Haemolymph Protein Profile Research Articles

DETERMINATION OF TEMPERATURE SENSITIVE DIAPAUSE TERMINATION STATE OF DABA TRIVOLTINE ECORACE OF ANTHERAEA MYLITTA DRURY

The Daba trivoltine ecorace of tropical tasar silkworm Antheraea mylitta Drury is one ofthe most commercially exploited non-mulberry silkworms in tropical India. It is generally grown inthe latitude range of 20°N to 25°N in Eastern India, especially, in parts of West Bengal, Jharkhand,Orissa, Chhattisgarh and Andhra Pradesh. The pupae of this ecorace remain in facultative pupaldiapause state for five to six months (January-June). During this long period of diapause losses occurdue to un-seasonal and unsynchronised emergence of male and female moths which is estimated tothe tune of 25-30% leading to reduction of seed production in grainages. The losses become moreprevalent when dry conditions prevail soon after pre-monsoonal showers. This can be avoided byconsigning the pupae of diapause termination state to low temperature. In the present study, thespecific age of diapause termination state has been worked out on the basis of the presence ofhaemolymph biochemical constituents like glycerol, trehalose, glycogen, quantitative total proteinsand free amino acids and protein profile comparing them with non-diapausing pupae harvestedduring first and second crop. Among diapausing pupae, the trehalose concentration always remainedat its low level during diapause and an increase in concentration was observed at the fag end ofdiapause when pupae attained the age of 145 to 150 days. Contrary to this, the level of glycerol andglycogen was always higher through out the diapause period and a down surge in the concentrationwas noticed when pupae were 145 to 150 days old. The level of protein was higher in nondiapausinggeneration. However, the level of protein and amino acids showed a fluctuating trendthrough out diapause development. The haemolymph protein profile of the diapausing pupae showedthe presence of a diapause specific protein band of 16kD which remained in its full intensity till thepupae attained the age of 145-150 days, thereafter, its intensity went down and protein profile ofdiapausing pupae looked similar to non-diapausing pupae. Therefore, it is confirmed that the diapausetermination state in Daba pupae occurs when pupae become 145-150 days old. The diapausingpupae of this age can be further exploited by working out low temperature treatment schedule toavoid losses in grainages

Proteomic profile of Ostrea edulis haemolymph in response to bonamiosis and identification of candidate proteins as resistance markers.

European flat oyster Ostrea edulis populations have suffered extensive mortalities caused by bonamiosis. The protozoan parasite Bonamia ostreae is largely responsible for this disease in Europe, while its congener B. exitiosa has been detected more recently in various European countries. Both of these intracellular parasites are able to survive and proliferate within haemocytes, the main cellular effectors of the immune system in molluscs. Two-dimensional electrophoresis was used to compare the haemolymph protein profile between Bonamia spp.-infected and non-infected oysters within 3 different stocks, a Galician stock of oysters selected for resistance against bonamiosis, a non-selected Galician stock and a selected Irish stock. Thirty-four proteins with a presumably relevant role in the oyster-Bonamia spp. interaction were identified; they were involved in major metabolic pathways, such as energy production, respiratory chain, oxidative stress, signal transduction, transcription, translation, protein degradation and cell defence. Furthermore, the haemolymph proteomic profiles of the non-infected oysters of the 2 Galician stocks were compared. As a result, 7 proteins representative of the non-infected Galician oysters selected for resistance against bonamiosis were identified; these 7 proteins could be considered as candidate markers of resistance to bonamiosis, which should be further assessed.

Short-term heat shock affects the course of immune response in Galleria mellonella naturally infected with the entomopathogenic fungus Beauveria bassiana

We aimed to investigate how exposition of infected insects to short-term heat shock affects the biochemical and molecular aspects of their immune response. Galleria mellonella larvae were exposed to 43°C for 15min, at the seventy second hour after natural infection with entomopathogenic fungus Beauveria bassiana. As a result, both qualitative and quantitative changes in hemolymph protein profiles, and among them infection-induced changes in the amount of apolipophorin III (apoLp-III), were observed. Heat shock differently affects the expression of the tested immune-related genes. It transiently inhibits expression of antifungal peptides gallerimycin and galiomicin in both the fat body and hemocytes of infected larvae. The same, although to a lesser extent, concerned apoLp-III gene expression and was observed directly after heat shock. Nevertheless, in larvae that had recovered from heat shock, apoLp-III expression was higher in comparison to unshocked larvae in the fat body but not in hemocytes, which was consistent with the higher amount of this protein detected in the hemolymph of the infected, shocked larvae. Furthermore, lysozyme-type activity was higher directly after heat shock, while antifungal activity was significantly higher also in larvae that had recovered from heat shock, in comparison to the respective values in their non-shocked, infected counterparts. These results show how changes in the external temperature modulate the immune response of G. mellonella suffering from infection with its natural pathogen B. bassiana.

Identification of tick species and disseminate pathogen using hemolymph by MALDI-TOF MS

BackgroundMatrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is increasingly emerging tool for identification of arthropods including tick vectors using whole or body part of specimens. The challenges of the present study were to assess MALDI-TOF MS profiling for the both identification of tick species and Rickettsia spp. in infected ticks using hemolymph as protein mixture. MethodsFirstly, hemolymph protein mixture from legs of 5 tick species, Rhipicephalus sanguineus, Rhipicephalus bursa, Dermacentor marginatus, Hyalomma marginatum rufipes and Amblyomma variegatum infected by Rickettsia africae were submitted to MALDI-TOF MS to assess tick species identification ability. Secondly, hemolymph MS spectra from Rh. sanguineus infected or not by Rickettsia c. conorii were compared to detect protein profiles changes. Finally, leg hemolymph MS spectra from new specimens of the 5 tick species were tested blindly including ticks infected by R. c. conorii. Discriminating mass peaks distinguishing the R. c. conorii infected and non-infected Rh sanguineus were determined. ResultsConsistent and reproducible MS profiles were obtained into each tick species. Comparison of MS spectra revealed distinct hemolymph protein profiles according to tick species. MS spectra changes were observed between hemolymphs from R. c. conorii-infected and non-infected Rh. sanguineus specimens, revealing 17 discriminating mass peaks. Clustering analysis based on MS protein profiles highlighted that hemolymph samples were grouped according to tick species. All tick hemolymph samples blindly tested against our home-made arthropod MS reference database were correctly identified at the species distinguishing also R. c. conorii-infected from Rickettsia-free Rh. sanguineus specimens. ConclusionThe present study demonstrated the use of hemolymph MS profiles for dual identification of tick species and associated pathogens. This concomitant identification could be helpful for tick entomological diagnosis, notably for specimens removed directly on patients.

Influence of host plant nitrogen fertilization on hemolymph protein profiles of herbivore Spodoptera exigua and development of its endoparasitoid Cotesia marginiventris

Nitrogen has complex effects on plant–herbivore–parasitoid tritrophic interactions. The negative effects of low nitrogen fertilization in host plants on insect herbivores can be amplified to the higher trophic levels. In the present study, we examined the impact of varying nitrogen fertilization (42, 112, 196, and 280ppm) of cotton plants (Gossypium hirsutum L.) on the interactions between the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), and the hymenopteran endoparasitoid Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae). We predicted that the development and fitness of C. marginiventris would be adversely affected by low host plant nitrogen fertilization through the herbivore S. exigua. The percentage of C. marginiventris offspring developing to emerge and spin a cocoon, and total mortality of parasitized S. exigua larvae were unaffected by nitrogen level. The developmental time of C. marginiventris larvae in S. exigua larvae feeding on low (42ppm) nitrogen cotton plants was approximately 30% longer than that of those feeding on higher (112, 196, and 280ppm) nitrogen plants. Parasitoid size (length of right metathoracic tibia), a proxy for fitness, of C. marginiventris males was positively affected by nitrogen level. Total amounts of S. exigua hemolymph proteins were not affected by nitrogen level, but were reduced by parasitism by C. marginiventris. Two proteins with molecular weights of ca. 84 and 170kDa dominated the S. exigua larval hemolymph proteins. Concentrations of the 170kDa hemolymph protein were unaffected by nitrogen treatment, but parasitism reduced concentrations of the 170kDa protein. Concentrations of the 84kDa protein, on the other hand, were interactively affected by parasitism and nitrogen treatment: higher nitrogen fertilization (112, 196, and 280ppm) increased protein concentrations relative to the 42ppm treatment for unparasitized S. exigua larvae, whereas nitrogen treatment had no effects on parasitized larvae. For S. exigua larvae feeding on 42ppm nitrogen plants, parasitism increased concentration of the 84kDa protein, while for those feeding on 112, 196, and 280ppm nitrogen plants, parasitism decreased concentrations of the protein. Possible mechanisms and ecological consequences for the extended development of C. marginiventris on S. exigua hosts grown on low-nitrogen plants are discussed.

Innate immune response in the hemolymph of an ascidian, Halocynthia roretzi, showing soft tunic syndrome, using label-free quantitative proteomics

Soft tunic syndrome of Halocynthia roretzi manifests as soft, weak, and rupturable tunics, causing mass mortality. Utilizing liquid chromatography–tandem mass spectrometry (LC–MS/MS), innate immune response was established by comparing hemolymph protein profiles of ascidians with healthy or softened tunics. Of 100 proteins in each individual ascidian, 59 proteins from healthy and 56 proteins from diseased ascidians were functionally classified. Proteins found only in diseased individuals included trypsin inhibitor and Hr-29, and with high exponentially modified protein abundance index (emPAI) values. From 41 proteins identified to be common to both healthy and diseased ascidians, 15 were associated with innate immune response. Ficolin 3, a component of the lectin-complement system, was significantly decreased in diseased ascidians, but a cell surface protein, type II transmembrane serine protease-1 (TTSP), was considerably elevated. These results suggest that trypsin inhibitor, ficolin 3, and TTSP are probably involved in the innate immune response related to this tunic disease. Beside, Hr-29 could be suggested as a biomarker for soft tunic syndrome.

Effect of RH-2485 on development, metamorphosis and synthesis of major proteins in female silkworm Bombyx mori

Toxicological data on silkworm Bombyx mori are quite comparable to those of other lepidopteran pest insects, therefore, it is considered as a suitable model for exploring effects of any new synthetic formulations. In this study, female V instar larvae of silk moth B. mori were chosen to evaluate the lethal and sublethal toxicity effects of RH-2485 (methoxyfenozide), a non-steroidal ecdysteroid agonist and to substantiate the ecdysteroid mimicking action of RH-2485 on ovary development, vitellogenin incorporation and egg production in isolated pupal abdomen (IPA). Probit analysis was carried out to find the median lethal dose (LD50) from 96 h cumulative mortality percent. Protein profile of haemolymph, fat body, ovary and eggs were separated in SDS-PAGE. Western blot analysis was carried out to confirm vitellogenin in the ovary. Sublethal effects on feeding, cocoon spinning, pupation, adult emergence and egg production were studied at doses of 1/5th, 1/10th and 1/20th of LD50. Significant changes were observed in all these parameters at all three sublethal doses. The morphological effects were related to underlying biochemical changes by finding the changes in haemolymph, fat body, ovary and egg protein profile. Marked changes were observed in storage proteins (80 kDa) and 30 kDa proteins in the haemolymph at all three sublethal doses. The larvae that escaped the sublethal effects at a dose of 1/20 of LD50 and emerged as adults with malformed wings produced significantly lower number of eggs. The isolated pupal abdomen (IPA) treated with RH-2485 did not metamorphose into adult but the oocyte development and vitellogenesis were normal but the egg precursor processing was incomplete leading to failure in choriogenesis.

Haemolymph Protein and Lipid Profile of Rhipicephalus (Boophilus) microplus Infected by Fungi

The current study evaluates the protein and lipid profile of haemolymph of Rhipicephalus (Boophilus) microplus engorged females infected by Metarhizium anisopliae, Beauveria bassiana or Fusarium oxysporum. Ticks were immersed or inoculated with conidial suspension. Haemolymph was collected from the dorsal surface of engorged females. The results showed altered total protein amounts; however, no significant difference was observed on electrophoretic profile among haemolymph samples. In addition, altered lipid profile was detected in haemocyte samples from ticks treated with Beauveria and Metarhizium.

Effect of immunostimulants on the haemolymph haemagglutinins of tiger shrimpPenaeus monodon

The levels of haemagglutinins in Penaeus monodon, following administration of immunostimulants, β-glucans and/or vibrio bacterin either orally or by immersion, were studied. The freshly drawn haemolymph was incubated with microbial materials like β-glucans/vibrio bacterin and serum obtained after coagulation was administered to naïve animals. The immunostimulant treatments either via immersion, feeding or injection were found to cause an increase (P<0.006) in haemagglutination activity (HA) of the haemolymph against mouse erythrocytes. Injection of saline or heterologous haemolymph caused an increase in the HA, but injection of haemolymph serum obtained by clotting haemolymph in the presence of vibrio bacterin or glucan did not bring about an increase in HA. There was no change in the haemolymph protein profile of the groups receiving immunostimulants through immersion or feed. Two protein bands (27 and 30 kDa), which were present in the uninjected group, were found to be overexpressed in the haemolymph-injected groups. Three bands of 17, 21 and 23 kDa, which were absent in control or saline-injected groups, were present in all the haemolymph serum-injected groups. The study indicates that modulation of HA may partly account for the immunomodulatory activity of immunostimulants like β-glucan or vibrio bacterins.

Haemolymph electrophoretic pattern of Ascia monuste orseis larvae (Lepidoptera: pieridae) parasitized by Cotesia glomerata (Hymenoptera: braconidae)

Cotesia glomerata is a natural enemy of the vegetable plague Ascia monuste orseis and preferably parasites 2nd, 3rd and 4th instar larvae. Parasitism effects on the haemolymph protein profile of Ascia monuste orseis larvae from the 2nd to 7th days were analyzed both qualitatively and quantitatively by SDS-PAGE and Coomassie-Blue binding methods. Quantitative analysis showed a progressive increase in the protein content of about 6.5 and 12.5 times in parasitized and non-parasitized larvae from the 2nd to 5th days, respectively. On the 6th day, a decrease in protein content was observed in both groups, although this decrease was significantly less than the control group that continued to metamorphosis. Meanwhile, parasitized larvae had one more day (7th day) in their larval period to complete parasitoid development, justified by the fact that parasitoid is koinobiont and allows host feeding. On this day, a drastic increase in protein content was detected when the parasitoids left the host. The SDS-PAGE showed proteins of high molecular weight (>120 kDa) on the 5th day of the non-parasitized larvae when they entered pre-pupa stage and on the 7th day of parasitized larvae. Proteins with MW lower than 62 kDa and higher than 27 kDa were absent on the 5th day in control larvae (pre-pupa phase), but present in parasitized larvae. This could indicate a possible relation between these proteins and the host juvenile hormone. Therefore, the presence of C. glomerata influences Ascia monuste orseis development, but its own physiological development is apparently independent of the host, which tends to die when parasitism succeeds.

126 Dopaminergic modulation by dopamine in medium spiny neurons in the striatum

Soft tunic syndrome of Halocynthia roretzi manifests as soft, weak, and rupturable tunics, causing mass mortality. Utilizing liquid chromatography–tandem mass spectrometry (LC–MS/MS), innate immune response was established by comparing hemolymph protein profiles of ascidians with healthy or softened tunics. Of 100 proteins in each individual ascidian, 59 proteins from healthy and 56 proteins from diseased ascidians were functionally classified. Proteins found only in diseased individuals included trypsin inhibitor and Hr-29, and with high exponentially modified protein abundance index (emPAI) values. From 41 proteins identified to be common to both healthy and diseased ascidians, 15 were associated with innate immune response. Ficolin 3, a component of the lectin-complement system, was significantly decreased in diseased ascidians, but a cell surface protein, type II transmembrane serine protease-1 (TTSP), was considerably elevated. These results suggest that trypsin inhibitor, ficolin 3, and TTSP are probably involved in the innate immune response related to this tunic disease. Beside, Hr-29 could be suggested as a biomarker for soft tunic syndrome.

The effect of photoperiod on the haemolymph protein profile in the silkworm, Bombyx mori L.

—The effect of photoperiod on the haemolymph protein profile in the silkworm, Bombyx mori L. larvae was investigated at photoperiod treatments of light to dark (L:D) ratios of 13:11 (control), 24:0, 18:6, 12:12, 6:18 and 0:24 hours using fifth instar larvae. The intensity of protein bands in haemolymph extracts from larvae was measured with a densitometer. The intensity was highest in the haemolymph from larvae in the 24:0 (L:D) h photoperiod. Strongly reactive antigens of SP-1 and SP-2 and weakly reactive bands of storage proteins for both male and female larvae were observed in the 24:0 (L:D) h photoperiod.

Concentration of hemolymph proteins during postembryonic worker development of Africanized honey bees in Brazil and Carniolans in Europe

The profiles of hemolymph protein content during postembryonic development of honey bees (Apis mellifera) were determined for the first time. Larval and pupal worker stages of Africanized honey bees in Brazil and Carniolans in Germany were studied. A rapid increase in protein concentration was found in the late larval stages, followed by a decrease in prepupae and a minimum amount near the pupal moult period. In the early pupal stages, the protein concentration increased again, and then decreased until adult eclosion. Only the first protein peak in spinning L5 was more pronounced in the Africanized honey bees. No differences between the hemolymph protein profiles of Africanized bees and Carniolans could be detected during pupal development. The duration of several larval and pupal stages was shorter in Africanized than in Carniolan workers. The data reflect the general characteristics of the Africanized biotype reared in tropical conditions exhibiting accelerated patterns of development and lower body weight at emergence in comparison to the European Apis subspecies reared in temperate conditions. The ontogenetic patterns described for hemolymph protein concentration in preimaginal honey bee development are also discussed, specifically in regard to the regulation of metamorphosis in holometabolous insects

Development of imaginal competence to adipokinetic hormone in Locusta: Lipophorin conversions in precocene-induced adultiforms and in azadirachtin-induced over-aged nymphs

Abstract 1. 1. In precocene III-induced fifth instar adultiforms of Locusta migratoria, both the haemolymph protein profile and formation of low density lipophorin (LDLp) upon injection of the insects with adipokinetic hormone (AKH 1) differ markedly from normal fifth (last) instar nymphs and closely resemble adult characteristics. 2. 2. The profile of the haemolymph proteins from azadirachtin-induced fifth instar over-aged nymphs, however, is intermediate between normal fifth instar nymphal and adult locusts with respect to the concentration of apolipophorin III (apoLp-III). Injection of AKH I results in the formation of a lipophorin intermediate in size between HDLp and LDLp. 3. 3. The incomplete formation of LDLp in the azadirachtin-treated insects could be related to a low level of the apoLp-III required; the presence of sufficient amounts of apoLp-III in the chemically allatectomized adultiforms, however, would suggest that juvenile hormone is not the major factor involved in regulating the apoLp-III gene during normal adult maturation.

Significance of haemolymph protein patterns in biosystematic studies of some grouse locusts (Tetrigidae: Orthoptera)

The grouse locusts (Tetrigidae) represent a group of small sized Caeliferan Orthoptera. These are considered to be primitive orthopterans related to Tridactylids on one hand to Acridids on the other. This family is represented in Indian subcontinent by about 90 species belonging to 35 genera. In the present study haemolymph protein profiles from 7 species from this family have been studied as an additional parameter to understand interrelationship amongst them. It is noted that the speciesParatettix dorsifer andEuparatettix personatus belonging to the same subfamily Tetriginae show remarkable similarity in the haemolymph protein profile, thus justifying their classical taxonomical grouping. In case of subfamily Scelimeninae, however, of the 4 species studied 3 species vizEucriotettix flavopictus, Criotettix latifrons andThoradonta pruthii show a marked similarity, howeverEucriotettix harpago shows a pattern closer to that observed in the subfamily Cladonotinae. This implies that though by classical taxonomic criteria the 4 species are close to each other this may not be a very natural grouping.