol oxidation. Dominin susceptibility to oxidation was confirmed in vitro by exposure to peroxides and hypochlorous acid, and 2 cysteine residues were identified as potential sites of oxidation. Overall, these data indicate that urban sewage contamination in local waters has a toxic potential and that protein thiol oxidation in hemolymph could be a useful biomarker of oxidative stress in bivalves exposed to contaminants. Environ Toxicol Chem 2017;36:1833-1845. © 2016 SETAC.

Effects of Scleroderma sichuanensis Xiao (Hymenoptera: Bethylidae) venom and parasitism on nutritional content regulation in host Tenebrio molitor L. (Coleoptera: Tenebrionidae).

To explore the mechanisms by which the wasp Scleroderma sichuanensis Xiao regulates the physiology and biochemistry of its host, effects of S. sichuanensis venom and parasitism on host the Tenebrio molitor L. pupae were examined. Significant differences in nutritional content were noted between parasitized and non-parasitized pupae and between venom- and phosphate buffered saline-injected pupae. When pupae were injected with venom, the fat body could not be disintegrated into granules; however, when pupae were parasitized, fat-body disintegration occurred. Electrophoresis showed no differences in hemolymph protein content between parasitized pupae and those injected with venom, indicating that the wasp did not have narrow-spectrum peptides. These findings confirmed that S. sichuanensis was a typical idiobiont ectoparasitoid wasp, and that nutrient regulation was similar between idiobiont and koinobiont wasps. The strong similarities between the two treatments suggest that venom injection is a major factor responsible for changes in host nutrient content. The wasp fed mainly on reducing sugars, free amino acids, and fat-body tissues; larval fat bodies were derived from hemolymph and from host tissue. Our findings suggest that lipid catabolism might be accelerated, and that lipid biosynthesis might be inhibited, when host pupae are parasitized or injected with venom. In addition to venom, physiological and biochemical changes that occur during the parasitic process might be caused by venom, ovarian proteins, saliva, or secretions.

Alterations in protein and amino acid metabolism in honeybees (Apis mellifera) fed different l-leucine diets during the larval stage

l-Leucine is an important factor for growth and development in animals. However, very little is known about the nutritional role and physiological function of l-leucine in honeybees. In this study, we determined the effects of l-leucine supplementation on protein synthesis and amino acid balance in honeybee larvae. First instar larvae were fed different l-leucine diets (6.45, 8.45 and 10.45mg/g) during the entire larval stage. After the larvae egested urate crystals, we analyzed the protein and amino acid metabolism indexes in the larvae. There was little effect of l-leucine on larval body weight and body protein content, but the hemolymph protein concentrations were higher in 8.45 and 10.45mg/g groups than 6.45mg/g group. Compared to the 6.45mg/g group, l-leucine significantly increased the contents of Ala, Leu, Lys and Met in 8.45mg/g group and continued to be increased in 10.45mg/g group. l-Leucine affected many TOR signaling pathway- and l-leucine metabolism-related genes, including tor, 4Ebp1, bcaat et al. Moreover, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK) and lactate dehydrogenase (LDH) activities as well as the triglycerides (TG), total cholesterol (TCHO), uric acid (UA) and urea nitrogen (URER) contents in haemolymph were significantly different among treatments. These results suggested that many factors involved in the metabolism of l-leucine and larval hemolymph play important roles in honeybee protein synthesis and amino acid balance.

Lysine acetylation stabilizes SP2 protein in the silkworm Bombyx mori

Lysine acetylation (Kac) is a vital post-translational modification that plays an important role in many cellular processes in organisms. In the present study, the nutrient storage proteins in hemolymph were first found to be highly acetylated—particularly SP2 protein, which contains 20 potential Kac sites. Further results confirmed that lysine acetylation could stabilize and up-regulate the protein level of anti-apoptosis protein SP2, thereby improving the survival of H2O2-treated BmN cells and suppressing the apoptosis induced by H2O2. The potential mechanism involved in the inhibition of ubiquitin-mediated proteasomal degradation by crosstalk between lysine acetylation and ubiquitination. Our results showed that the increase in the acetylation level by TSA could decrease the ubiquitination and improve the protein level of SP2, indicating that lysine acetylation could influence the SP2 protein level through competition between ubiquitination and the suppression of ubiquitin-mediated proteasomal degradation, thereby stabilizing the protein. SP2 is a major nutrient storage protein from hemolymph for amino acid storage and utilization. The crosstalk between lysine acetylation and ubiquitination of SP2 might imply an important role of lysine acetylation for nutrient storage and utilization in silkworm.

Interactions of cationic polystyrene nanoparticles with marine bivalve hemocytes in a physiological environment: Role of soluble hemolymph proteins

The bivalve Mytilus galloprovincialis has proven as a suitable model invertebrate for evaluating the potential impact of nanoparticles (NPs) in the marine environment. In particular, in mussels, the immune system represents a sensitive target for different types of NPs. In environmental conditions, both NP intrinsic properties and those of the receiving medium will affect particle behavior and consequent bioavailability/uptake/toxicity. However, the evaluation of the biological effects of NPs requires additional understanding of how, once within the organism, NPs interact at the molecular level with cells in a physiological environment. In mammalian systems, different NPs associate with serum soluble components, organized into a “protein corona”, which affects particle interactions with target cells. However, no information is available so far on the interactions of NPs with biological fluids of aquatic organisms.In this work, the influence of hemolymph serum (HS) on the in vitro effects of amino modified polystyrene NPs (PS-NH2) on Mytilus hemocytes was investigated. Hemocytes were incubated with PS-NH2 suspensions in HS (1, 5 and 50µg/mL) and the results were compared with those obtained in ASW medium. Cell functional parameters (lysosomal membrane stability, oxyradical production, phagocytosis) were evaluated, and morphological changes were investigated by TEM. The activation state of the signalling components involved in Mytilus immune response (p38 MAPK and PKC) was determined. The results show that in the presence of HS, PS-NH2 increased cellular damage and ROS production with respect to ASW medium. The effects were apparently mediated by disregulation of p38 MAPK signalling. The formation of a PS-NH2-protein corona in HS was investigated by centrifugation, and 1D- gel electrophoresis and nano-HPLC-ESI-MS/MS. The results identified the Putative C1q domain containing protein (MgC1q6) as the only component of the PS-NH2 hard protein corona in Mytilus hemolymph. These data represent the first evidence for the formation of a NP bio-corona in aquatic organisms and underline the importance of the recognizable biological identity of NPs in physiological exposure medium when testing their potential impact environmental model organisms. Although the results obtained in vitro do not entirely reflect a realistic exposure scenario and the more complex formation of a bio-corona that is likely to occur in vivo, these data will contribute to a better understanding of the effects of NPs in marine invertebrates.

Conversion of protein from supplements into protein of hemolymph and fat bodies in worker honey bees (Apis mellifera L)

Adequate supplementation or replacement of pollen is necessary for maintaining healthy honey bee colonies during periods with limited pollen. Different supplemental protein diets could be metabolized in different ways by bees. We compared how protein supplements elaborated with free amino acids, with a soy-based diet, and with pollen, influence protein stores within individual honey bees. Caged bees were fed with one of three diets, and measurements were made based on protein content of hemolymph and fat bodies. The results show that these diets were metabolized in different way by bees. Bees fed with the free amino acid diet had significantly more proteins in hemolymph than bees fed with soya patty and pollen as they aged. The major amount of protein transported into the hemolymph by 27-day-old bees fed with free amino acid diet promoted the development of wax glands, and probably, it was the cause of a reduced storage of protein in fat bodies. Overall, our results suggest a connection between diets and physiological condition of honey bees.