Hemolymph Extracts Research Articles

Immune response of Galleria mellonella after injection with non-lethal and lethal dosages of Candida albicans

The immune response of Galleria mellonella to injection with non-lethal and lethal dosages of Candida albicans was compared. Larvae infected with the non-lethal dosage (2 × 104 cells/larva) did not show significant morphological changes, while those infected with the lethal dosage (2 × 105 cells/larva) showed inhibition of motility and cocoon formation and became darker around the area of injection after 24 h. While the administration of the lower dosage caused approx. 5- and 20-fold induction of genes for gallerimycin and galiomycin, respectively, the injection with the higher dosage induced approx. 25 and 120-fold expression of the respective genes. Similar differences were obtained for the insect metalloproteinase inhibitor (IMPI) and hemolin gene transcripts. The relatively low level of immune gene expression was confirmed by an assay of hemolymph antifungal activity, which was detected only in larvae infected with lethal dosage of C. albicans. Furthermore, greater amounts of immune-inducible peptides were detected in the hemolymph extracts in the same group of larvae. The stronger humoral immune response was not correlated with survival. Phenol oxidase (PO) activity was induced only in the hemolymph of larvae infected with the non-lethal dose; injection of the lethal dose resulted in strong inhibition of this enzyme after 24 h. We showed that PO is susceptible to regulation by immune priming with the non-lethal dose of C. albicans. The activity of this enzyme was enhanced in primed larvae at the time of re-injection. When both primed and non-primed larvae received 2 × 105 cells, the inhibition of PO was stronger in the primed group. G. mellonella infected with the lethal dose of C. albicans died despite the strong induction of humoral defence mechanisms. The priming-enhanced activity of PO was correlated with increased resistance to subsequent infection.

Biologically active metabolite(s) from haemolymph of red-headed centipede Scolopendra subspinipes possess broad spectrum antibacterial activity

The discovery of novel antimicrobials from animal species under pollution is an area untapped. Chinese red-headed centipede is one of the hardiest arthropod species commonly known for its therapeutic value in traditional Chinese medicine. Here we determined the antibacterial activity of haemolymph and tissue extracts of red-headed centipede, Scolopendra subspinipes against a panel of Gram-positive and Gram-negative bacteria. Lysates exhibited potent antibacterial activities against a broad range of bacteria tested. Chemical characterization of biologically active molecules was determined via liquid chromatography mass spectrometric analysis. From crude haemolymph extract, 12 compounds were identified including: (1) l-Homotyrosine, (2) 8-Acetoxy-4-acoren-3-one, (3) N-Undecylbenzenesulfonic acid, (4) 2-Dodecylbenzenesulfonic acid, (5) 3H-1,2-Dithiole-3-thione, (6) Acetylenedicarboxylate, (7) Albuterol, (8) Tetradecylamine, (9) Curcumenol, (10) 3-Butylidene-7-hydroxyphthalide, (11) Oleoyl Ethanolamide and (12) Docosanedioic acid. Antimicrobial activities of the identified compounds were reported against Gram-positive and Gram-negative bacteria, fungi, viruses and parasites, that possibly explain centipede’s survival in harsh and polluted environments. Further research in characterization, molecular mechanism of action and in vivo testing of active molecules is needed for the development of novel antibacterials.

Identification of Glutamic Acid as a Host Marking Pheromone of the African Fruit Fly Species Ceratitis rosa (Diptera: Tephritidae).

Host marking pheromones (HMPs) deposited by female fruit flies deter other females from overexploiting the same fruit for egg laying. Using a bioassay-guided approach, we identified the HMP of the Natal fruit fly species Ceratitis rosa as glutamic acid, 1, from the aqueous fecal matter extract of ovipositing females by liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS). Dual choice oviposition assays showed that both the fecal matter extract and 1 significantly reduced oviposition responses in conspecific females of C. rosa. Glutamic acid levels were 10-20 times higher in fecal matter than in the ovipositor or hemolymph extracts of females. Identification of 1 as a host marking pheromone in females of C. rosa improves our understanding of fruit fly chemical ecology and provides evidence that it could be used as a potential component in the integrated management of this fruit fly species.

Identification of the Ubiquitous Antioxidant Tripeptide Glutathione as a Fruit Fly Semiochemical.

Many insects mark their oviposition sites with a host marking pheromone (HMP) to deter other females from overexploiting these sites. Previous studies have identified and used HMPs to manage certain fruit fly species; however, few are known for African indigenous fruit flies. The HMP of the African fruit fly, Ceratitis cosyra, was identified as the ubiquitous plant and animal antioxidant tripeptide, glutathione (GSH). GSH was isolated from the aqueous extract of adult female fecal matter and characterized by LC-QTOF-MS. GSH level increased with increasing age of female fecal matter, with highest concentration detected from 2-week-old adult females. Additionally, GSH levels were 5-10-times higher in fecal matter than in the ovipositor or hemolymph extracts of females. In bioassays, synthetic GSH reduced oviposition responses in conspecifics of C. cosyra and the heterospecific species C. rosa, C. fasciventris, C. capitata, and Zeugodacus cucurbitae. These results represent the first report of a ubiquitous antioxidant as a semiochemical in insects and its potential use in fruit fly management.

Enhanced cuticular penetration as the mechanism of synergy for the major constituents of thyme essential oil in the cabbage looper, Trichoplusia ni

For the past two decades, the insecticidal activity of botanicals, particularly plant essential oils has been gaining broad research interest. Due to the diverse chemical composition of essential oils, they often exhibit complex interactions among their major constituents, for example, synergistic or antagonistic relationships. Several hypotheses have been proposed for synergistic toxicity, including multiple modes-of-action and inhibition of metabolism, but the mechanism underlying synergy has not been fully elucidated. In the present study, interactions among the four major constituents of thyme (Thymus vulgaris) essential oil against the larvae of the cabbage looper were examined following topical administration. Two statistical models were used to determine the interactions, and several synergistic relationships were identified in natural proportional and equivalent blending ratios, but no antagonistic effects were observed. Among binary mixtures, thymol and p-cymene was synergistic in a topical application assay. GC–MS analyses showed increased recovery of thymol in the mixture both in vivo and in vitro from hemolymph extracts and receiver solutions, respectively, indicating enhanced penetration of thymol through the integument by p-cymene. An injection assay in fifth instar larvae and an in vitro cytotoxicity assay in an ovarian cell line of the cabbage looper showed no internal boosting effect of thymol by p-cymene, suggesting p-cymene facilitates the penetration of thymol, but does not directly contribute to increased toxicity of the mixture. A divided application method confirmed that the increased toxicity of the mixture can be produced only if the two compounds were applied as a mixture, proving p-cymene’s role as a penetration enhancer. Contact angle measurement on a waxy surface showed lowered surface tension of thymol in the mixture, suggesting the surface tension can directly influence the penetration and efficacy of a toxicant in pesticide formulation design.

Protective effects of silkworm hemolymph extract and its fractions on UV-induced photoaging

Ultraviolet (UV) irradiation induces skin photoaging by generating reactive oxygen species (ROS). ROS caused by UV-irradiation results in loss of skin cells and degradation of extracellular matrix. A number of antioxidants have been chemically synthesized or naturally extracted to prevent ROS-mediated skin photoaging. In our previous work, silkworm hemolymph extract (SHEX) was prepared, and its antioxidant activity was tested by free radical-scavenging assay. This study assessed the protective effects of SHEX on UV-induced photoaging of human immortalized keratinocytes (HaCaT). UVA (365 nm)-induced ROS generation was inhibited by supplementation of silkworm hemolymph (SH). Treatment with SHEX prepared by boiling SH inhibited death of HaCaT cells caused by UVB (315 nm) and UVA irradiation in a dose-dependent manner. Seven fractions were obtained by separating SHEX by gel permeation chromatography and the antioxidant activity of the fractions was examined. The fraction showing the highest protective efficacy on UV-induced cell damage corresponded to the lutein-containing fraction isolated in our previous study. Moreover, the SHEX fraction suppressed the expression of MMP-1 (matrix metalloproteinase-1), a matrix-degrading enzyme, suggesting that the active constituent of SHEX has the potential to inhibit skin photoaging. These results suggest that SHEX can be developed as a dietary and cosmetic supplement for prevention of skin photoaging.

Antibacterial Effect of Haemolymph Extracts of Edible Snail on Multi-drug Resistant Bacteria

Antibacterial Effect of Haemolymph Extracts of Edible Snail on Multi-drug Resistant Bacteria

Evaluation of Some Biochemical Parameters in Haemolymph of Bulinus specie in Ajiwa Reservoir Katsina State, Nigeria

Evaluation of some biochemical parameters was conducted in haemolymph of Bulinus sp in Ajiwa reservoir, Katsina state, Nigeria from September, 2014 to August, 2015. Four samples of Bulinus spwere collected at 5 different sampling locations every 3 months and were transferred into labelled plastic storage bottle and transported into laboratory for haemolymph extraction and analysis. Glucose, Aspartate amino transferase (AST) and Alanine amino transferase (ALT) were determined using standard methods, procedures and instruments. The mean value of the biochemical parameters are Glucose (36.28±1.76mg/dl), AST (19.21±0.24U/L) and ALT (13.91±0.62U/L). The level of the parameters were slightly increasing in the following order of magnitude; station 3 > station 2 > station 4> station 1 and station 5 recorded the lowest value. Seasonal variation of the biochemical parameters were clearly observed in the Bulinus sp. Glucose, AST and ALT levels were higher during the wet season compared to dry season (45.83±1.83mg/dl to 27.49±0.93mg/dl), (24.61±0.16 to 13.80±0.13U/L) and (19.33±0.78 to 10.44±0.21U/L) respectively. This may be due to high influx of nutrients from the surrounding farm lands and other domestic activities like bush burning, washing clothes, bathing and cattle rearing within the vicinity of the reservoir. Alterations in these biochemical parameters (Glucose, AST and ALT) in the haemolymph, indicate disturbance of the oxidative carbohydrate and protein metabolism. Apart from evaluating energy content as biomarkers, this experiment shows how Bulinus sp undergoes metabolic alteration to overcome stress.

Changes in energetic metabolism of Biomphalaria glabrata (Mollusca, Planorbidae) in response to exogenous calcium.

Calcium is considered an essential element for the metabolism of aquatic snail Biomphalaria glabrata (Say, 1818), intermediate host of Schistosoma mansoni Sambon, 1907 in Brazil, and represents a limiting factor to its distribution and adaptation to the environment. This study investigated the effect of different concentrations of exogenous CaCO3 on the energetic metabolism of B. glabrata for better understanding the physiological interference of chemical elements dissolved in the environment with the physiology of this species. Sixty-day-old snails were distributed into six groups, five exposed to different concentrations of CaCO3 (20, 40, 60, 80 and 100 mg/L) and a control group. The exposure to CaCO3 was assessed over time, with analysis of 15 snails of each group in the following intervals: 1, 14, 21 or 30 days for hemolymph extraction. Concentrations of calcium and glucose in the hemolymph were determined by commercial kits, and organic acids were extracted using an ion exchange column and analyzed by high-performance liquid chromatography. Concentration of calcium in the hemolymph showed no significant difference (p>0.05) from the control group and between the concentrations tested. Concentration of glucose decreased (p<0.05) in the treatments of exposure to 20 and 40 mg/L and increased when exposed to 80 and 100 mg/L CaCO3 compared to control and to other concentrations tested over 30 days. The organic acids pyruvate, oxaloacetate, citrate, succinate, fumarate, beta-hydroxybutyrate and lactate presented increased concentrations, while propionate and acetoacetate, decreased concentrations, when exposed to CaCO3 compared to control. Considering the influence of different periods of exposure to CaCO3, on the 14th day, there were stronger alterations in the metabolism of B. glabrata. In conclusion, exposure to CaCO3 reduced the concentration of glucose, which is metabolized into pyruvate, the final product of glycolysis, and also influenced the energetic metabolism pathways, indicating an aerobic or partially anaerobic functioning.

Proteomic profile of hemolymph and detection of induced antimicrobial peptides in response to microbial challenge in Diatraea saccharalis (Lepidoptera: Crambidae)

Insects are organisms extremely well adapted to diverse habitats, primarily due to their innate immune system, which provides them with a range of cellular and humoral responses against microorganisms. Lepidoptera hemolymph proteins involved in humoral responses are well known; however, there is a lack of knowledge about the sugarcane borer Diatraea saccharalis. In this present work, the hemolymph proteins of this pest insect were studied by applying proteomic methodologies. Two-dimensional electrophoresis (2-DE) gels of proteins extracted from naive larvae and larvae challenged with Escherichia coli (ATCC 11224) and Bacillus subtilis (ATCC 6623) showed an average of 300 spots, and 92 of these spots corresponded in all three 2-DE gels. Forty-one spots were excised and digested with trypsin and analyzed using mass spectrometry. After analysis, 10 proteins were identified, including some proteins of the immune system: β-defensin-like protein, Turandot A-like protein, attacin-like protein, peptidoglycan recognition protein and cyclophilin-like protein. Nine proteins were present in both experimental conditions; however, β-defensin-like protein was present only in hemolymph challenged by B. subtilis. Notably, attacin-like protein was strongly induced by challenge with E. coli, suggesting an immune response against the infection. However, antimicrobial activity was observed in the test zone of microbial growth inhibition of B. subtilis solely with the hemolymph extract of the larvae challenged with B. subtilis. We made for the first time a proteomic profile of the hemolymph of D. saccharalis in which it was possible to identify the presence of important proteins involved in the immune response.

A Critical Assessment of Bombyx mori Haemolymph Extract on Staphylococcus aureus an In vitro and In silico Approach

The newly reemerging drug-resistant Gram-positive pathogens require the discovery of new drug targets and the development of new therapeutics. We focus on antimicrobial proteins which inhibits growth of Staphylococcus aureus. In the present study fifth instar Bombyx mori larvae was used and infected with S. aureus by intrahaemocoelic injection of bacterial sample. The haemolymph was collected from the healthy and infected larvae after 24 h of infection and stored at -4°C in eppendorf tubes until use. Haemolymph extract was prepared and antimicrobial activity was done by the classical well diffusion method. Haemolymph extract prepared form infected larvae exhibit maximum zone of inhibition on S. aureus when compared with healthy haemolymph extract. Bioinformatics tools were used to find out the molecular interaction and binding mode of antimicrobial peptides from B. mori such as Moricin and Cecropin on three different drug target proteins Glycerol phosphate lipoteichoic acid synthase (PDB: 2W5Q), ABC transporter (PDB: 1P99) and DNA Gyrase (PDB: 2XCO) from S. aureus. Hex Docking software was used for the docking studies. The interaction mode and binding results can be represented in terms of docking energy. The best binding energies were obtained from the docking of DNA gyrase with Moricin and Cecropin (-702.13 Kcal/mol and -639.39 Kcal/mol) respectively.

Metabolomic Analysis of Diapausing and Noni-diapausing Larvae of the European Corn Borer Ostrinia nubilalis (Hbn.) (Lepidoptera: Crambidae)

In this study, an (1)H-NMR -based metabolomic approach was used to investigate the biochemical mechanisms of diapause and cold hardiness in diapausing larvae of the European corn borer Ostrinia nubilalis. Metabolomic patterns in polar hemolymph extracts from non-diapausing and diapausing larvae of O. nubilalis were compared. Analysis indicated 13 metabolites: 7 amino acids, glycerol, acetate, citrate, succinate, lactate and putrescine. Results show that diapausing larvae display different metabolomic patterns compared to active non-diapausing larvae, with predominant metabolites identified as glycerol, proline and alanine. In specific diapausing larvae initially kept at 5 °C then gradually chilled to –3 °C and –16 °C, alanine , glycerol and acetate were predominant metabolites. (1)H-NMR spectroscopy provides new insight into the metabolomic patterns associated with cold resistance and diapause in O. nubilalis larvae, suggesting distinct metabolomes function in actively developing and diapausing larvae.

Evaluation of methods for assessing physiological biomarkers of stress in freshwater mussels

Freshwater mussel populations are highly susceptible to environmental alterations because of their diminished numbers and primarily sessile behaviors; nonlethal biomonitoring programs are needed to evaluate the health of populations prior to mass mortality events. Our objectives were to determine (i) which biochemical parameters in freshwater mussel hemolymph could be consistently quantified, (ii) how hemolymph parameters and tissue glycogen respond to a thermal stress gradient (25, 30, and 35 °C), and (iii) the effects of tissue and hemolymph extraction on long-term growth and survival of smaller- and larger-bodied mussel species. Glucose exhibited elevated expression in both species with increasing water temperature. Two transaminase enzymes had elevated expression in the 30 °C treatment. The effects of hemolymph extraction and tissue biopsies were evaluated with a large-bodied species, Elliptio crassidens, and a smaller species, Villosa vibex. Individuals were monitored for 820 to 945 days after one of four treatments: hemolymph extraction, tissue biopsy, tissue and hemolymph extraction, and control. Hemolymph extraction and tissue biopsy adversely affected survival of V. vibex, suggesting that these extraction methods may add some risk of reduced survival to smaller-bodied species. Survival of E. crassidens was not impaired by any of the treatments, supporting the use of these techniques in nonlethal biomonitoring programs for larger-bodied mussel species.

THE ENDOPARASITOID Campoletis chlorideae INDUCES A HEMOLYTIC FACTOR IN THE HERBIVOROUS INSECT Helicoverpa armigera

Although lysis of invading organisms is a major innate form of immunity used by invertebrates, it remains unclear whether herbivorous insects have hemolysin or not. To address this general question, we tested the hemolytic (HL) activity of the hemolymph and tissue extracts from various stages of the polyphagous insect Helicoverpa armigera (Hübner) against the erythrocytes from chicken, duck, and rabbit. An HL activity was identified in the hemolymph of H. armigera larvae. Further studies demonstrated that the HL activity is proteinaceous as it was precipitable by deproteinizing agents. Hemolysins were found in Helicoverpa egg, larva, pupa, and adult, but the activity was higher in feeding larvae than in molting or newly molted larvae. Hemolysins were distributed among a variety of larval tissues including salivary gland, fat body, epidermis, midgut, or testes, but the highest activity was found in salivary gland and fat body. Relative to nonparasitized larvae, parasitization of H. armigera larvae by the endoparasitoid Campoletis chlorideae Uchida induced a 3.4-fold increase in the HL activity in the plasma of parasitized host at day two postparasitization. The present study shows the presence of a parasitoid inducible HL factor in the parasitized insect. The HL activity increased significantly in H. armigera larvae at 12 and 24 h postinjection with Escherichia coli. We infer the HL factor(s) is inducible or due to de novo synthesis, which means that the HL factor(s) is associated with insect immune response by inhibiting or clearance of invading organisms.

Expression of RXR, EcR, E75 and VtG mRNA levels in the hepatopancreas and ovary of the freshwater edible crab, Oziothelphusa senex senex (Fabricius, 1798) during different vitellogenic stages.

The objective of the present study was to investigate the expression profile of retinoid X receptor (RXR), ecdysone receptor (EcR) and ecdysone inducible gene (E75) in the hepatopancreas and ovary of Oziothelphusa senex senex during different vitellogenic stages. RXR, EcR and E75 complementary DNAs (cDNAs) were isolated from the ovaries, while vitellogenin (VtG) cDNA was isolated from the hepatopancreas of vitellogenic female crab. Deduced amino acid sequence of the messenger RNAs (mRNAs) of RXR, EcR and E75 showed more than 80% identity with their respective mRNAs of other brachyurans. VtG mRNA was not detected in the ovary throughout vitellogenic stages. RXR and EcR were significantly increased in the ovaries during vitellogenic stage I. The levels of EcR, E75 and VtG in the hepatopancreas elevated significantly during vitellogenic stages I and II, whereas the levels of RXR elevated only in vitellogenic stage I. During vitellogenic stage III, the levels of RXR, EcR and VtG in the hepatopancreas were significantly decreased. Immunoprecipitation analysis revealed the presence of VtG in the haemolymph, hepatopancreas and ovary extracts from the females but absent in haemolymph and hepatopancreas extract of males. It can be inferred that RXR, EcR and E75 are involved in the regulation of synthesis of VtG in hepatopancreas, whereas in ovary, it is hypothesized that they play an important role in the uptake of VtG from the haemolymph, probably by regulating the levels of vitellogenin receptor. These are the first data showing an association between the expression levels of RXR, EcR and E75 and vitellogenesis and provide an alternative molecular intervention mechanism to the traditional eyestalk ablation to induce vitellogenesis and ovarian maturation in crustaceans.

Evaluating the effect of Sarconesiopsis magellanica (Diptera: Calliphoridae) larvae-derived haemolymph and fat body extracts on chronic wounds in diabetic rabbits.

We evaluated extracts taken from S. magellanica third instar larvae fat body and haemolymph using a diabetic rabbit model and compared this to the effect obtained with the same substances taken from Lucilia sericata larvae. Alloxan (a toxic glucose analogue) was used to induce experimental diabetes in twelve rabbits. Dorsal wounds were made in each animal and they were infected with Staphylococcus aureus and Pseudomonas aeruginosa. They were then treated with haemolymph and lyophilized extracts taken from the selected blowflies' larvae fat bodies. Each wound was then evaluated by using rating scales and histological analysis. More favourable scores were recorded on the PUSH and WBS scales for the wounds treated with fat body derived from the larvae of both species compared to that obtained with haemolymph; however, wounds treated with the substances taken from S. magellanica had better evolution. Histological analysis revealed that treatment led to tissue proliferation and more effective neovascularisation in less time with both species' fat body extracts compared to treatment with just haemolymph. The results suggest the effectiveness of the substances evaluated and validate them in the animal model being used here as topical agents in treating chronic wounds.

Evidence that the major hemolymph protein of the Pacific oyster, Crassostrea gigas, has antiviral activity against herpesviruses

Viruses belonging to the family Malacoherpesviridae currently pose a serious threat to global production of the Pacific oyster, Crassostrea gigas. Hemolymph extracts from C. gigas are known to have potent antiviral activity. The compound(s) responsible for this broad-spectrum antiviral activity in oyster hemolymph have not been identified. The objective of this study was to identify these antiviral compound(s) and establish whether hemolymph antiviral activity is under genetic control in the Australian C. gigas population. Hemolymph antiviral activity of 18 family lines of C. gigas were assayed using a herpes simplex virus type 1 (HSV-1) and Vero cell plaque reduction assay. Differences in anti-HSV-1 activity between the family lines were observed (p<0.001) with heritability estimated to be low (h2=0.21). A glycoprotein that inhibits HSV-1 replication was identified by resolving oyster hemolymph by native-polyacrylamide gel electrophoresis (PAGE) and assaying extracted protein fractions using the HSV-1 and Vero cell plaque assay. Highest anti-HSV-1 activity corresponded with an N-linked glycoprotein with an estimated molecular mass of 21kDa under non-reducing SDS–PAGE conditions. Amino acid sequencing by tandem mass spectrometry revealed this protein matched the major hemolymph protein, termed cavortin. Our results provide further evidence that cavortin is a multifunctional protein involved in immunity and that assays associated with its activity might be useful for marker-assisted selection of disease resistant oysters.

Comet Assay on Daphnia magna in eco-genotoxicity testing

Detection of potentially hazardous compounds in water bodies is a priority in environmental risk assessment. For the evaluation and monitoring of water quality, a series of methodologies may be applied. Among them, the worldwide used toxicity tests with organisms of the genus Daphnia is one of the most powerful. In recent years, some attempts were made to utilize Daphnia magna in genotoxicity testing as many of the new environmental contaminants are described as DNA-damaging agents in aquatic organisms. The aim of this research was to develop a highly standardized protocol of the Comet Assay adapted for D. magna, especially regarding the isolation of cells derived from the same tissue (haemolymph) from newborn organisms exposed in vivo. Several methods for haemolymph extraction and different Comet Assay parameters were compared. Electrophoretic conditions were adapted in order to obtain minimum DNA migration in cells derived from untreated organisms and, at the same time, maximum sensitivity in specimens treated with known genotoxicants (CdCl2 and H2O2). Additional tests were performed to investigate if life-history traits of the cladoceran (such as the age of adult organisms that provide newborns, the clutch size of origin, the number of generations reared in standard conditions) and the water composition as well, might influence the response of the assay. This study confirms the potential application of the Comet Assay in D. magna for assessing genotoxic loads in aqueous solution. The newly developed protocol could integrate the acute toxicity bioassay, thus expanding the possibility of using this model species in freshwater monitoring (waters, sediment and soil elutriates) and is in line with the spirit of the EU Water Framework Directive in reducing the number of bioassays that involve medium-sized species.

Improved replication of the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) in vitro using proteins from Lonomia obliqua hemolymph.

The baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV), a member of the family Baculoviridae, has been widely applied as a biopesticide for the control of the velvetbean caterpillar, a pest of soybean crop field. Baculoviruses are considered safe and efficient agents for this purpose, because they do not infect vertebrates, being safe for the health of humans and animals, as well as to the environment. The objective of this work was to identify proteins obtained from Lonomia obliqua hemolymph with potential application in the optimization of baculovirus AgMNPV replication in Sf9 insect cell culture. In this work the improvement of the cell culture and viral replication of the AgMNPV baculovirus was observed when Grace medium was supplemented with 10% (v/v) Fetal Bovine Serum (FBS), 1% (v/v) hemolymph extract, or 3% (v/v) of hemolymph fractions or hemolymph sub-fractions obtained by purifying hemolymph through High Performance Liquid Chromatography. Hemolymph presented a positive effect on the synthesis of polyhedra and enhanced baculovirus replication in Spodoptera frugiperda (Sf9) cells (TCID50/mL), and led to Sf9 cell culture improvement. Grace medium supplemented with 10% (v/v) FBS and 1% (v/v) hemolymph provided an increase of baculovirus replication, when the cells were infected with multiplicity of infection of 1. In this case, the baculovirus replication was 6,443.91 times greater than that obtained with the control: Grace medium supplemented with 10% (v/v) FBS. In addition, this work suggests that hemolymph from L. obliqua could have an interesting application in biotechnology, due to an increase in the viability of the cells and virus replication.

Hepatopancreas but not ovary is the site of vitellogenin synthesis in female fresh water crab, Oziothelphusa senex senex

The objective of the present study was to explore the site of synthesis of vitellogenin (Vtg) in fresh water edible crab, Oziothelphusa senex senex. Vtg cDNA fragments were isolated from the hepatopancreas of female crabs using RT-PCR method, and the deduced amino acid sequence of O. senex senex showed more than 60% identity with other brachyuran Vtg sequences. RT-PCR analysis showed that Vtg mRNA can be detected only in hepatopancreas of female Oziothelphusa but not in other tissues including eyestalks, Y-organs, mandibular organs, thoracic ganglion, hypodermis and ovary. Antibodies were raised against vitellin purified from the ovary of O. senex senex. Immunoprecipitation analysis revealed the presence of Vtg in the hepatopancreas of vitellogenic stage I females and in the hemolymph, hepatopancreas and ovary extracts from vitellogenic stage II females but absent in hemolymph and hepatopancreas extract of males. These results suggest that Vtg is synthesized only in hepatopancreas but not in the ovaries of O. senex senex. In addition, Vtg synthesized in hepatopancreas is transported to ovary through hemolymph.