Total Hemolymph Protein Research Articles

Physiological changes in the Norway lobster Nephrops norvegicus (L.) escaping and discarded from commercial trawls on the West Coast of Scotland : 1. Body fluid volumes and haemolymph composition after capture and during recovery

The Norway Lobster Nephrops norvegicus (L.) was captured by trawling at depths of 120 to 150 m off the West Coast of Scotland using commercial fishing gears. Discarded animals (normally <25–35 mm carapace length) were sampled immediately after capture, and following emersion (exposure to air) on deck for 1 or 2 h. Their recovery from, and survival of, the stresses of being fished were examined in separate groups of animals transferred by SCUBA divers to underwater (u/w) cages on the seabed at a mean depth of 24 m. “Escaped” N. norvegicus were collected in a small-meshed (40 mm) “cover” around the cod-end and sampled without emersion. It was assumed that the physiological state of these animals would be similar to those passing through the cod-end meshes directly back into the surrounding sea during normal fishing operations. Groups of escaped animals were also transferred to u/w cages for recovery and survival studies. Control animals (creel-caught from the same fishing grounds) were sampled after recovery in u/w cages. Total body water content, haemolymph (blood) volume and composition were examined at various stages after capture and during recovery of control, discarded and escaped animals. Discarded animals showed significant reductions in total body water content compared to controls but recovered after 24 h. Controls that had been artificially damaged by puncturing also showed a reduction in water content. Escaped animals showed significantly higher mean water content compared to discards. Haemolymph volumes were also reduced in discarded animals suggesting that significant blood loss occurs during capture. There was also evidence of a shift of fluid from haemolymph to tissues in discarded animals. Pericardial (haemolymph) pressures were much reduced in discards compared to undisturbed control animals but showed significant recovery in surviving animals. Pressure reduction was less in escapes but was seen in control animals that had been exercised to exhaustion. Haemolymph osmotic concentrations, [Na +] and [K +] showed increases after discarding, notably after emersion, but haemocyanin [Hcy] and total haemolymph protein concentrations were less affected. The effect of these changes on longer term survival and recovery from the stress of capture are discussed.

Host physiological changes due to parasitism of a braconid wasp, Cotesia plutellae, on diamondback moth, Plutella xylostella

Braconid wasps, Cotesia plutellae (Kurdjumov), were collected from parasitized host larvae of diamondback moth, Plutella xylostella (L.) in Korea. Virus particles were found in the oviduct lumen of C. plutellae females. Multiple nucleocapsids with ∼30-nm diameter and variable length (30–80 nm) were surrounded with a single unit membrane envelope. The parasitization of C. plutellae completely inhibited pupal metamorphosis. The parasitized larvae showed significant decrease in feeding activity and total hemolymph proteins, especially as larval storage proteins. They also showed a significant decrease in immune capacity as evidenced by reduced ability to form hemocyte nodules and reduced phenoloxidase and lysozyme activity. Here, we show that C. plutellae has an endosymbiotic virus like other reported species in Microgastrinae, and suggest that it causes host developmental arrest and immune-depression at parasitization.

Estimating disease resistance in insects: phenoloxidase and lysozyme-like activity and disease resistance in the cricket Gryllus texensis

An animal’s ability to resist disease is usually estimated by measuring one or more components of the immune system. There is an assumption that these assays of immunity measure an animal’s ability to mount an effective immune response. This paper tests this assumption by examining the relationship between two common estimates of insect immunocompetence, phenoloxidase and lysozyme-like enzyme activity, and resistance to three common insect bacterial pathogens: Serratia marcescens, Serratia liquefaciens, and Bacillus cereus. There was a correlation (Spearman’s r s=0.33, p<0.001, n=190 pairs) between total phenoloxidase and baseline lysozyme-like activity within individuals. However, total phenoloxidase and baseline lysozyme-like activity levels did not predict which male crickets would survive any of the three bacterial challenges. Lysozyme-like activity increased after an immune challenge (Friedman, 33.72, p<0.001), and the greater the increase, the greater the chance that the cricket would survive S. marcescens (slope=0.15, χ 2=8.2, p=0.005) or B. cereus (slope=0.8, χ 2=6.4, p=0.01). The crickets with a greater total hemolymph protein concentration were also more likely to survive a challenge with any of the three bacterial pathogens than the crickets with lower total hemolymph protein concentrations ( S. liquefaciens: slope=0.02, χ 2=9.2, p=0.002; B. cereus: slope=0.02, χ 2=6.5, p=0.01; S. marcescens: slope=0.03, χ 2=7.8, p=0.005). Because of the complexity of the immune system, empirical tests of the relationship between assays of immunity and resistance to a range of actual pathogens are important for correctly interpreting these measures.

A multibiomarker approach to environmental assessment.

Incorporation of ecologically relevant biomarkers into routine environmental management programs has been advocated as a pragmatic means of linking environmental degradation with its causes. Here, suites of biomarkers, devised to measure molecular damage, developmental abnormality and physiological impairment, were combined with chemical analysis to determine exposure to and the effects of pollution at sites within Southampton Water (UK). Test species included a filter feeder, a grazer, and an omnivore to determine the sensitivity of organisms occupying different trophic levels. Linear regression confirmed a significant association between incidence of intersex in Littorina littorea and tributyltin (TBT) concentrations (R2 = 0.954) and between PAH metabolites in Carcinus maenas urine and PAHs in sediments (R2 = 0.754). Principal component analysis revealed a gradient of detrimental impact to biota from the head to the mouth of the estuary, coincident with high sediment concentrations of heavy metals, PAHs, and biocides. Multidimensional scaling identified C. maenas as the organism most sensitive to contamination. Carboxylesterase activity, metallothionein and total haemolymph protein were the most discriminating biomarkers among sites. This holistic approach to environmental assessment is encouraged as it helps to identify the integrated impact of chemical contamination on organisms and to provide a realistic measure of environmental quality.

Acetylcholinesterase inhibition as a biomarker of adverse effect: A study of Mytilus edulis exposed to the priority pollutant chlorfenvinphos

The inhibition of acetylcholinesterase (AChE) activity has been used widely as a biomarker of exposure to organophosphorous pesticides (OPs). However, scientific uncertainty about the risk assessment implications of data describing inhibition of cholinesterases in diverse species and tissues has hampered the use of AChE activity as a biomarker of adverse effect. Here, haemolymph AChE activity was combined with biomarkers of cellular integrity, immunotoxicity and physiological status in order to measure exposure to and the effects of the priority pollutant chlorfenvinphos. Laboratory exposures of the blue mussel Mytilus edulis to commercial grade chlorfenvinphos (Sapecron ®) were conducted over 24, 48 and 96 h. AChE activity in haemolymph of M. edulis was highly variable and bore no relationship to either sublethal effects or lethality over the range 0.003–0.03 mg/l chlorfenvinphos. In comparison, concentration dependent inhibition was evident for each of the remaining biomarkers (phagocytic activity, spontaneous cytotoxicity, neutral red retention time, total haemolymph protein). Mussels at the highest exposure concentration showed visual signs of neurotoxicity (impaired neuromuscular control). Haemocyte phagocytic activity and spontaneous cytotoxicity responses were highly sensitive to chlorfenvinphos with significant modulation evident after 24 h exposure to environmentally realistic concentrations of 0.007 mg/l ( P=0.0003). Thus the immune function and well being of the mussels was significantly impacted in the absence of measurable inhibition of haemolymph AChE.

Differential sensitivity of three marine invertebrates to copper assessed using multiple biomarkers

Understanding how biomarkers relate to each other on exposure to particular contaminants in different species is key to their widespread application in environmental management. However, few studies have systematically used multiple biomarkers in more than a single species to determine the variability of sublethal effects of a particular contaminant. In this study, three marine invertebrates, the shore crab Carcinus maenas, the common limpet Patella vulgata and the blue mussel Mytilus edulis, were exposed over 7 days in the laboratory to environmentally realistic concentrations of the priority pollutant copper. A combination of molecular, cellular and physiological biomarkers was measured in each organism to detect the toxic effects of copper. Biomarkers included lysosomal stability (neutral red retention), neurotoxicity (acetylcholinesterase activity), metabolic impairment (total haemolymph protein), physiological status (heart rate) and induction of protective metallothionein proteins. P. vulgata was the most sensitive to copper with significant effects measured in all biomarkers at concentrations of 6.1 μg Cu l −1. In C. maenas, cellular and neurotoxic endpoints were affected significantly only at 68.1 μg Cu l −1. Exposure to copper also induced metallothionein production in crabs. Over a 7-day exposure period, M. edulis was the most tolerant species to copper with significant effects being observed at the cellular level only at 68.1 μg Cu l −1. In all three species, cellular and neurotoxic pathways were more sensitive to disruption than physiological processes (protein and heart rate). Results illustrate how a suite of biomarkers applied to different sentinel species can provide a ‘diagnosis of stress’, whereby, effects at the molecular level can be used to interpret the level of physiological impairment of the organism.

The role of phenoloxidase suppression in QX disease outbreaks among Sydney rock oysters (Saccostrea glomerata)

Abstract The paramyxean protozoan, Marteilia sydneyi , is the etiological agent of QX disease in Sydney rock oysters ( Saccostrea glomerata ). QX disease affects the farming of oysters in Queensland and on five rivers in New South Wales (NSW), Australia. Disease outbreaks occur during summer months (January to April) and are associated with high mortality rates (up to 98%), which limit the growing out period for oyster production. This study investigated the relationship between oyster host defense systems and QX disease. Oysters from the same brood stock were harvested from QX prone and QX free growing areas over the course of the 2000–2001 outbreak. A variety of parameters—infection intensity in the digestive diverticulum, total hemolymph protein content, condition index and phenoloxidase (PO) activity—were measured in these oysters. Phenoloxidase activity, which is often associated with host defense, was significantly suppressed in oysters from the QX prone area when compared to those from the QX free river. There was also a strict negative correlation between phenoloxidase activity and the intensity of parasitic infection ( p =0.0038). In contrast, neither total protein content nor condition indexes differed between oysters from the QX prone and QX free rivers. These data suggest that inhibition of prophenoloxidase cascade may facilitate lethal infection by M. sydneyi .

Effects of dissolved oxygen on hemolymph parameters of freshwater giant prawn, Macrobrachium rosenbergii (de Man)

Abstract Profiles of changes in physiological parameters of freshwater giant prawns, Macrobrachium rosenbergii, exposed to various dissolved oxygen (DO) levels of 7.75, 4.75, 2.75, and 1.75 mg l−1 are reported. The parameters involved in osmoregulation and oxygen transport were monitored for a 6-day period. Notable depressions in hemolymph osmolality, Na+, K+, and Cl− contents were observed within 24 h after exposure to hypoxia at 2.75 and 1.75 mg O2 l−1, and thereafter remained at rather steady levels, which were significantly lower than those under normoxic conditions (4.75–7.75 mg O2 l−1). The extent of depression of osmotic-related constituents, hemolymph osmolality and Cl− in particular, increased with decreased DO. Oxyhemocyanin constituted 65.46–65.84% of total hemolymph proteins under the various DO levels examined; both hemolymph oxyhemocyanin and proteins showed notable elevations 24 h after exposure to hypoxic conditions, and reached the highest and constant level by 48 h after exposure. The compensatory responses of prawns to reduced O2 were manifested by increased O2 uptake through augmentation of hemocyanin, which results in enhancement of oxygen binding capacity of the hemolymph. In the same period, a significant surge of the respiratory products, PCO2 and HCO3−, was also demonstrated 6 h after exposure to hypoxic conditions which resulted in hemolymph alkalosis. These processes likely resulted in an increase in water influx and consequent declines in hemolymph osmolality and ion composition. Furthermore, hyperventilation and respiratory alkalosis, indicated by increased oxyhemocyanin and pH, respectively, were found to be predominant responses of M. rosenbergii to hypoxic stress.

Maximum titers of vitellogenin and total hemolymph protein occur during the canalized phase of grasshopper egg production.

Many organisms exhibit developmental plasticity only in sensitive phases and cannot respond to environmental perturbations at other times. However, we know little about the physiological events that define plastic and canalized phases. During egg production in insects, vitellogenin (Vg) accumulates first in the hemolymph and then in the eggs. In addition, storage proteins may be important resources for egg production. Therefore, we tested hypotheses on the relationships of Vg and TP (total hemolymph protein minus Vg) titers to the transition from flexible to inflexible development during egg production. In lubber grasshoppers, approximately 70% of TP is contained in three proteins that range from 68 to 83 kDa. We maintained females on food treatments that produced defined plastic and canalized periods, collected hemolymph every approximately 4 d, and determined the ages at which oviposition and the maximum Vg and TP titers occurred. Both Vg(max) titer and especially TP(max) titer were predictors of the number of eggs produced. The time from eclosion to Vg(max) was significantly affected by diet, but the time from Vg(max) to oviposition was not. Similarly, the time from eclosion to TP(max) was significantly affected by diet, while the time from TP(max) to oviposition was not. Hence, Vg(max) and TP(max) are physiological landmarks that occur during the canalized phase of egg production.

Response of the tick Dermacentor variabilis (Acari: Ixodidae) to hemocoelic inoculation of Borrelia burgdorferi (Spirochetales).

When Borrelia burgdorferi B31 low passage strain spirochetes were directly injected into the hemocoel of Dermacentor variabilis (Say) females, the bacteria were cleared from the hemocoel within < 24 h. Viable spirochetes were not found in hemolymph, salivary gland, or ovary tissues by subculturing or by IFA. The hemocyte population increased approximately 6 times within the first 6 h after inoculation compared with the uninoculated controls. In contrast, the soluble total hemolymph protein content decreased inversely with the increase in hemocytes. Borreliacidal activity was demonstrated with cell-free hemolymph from D. variabilis. In vitro antimicrobial assays using hemolymph from borrelia-challenged and nonchallenged ticks resulted in 72% spirochete reductions compared with only 11.5%, respectively, within 1 h. Additional evidence of induced antimicrobial hemolymph protein activity was demonstrated by SDS-PAGE, which revealed upregulation of a lysozyme-like peptide (approximately 15 kDa) (22% increase) and the induction of a approximately 5.8 kDa peptide in the B. burgdorferi-challenged ticks. In contrast with the nonvector borne Bacillus subtilis, D. variabilis presented a rapid and robust response to challenge with cultured B. burgdorferi spirochetes and lead to their early elimination. The role of the tick immune system, including possible differences between vector and nonvector ticks, in determining the success of invasive bacteria is discussed.

Molecular and physiological responses in shore crabs Carcinus maenas following exposure to copper

The effects of acute (24 h) and chronic (four weeks) copper exposure and subsequent recovery in ‘clean’ conditions on cardiac activity were examined in shore crabs ( Carcinus maenas). Cardiac activity was monitored by Automated Interpulse Duration Assessment (AIDA) which enables detection of irregularities in cardiac rhythms in selected invertebrates. Mean interpulse durations, maximum interpulse durations and coefficients of variation were significantly affected by exposure to copper for 24 h and four weeks ( p < 0.05). Recovery of normal cardiac activity was evident following a six week recovery period in ‘clean’ conditions. However, high levels of mortality were seen among crabs exposed to the two highest copper concentrations (0.6 and 0.8 mgl −1 Cu).The extent of recovery was assessed by subjecting the surviving crabs to a physiological challenge. There were no apparent differences in circulatory response to physiological challenge between crabs previously exposed to copper and control crabs ( p > 0.05). Gill copper concentrations were significantly elevated in crabs that had previously been exposed to copper, despite their return to ‘clean’ seawater for six weeks ( p < 0.01). Midgut gland wet weight/dry weight (WW/DW) ratios, haemolymph total protein and midgut gland metallothionein concentrations did not reflect the previous copper exposure. The findings of the present study indicate that AIDA may be a valuable tool for use in toxicity testing.

Automated interpulse duration assessment (AIDA) in the shore crab Carcinus maenas in response to copper exposure

The effects of acute (24 h) and chronic (4␣weeks) copper exposure and subsequent recovery in “clean” conditions on cardiac activity were examined in shore crabs ( Carcinus maenas). Cardiac activity was monitored by automated interpulse duration assessment (AIDA) which permits detection of irregularities in cardiac rhythms in selected invertebrates. Mean interpulse durations, maximum interpulse durations and coefficients of variation were significantly affected by exposure to copper for 24 h and 4 weeks. Mean interpulse durations decreased reflecting an increase in heart rate, whereas maximum interpulse durations and coefficients of variation increased with increasing exposure concentration. Recovery of normal cardiac activity was evident following a 6-week recovery period in “clean” conditions. However, high levels of mortality were seen among crabs exposed to the two highest copper concentrations (0.6 and 0.8 mg l−1 Cu). The extent of recovery was assessed by subjecting the surviving crabs to a physiological challenge. There were no apparent differences in circulatory response to physiological challenge between crabs previously exposed to copper and control crabs. Gill copper concentrations were significantly elevated in crabs that had previously been exposed to copper, despite their return to “clean” sea water for 6 weeks. Midgut gland wet weight/dry weight ratios, haemolymph total protein and midgut gland metallothionein concentrations did not reflect the previous copper exposure. The findings of the present study indicate that AIDA may be a valuable tool for use in toxicity testing.

Effects of ultralow doses of fenoxycarb on juvenile hormone-regulated physiological parameters in the silkworm,Bombyx mori L.

Effects of fenoxycarb at ultralow doses were investigated on juvenile hormone (JH)–regulated parameters in the silkworm, B. mori. Like JH, this non-terpenoid carbamate is able to induce permanent larvae in the last larval instar. However, whereas micrograms of JH are needed to produce this effect, only a few picograms of fenoxycarb are necessary to induce the same effect. The effects of fenoxycarb observed in this study were only visible from day 4 of the last larval instar—that is, when the JH titer has dropped to undetectable levels and JH-repressed physiological parameters would naturally be expressed. We observed that the permanent larvae induced with low doses of fenoxycarb (100 pg/larva) had no 20-hydroxyecdysone (20E) peak. Their prothoracic glands (Pgs) were completely inactive and very weakly sensitive to prothoracicotropic hormone (PTTH). Fenoxycarb at doses of 1 ng/larva also significantly inhibited silk gland growth and coloration, whereas carotenoid content of the hemolymph was maintained at high levels, which could reflect an inhibition of its uptake by the silk glands. Total hemolymph protein levels in last instar larvae were also depressed at these doses. So, it seems that low doses of fenoxycarb are sufficient to maintain in a juvenilized status the physiological parameters that are normally expressed when JH titer has declined. Moreover, from an endocrinological viewpoint, we demonstrated that the corpora allata (CA) are not necessary for fenoxycarb to induce those effects and discussed its possible mode of action. Arch. Insect Biochem. Physiol. 37:178–189, 1998. © 1998 Wiley-Liss, Inc.

Changes in protein content and free amino acid composition in metal-contaminated gypsy moth larvae ( Lymantria dispar L., Lymantriidae, Lepidoptera)

Larvae of Lymantria dispar (Lymantriidae, Lepidoptera) were either reared on oak foliage or on an uncontaminated and metal-contaminated wheat germ diet from hatching to the end of experiment. Cd, Pb, Cu and Zn were separately applied, each at two concentrations in the range of no-observed-effect concentrations recently published for this species. Hemolymph was collected from day 3 fourth instar larvae, and total protein content and free amino acid composition were determined along with total body protein content (TBP) of the corresponding larvae. Clear diet-dependent differences were observed: total protein content of the larvae and of the hemolymph was higher in foliage-fed (281.2 μg/mg; 13.6 mg/ml) than in diet-fed control individuals (200.5 μg/mg; 10.5 mg/ml), whereas the opposite applied to the total free amino acid (TFAA) concentration (64.78 mM, 108.81 mM). Chronic exposure to Cd, Cu and Zn in the diet generally resulted in a decrease of total hemolymph protein but enhanced TFAA concentrations and TBP compared with artificial-diet-fed control group. Sixteen free amino acids were identified and quantitated by high-performance liquid chromatography. Concentrations of single amino acids and/or their relative abundance depended on the diet and the metal applied. For example, the concentration of histidine, the most abundant amino acid (40–53% of total amino acids), was lower in the oak-leaf-fed group (26.04 mM, 40.8% of total amino acids) than in the wheat germ control group (47.89 mM, 44%), but in most metal-contaminated specimens histidine increased significantly (56.58–69.78 mM, 46–53%). The phenylalanine concentration was not diet dependent but was the only amino acid elevated in all contaminated groups.

A non-sex-specific hemolymph lipoprotein from the white shrimp Penaeus vannamei boone. Isolation and partial characterization

A procedure was designed to isolate a lipoprotein (Lp) from the hemolymph of juveniles of the white shrimp, Penaeus vannamei Boone, using two high-speed ultracentrifugation steps. The lipoprotein fraction presented a buoyant density of 1.1-1.17g/ml with a peak at 1.14 g/ml. The isolated Lp corresponds to 0.93 and 0.73% of the total female and male hemolymph protein content, respectively. The Lp from males and females consisted of an apoprotein with an apparent molecular weight of 98 kDa as was shown by electrophoresis under denaturing and reducing conditions and it is a glycoprotein that contains mannose. In the hemolymph of both males and females one main native form of the Lp was found. The isoelectric point of the Lp was 5.81, but minor forms of different isoelectric points were also found.

Rapid and efficient isolation of transferrin and ferritin from Manduca sexta

We report methods for the rapid purification of two iron-binding proteins from larval hemolymph of Manduca sexta. Ferritin was purified in two steps by density gradient ultracentrifugation. To accomplish this, we utilized the relatively high level of ferritin present in the hemolymph of this animal and augmented the density of the protein in vivo by injection of iron sulfate. Nitrocellulose blots analyzed by laser densitometry showed hemolymph from iron-injected insects contained about 0.4 mg of ferritin per ml (approximately 0.7% of total hemolymph protein); of this, 62% was found as pure ferritin in the pellet formed during ultracentrifugation. Following the density ultracentrifugation, we purified transferrin from the hemolymph subphase by immobilized metal ion affinity chromatography using a new gel, Novarose-SE1000/40 coupled to dipicolylamine (DPA) chelated with nickel. Higher capacity Ni 2+DPA-gel permitted good resolution of transferrin in the first chromatography; a lower capacity of the same gel allowed purification of transferrin in a second step. Overall transferrin recovery was 52%. Larval hemolymph contained 0.770 mg transferrin/ml, representing about 1.3% of the total protein.

Polydnavirus of Microplitis croceipes prolongs the larval period and changes hemolymph protein content of the host, Heliothis virescens

AbstractPolydnaviruses from certain parasitoid Hymenoptera have been reported to interfere with both host immunity and host development. Heliothis virescens larvae injected with either calyx fluid or sucrose gradient‐purified polydnavirus from Microplitis croceipes (McPDV) gained less weight than saline‐injected larvae. The active feeding portion of the fifth stadium larva (time to reach the burrowing‐digging stage) was doubled (7.0 vs. 3.4 days) when a 0.25 wasp equivalent (WE) of sucrose gradient‐purified McPDV was injected into a newly ecdysed fifth stadium host. Many of the treated larvae were unable to pupate, successfully and died at a point of incomplete larval‐pupal ecdysis. Pupae that did result from the treated larvae weighed significantly less than controls, even at 0.025 WE. The rate of weight gain and extent of delay of development were dose‐dependent; as little as 0.1 WE extended the time of active feeding by 1.5 days and yielded only 25% adults. A 0.05 WE dose yielded 78% adults compared to 95% for controls. The total protein content of hemolymph from individuals injected with McPDV was significantly less than that of controls at any McPDV dose equal to or greater than 0.1 WE. SDS‐PAGE profiles of hemolymph proteins from control and McPDV‐injected larvae revealed a marked inhibition of the normal accumulation of storage proteins during the fifth stadium and a lesser reduction of serine protease inhibitor protein. Thus, McPDV‐injected larvae exhibited some symptoms (less total hemolymph protein and reduced amounts of storage protein) similar to those shown by both parasitized larvae and by larvae injected with M. croceipes teratocytes. However, McPDV affected development during the active feeding stage of the larva, while teratocytes primarily impacted larvae at the time when larval‐pupal transformation processes are initiated. © 1995 Wiley‐Liss, Inc.

Juvenile hormone regulation of hemolymph juvenile hormone binding protein in the black strain of the tobacco hornworm, Manduca sexta

AbstractNumerous studies have demonstrated regulation of specific lepidopteran proteins by pharmacological doses of insect juvenile hormone (JH). In this study, topical application of a 1 pg dose of JH I to fourth stadium larvae of the black (bl) mutant strain of the tobacco hornworm, Manduca sexta, induced a 50% increase in the titer of hemolymph juvenile hormone binding protein (hJHBP). Radioimmunoassay confirmed that JH titers were lower in bl larvae than in wild‐type larvae at the time of JH treatment. Enzyme immunoassay analysis of hJHBP titers demonstrated that regulation by JH I was dose‐dependent at doses up to 10 pg and that the response was saturated above 100 pg. Western blotting and equilibrium dialysis confirmed these results and demonstrated that hJHBP from bl larvae had the same molecular mass and displayed the same affinity for JH I as hJHBP isolated from wild‐type larvae. Time course studies showed that regulation was complex: 1 2 h after JH I treatment, hJHBP titers were twofold lower in treated than in control bl larvae, while 44 h after treatment they were twofold higher. JH I regulation of hJHBP titers in bl larvae was independent of changes in total hemolymph protein. © 1995 Wiley‐Liss, Inc.

An Abundantly Expressed Hemolymph Glycoprotein Isolated from Newly Parasitized Manduca sexta Larvae Is a Polydnavirus Gene Product

Cotesia congregata polydnavirus (CcPDV) is essential for successful parasitism of Manduca sexta larvae by the braconid wasp C. congregata. CcPDV virions are present in large numbers in the oviducts of C. congregata and injected with eggs into the hemocoel of M. sexta larvae during parasitization. Injection of sucrose density purified virions into nonparasitized larvae causes several of the parasitism-induced alterations in the physiology of host larvae that occur as a result of natural parasitism, including the synthesis of novel hemolymph proteins and abrogation of the host's immune response against the developing parasites. One of these proteins, early-expressed protein 1 (EP1), is a 190-kDa molecule which constitutes up to 5% of the total hemolymph protein by 24 hr following oviposition by the wasp. Using N-terminal sequence data for EP1 to construct primers for use in the polymerase chain reaction, we amplified and cloned a cDNA corresponding to the gene encoding EP1. This cDNA hybridized to DNA of the CcPDV genome, but not to DNA isolated from M. sexta larvae, suggesting that EP1 is a CcPDV gene product. A cDNA clone was isolated from an expression library generated from RNA extracted from newly parasitized M. sexta larvae. Sequence analysis of the cDNA clone revealed the presence of an open reading frame of 819 bp encoding a protein of 30.7 kDa. In vitro transcription/translation of the cDNA clone produced a protein of approximately 31 kDa, which was immunoprecipitated by EP1-specific polyclonal antiserum generated against purified deglycosylated EP1. EP1-like sequences also were amplified from male wasp genomic DNA, suggestive of integration of EP1-like sequences in the genome. This report constitutes the first evidence that a specific protein isolated from a parasitized host insect is a wasp polydnavirus gene product.

The effects of Onchocerca lienalis infection on vitellogenesis in the British blackfly, Simulium ornatum.

We have previously described the major yolk protein, vitellin, in the British blackfly Simulium ornatum Meigen. Here we demonstrate that vitellogenin, synthesized in the fat body and secreted into the haemolymph, is composed of subunits with the same approximate molecular weight as vitellin, namely 200 and 68 kDa. Simulium ornatum is the natural vector for the cattle filarial nematode Onchocerca lienalis Stiles, which induces host fecundity depletion. A significant reduction in ovarian vitellin content was associated with infection by intrathoracic injection of 20 O. lienalis microfilariae immediately after blood-feeding. Fat body synthesis of vitellogenin was significantly reduced as early as 8 h post-infection in comparison with sham-injected flies. When total haemolymph protein from infected and sham injected flies was compared, titres were significantly depressed 6 h post-infection. However, later in the infection, titres were elevated by 30%, the major component being vitellogenin. The injection of dead microfilariae had no effect. An infection burden of a single parasite caused a significant reduction in ovarian protein content in comparison with shams, but no further significant decrease was observed as the parasite burden was increased from 5 to 20. Possible mechanisms underlying the disturbance of Simulium reproductive physiology are proposed.