Cyanoprotein: Developmental stage,sex and diapause-dependent expression,and synthesis regulation by juvenile hormone in the bean bug,Riptortus clavatus

Cyanoprotein (CP) synthesis was studied in nymphal and nondiapause adult, diapause adult, and juvenile hormone (JH) treated adult bean bugs, Riptortus clavatus. Hemolymph collected from bugs injected with [³⁵S]-methionine was analyzed by native polyacrylamide gel electrophoresis (PAGE) and fluorography, and CP synthesis was also determined quantitatively by rocket immunoelectrophoresis of hemolymph and counting. In the nymphal stages synthesis of CP-1, 2, 3, and 4 (with CP-4 synthesis predominant) reached a maximum at mid-instar and was not detected at each ecdysis, so that the synthetic activity of CPs changed cyclically in each instar. During the nymphal stages CP synthesis showed the same pattern and level in both females and males, and both diapause and nondiapause oriented bugs. In the adult stage, however, CP synthesis differed in the two sexes and nondiapause or diapause conditions. In nondiapause males CP synthesis was not detected in the adult, but in nondiapause females CP (only CP-1) was synthesized through the reproductive stages. CP-1 accumulated in the egg yolk together with vitellogenin. In diapause adults (both females and males) CP-1 to 4 were synthesized at a very low rate for over 2 months and accumulated in the hemolymph. JH or JH analog (JHA) methoprene and also long day condition, which terminate diapause, switched the main CP synthesis CP-4 to CP-1 in females. CP synthesis in diapause males stopped after JH(A) treatment. The activities of CP synthesis, thus, changed in developmental stages, sexes, and diapause. This is an excellent system for study of specific gene expression and switching controlled by insect hormones and sex. © 1992 Wiley-Liss, Inc.     

Cyanoprotein: Immunological properties and content changes during the development of non-diapause female bean bugs,Riptortus clavatus

Immunological properties and content changes of cyanoprotein (CP) were investigated in the bean bug, Riptortus clavatus. Anti-CPegg serum was prepared by immunizing a rabbit with CP purified from eggs (CPegg). In the Ouchterlony double diffusion test, the precipitin line between CPegg and anti-CPegg serum fused with that of non-diapause and diapause female hemolymph and anti-CPegg serum. Rocket immunoelectrophoresis (RIE) using anti-CP serum showed two types of rockets (A and B) depending on the samples. Namely, CPegg and non-diapause female adult hemolymph formed A rockets (heavy-stained with Coomassie Brilliant Blue) and early diapause female adult hemolymph formed B rockets (light-stained), but hemolymph from fifth instar nymphs formed both A and B rockets. Both rockets A and B were demonstrated by SDS-PAGE analysis of the precipitin lines to be formed from the same CP subunit (MW, 76,000). CP-1, 2 and 3 bands from native PAGE of nymphal hemolymph formed A rockets and CP-4 formed B rockets. The contents of CP-A (CP-1 to 3) and CP-B (CP-4) were separately determined by measuring the sizes of rocket A and B. CP-A and CP-B content were demonstrated to increase during the development of the last instar nymph and decrease at adult emergence by RIE analysis of non-diapause female whole body extracts. CP-B is predominant in the nymphal stage. In the early adult stage (day 2 and 3 after emergence), neither CP-A nor CP-B were detected. Only CP-A appeared again at day 4 after emergence and increased during development and vitellogenesis of non-diapause females.     

Xenobiotic absorption and binding by proteins in hemolymph of the weevil Diaprepes abbreviatus

A synthetic coumarin, 7-amino-3-phenyl coumarin (coumarin-10), was used to study the uptake of ingested xenobiotics into hemolymph. Larvae were forcefed coumarin-10 in peanut oil, and hemolymph was extracted and analyzed by fluorescence spectroscopy. Coumarin-10 entered hemolymph within 5 min, reaching a steady state of concentration within 1 h. Assayed 2 h after feeding, hemolymph titers of 1–5 ng/μl were proportional to log dose between 10 and 100 ng/mg body weight; hemolymph did not reach saturation. Fluorescence spectra of hemolymph in saline revealed that energy was readily transferred from hydrophobic residues of hemolymph proteins to coumarin-10. Ultracentrifugal density gradients revealed that 94% of absorbed coumarin-10 was bound to sedimenting proteins while 6% bound to lipophorin. Native polyacrylamide gel electrophoresis (N-PAGE) on minigels identified two major proteins responsible for binding. Though readily separated by native electrophoresis, these proteins were not fully separable by HPLC using a wide variety of columns. Gel permeation-HPLC of the sedimenting proteins from hemolymph revealed a single major peak of 480,000 M_r. When upper and lower electrophoretic bands were isolated by preparative N-PAGE, the upper band (band I) yielded subunits of 75,000 and 71,000 M_r, while the lower band (band II) yielded only one size subunit of 75,000 M_r on denaturing (SDS) PAGE. The fluorescent products bound by sedimenting proteins were identified by thin-layer chromatography and scanning fluorescence densitometry as coumarin-10 (80% of total) and a polar metabolite (20%). In addition, lipophorin-containing fractions contained an apolar metabolite (3% of total fluorescence). In vitro binding studies utilizing fluorescent energy transfer demonstrated saturation binding with a K_D of 1.5 μM.     

Molecular Weight Determination of Gliadin Fractions in Gel Filtration by SDS-Polyacrylamide Gel Electrophoresis and Sedimentation Equilibrium

Gliadin was fractionated into three fractions; ω-gliadin, Fraction III (γ-gliadin) and Fraction IV (α- and β-gliadin). The determination of the molecular weights (MW) of the three fractions was performed by both SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and sedimentation equilibrium. In SDS–PAGE, ω-gliadin gave three bands (MW 50,000, 54,000 and 64,000), Fraction III two bands (MW 38,000 and 46,000) and Fraction IV two bands (MW 33,000 and 38,000), The sedimentation analysis showed that each fraction was fairly homogeneous relative to molecular weight. The molecular weights obtained by sedimentation were 28,000 for Fraction III and 27,000 for both Fraction IV and ω-gliadin. The disagreement in molecular weight between sedimentation and gel electrophoresis was discussed.     

Properties and Subunit Characterization of Affinity Purified Sophora Japonica Lectin

The affinity purified Sophora japoniea lectin exhibits an anomalous behavior on polyacrylamide gel electrophoresis (PAGE). Electrophoresis at pH 8.9 produces three protein staining bands. Extraction and re-electrophoresis of the fastest and slowest migrating components demonstrates that the lectin solution is an equilibrium mixture of interconvertible forms. Addition of a bindable saccharide, D-galactose, during PAGE causes the equilibrium to be shifted toward a single form. As indicated by analytical gel filtration, sedimentation velocity ultracentrifugation and ion-exchange chromatography experiments, the equilibrium mixture consists of charge and not molecular weight variants of the native molecule of 132,800 g/m. Results from end-group and cysteine analyses and PAGE in sodium dodecyl sulfate indicate that the native lectin is composed of the non-covalent association of two dissimilar subunits. One subunit consists of two identical polypeptide chains attached by two disulfide bonds and the other subunit of two identical polypeptide chains stabilized by a single cysteine bridge.     

Tissue distribution and characterization of predominant hemolymph carrier proteins from Dermacentor variabilis and Ornithodoros parkeri

The tissue distribution of the predominant hemolymph protein found throughout tick development was examined in the hard tick, Dermacentor variabilis, and in the soft tick, Ornithodoros parkeri. In D. variabilis, the predominant (purified) hemolymph protein was a lipoglycoheme-carrier protein (DvCP) with a molecular weight of 200 K. A protein with a similar mobility on native-PAGE was found in fat body, salivary gland, muscle and ovary from partially fed females which was most abundant in the plasma and salivary gland. DvCP from plasma, salivary gland and fat body of partially fed females consisted of two subunits on SDS-PAGE (98 and 92 K). In replete females, only salivary gland exhibited protein subunits equivalent to hemolymph CP. CP in salivary gland and fat body stained positive for lipids. The concentration of CP in tissues varied between partially fed and replete females, indicating a difference in the expression and/or sequestration of CP during adult development. The predominant hemolymph carrier protein from O. parkeri (OpCP) was purified to homogeneity for the first time and is presumed to have similar functions to CP from D. variabilis. Purified OpCP exhibited a molecular weight of 668 K by native-PAGE. Unlike CP from D. variabilis, OpCP was not detected in fat body or salivary gland tissues but occurred abundantly in coxal fluid. By SDS-PAGE, purified hemolymph OpCP consisted of two major subunits (114 and 93 K) and a less abundant protein with an apparent molecular weight of 48 K. Purified native OpCP was a lipoprotein like DvCP. A spectral analysis of purified OpCP failed to demonstrate the presence of heme like that found for CP from D. variabilis, purified by the same methods. However, plasma from O. parkeri contained heme with a λ_max of 410 nm.     

Characterization of vitellin from the ovaries of the banana shrimp Litopenaeus merguiensis

Vitellin (Vt) was purified from ovary extracts of mature females of the banana shrimp Litopenaeus merguiensis using DEAE-Sephacel and Superdex 200 columns. Native Vt had an apparent molecular mass of 398 kDa as determined by native PAGE and by gel filtration chromatography. Under reducing and denaturing conditions (SDS-PAGE), Vt is composed of two major subunits of 87 and 78 kDa, although some faint bands were also detected. The N-terminal 10 amino acids sequence of the 78 kDa subunit is identical to that of Litopenaeus vannamei Vt and very similar to that of Litopenaeus japonicus vitellogenin (Vg) as well as Litopenaeus semisulcatus Vt, with an identity of 89%. Anti-Vt polyclonal antibody raised against purified Vt shows a high specificity with only ovarian Vt and hemolymph Vg of vitellogenic shrimps in double immunodiffusion and Western blot assays. Vg and Vt concentrations in hemolymph, hepatopancreas and ovaries were measured by ELISA. Vg concentrations increased in the hemolymph in the early stages of ovarian development and declined in the maturation stages. As there were undetectable concentrations of Vg in the hepatopancreas while an elevation of Vg levels occurred in the hemolymph, during the time that Vt was accumulating in the ovaries during oogenesis, this would suggest that the contribution of Vg synthesized by the hepatopancreas only might be not sufficient for adequate development of the oocytes in the banana shrimp L. merguiensis during vitellogenesis.     

Purification and Characterization of Acid Phosphatase from the Egg of the Lady Beetle, Harmonia axyridis (Coccinellidae: Coleoptera)

Acid phosphatase (AP) in the egg of the lady beetle, Harmonia axyridis, was purified and characterized. Ammonium sulfate precipitation, CM column and isoelectrofocusing (IEF) were applied to purify an estimated molecular weight of 66 kDa AP. The purity was checked by SDS PAGE, native PAGE and Western blot. AP was detected in the hemolymph of the female and the egg, but not in the male on the blotting. Km of AP for a substrate, p‐nitrophenyl phosphate (p‐NPP), was 1.64 x 10^‐4 M. AP had the optimum enzymatic activity at pH 3.5. In inhibition tests performed with various chemicals, ammonium molybdate suppressed 99% of the enzyme activity of AP even at the concentration of 5 x 10^‐4 mM. AP was stable up to 50°C.     

Malate dehydrogenase from Chlorobium vibrioforme, Chlorobium tepidum, and Heliobacterium gestii: purification, characterization, and investigation of dinucleotide binding by dehydrogenases by use of empirical methods of protein sequence analysis.

Malate dehydrogenase (MDH; EC 1.1.1.37) from strain NCIB 8327 of the green sulfur bacterium Chlorobium vibrioforme was purified to homogeneity by triazine dye affinity chromatography followed by gel filtration. Purification of MDH gave an approximately 1,000-fold increase in specific activity and recoveries of typically 15 to 20%. The criteria of purity were single bands on sodium dodecyl sulfate (SDS) and nondenaturing polyacrylamide electrophoresis (PAGE) and the detection of a single N terminus in an Edman degradation analysis. MDH activity was detected in purified preparations by activity staining of gels in the direction of malate oxidation. PAGE and gel filtration (Sephadex G-100) analyses showed the native enzyme to be a dimer composed of identical subunits both at room temperature and at 4 degrees C. The molecular weight of the native enzyme as estimated by gel filtration was 77,000 and by gradient PAGE was 74,000. The subunit molecular weight as estimated by SDS-gradient PAGE was 37,500. N-terminal sequences of MDHs from C. vibrioforme, Chlorobium tepidum, and Heliobacterium gestii are presented. There are obvious key sequence similarities in MDHs from the phototrophic green bacteria. The sequences presented probably possess a stretch of amino acids involved in dinucleotide binding which is similar to that of Chloroflexus aurantiacus MDH and other classes of dehydrogenase enzymes but unique among MDHs.     

Physicochemical study of rock crab lipoproteins

Physicochemical studies have been carried out on the hemolymph and egg lipoproteins of the rock crab (Cancer antennarius). Analytical ultracentrifugal analyses of vitellogenic female HDL₃ revealed the presence of two types of lipoproteins. The first with a sedimentation rate of 5.35 S was comparable to lipoproteins in male and non-vitellogenic female hemolymph. The second with a sedimentation rate of 10.74 S was comparable to the major lipoprotein of egg yolk. A similar comparison could be made following electrophoretic analyses in native polyacrylamide gels. Electrophoresis in SDS-polyacrylamide gels revealed three major apolipoproteins common to egg and vitellogenic HDL₃. A fourth apolipoprotein was found in both male and female HDL₃. In contrast to mammalian HDL, none of these crustacean apolipoproteins had a molecular weight less than 82000. One of these apolipoproteins appears to be comparable physicochemically to the enteric form of apolipoprotein B in mammals.     

Characterization of vitellogenin in the red imported fire ant, Solenopsis invicta (Hymenoptera: Apocrita: Formicidae)

Vitellin (VN) and vitellogenin (VG) profiles were analyzed in monogyne and polygyne colonies of the red imported fire ant, Solenopsis invicta. Non-denaturing and SDS-polyacrylamide gel electrophoresis (PAGE) analyses indicated that the native VN was likely 350 kDa and comprised of two subunits in the molecular size range of 170-185 kDa. SDS-PAGE of hemolymph showed that the relative mobilities and subunit patterns of VG and VN were similar. VG was present in the hemolymph of reproductive queens; alate, virgin queens; and workers, but not in males. Anti-VN, prepared from polygyne egg homogenates, reacted with egg homogenates and with hemolymph VG from reproductive, monogyne and polygyne queens and alate, virgin polygyne queens. Analysis of circulating VG and ovarian development in alate, virgin queens showed that low levels of VG appeared by five days following adult eclosion, but egg development was not observed until seven weeks. VG was evident in newly inseminated queens, and increased steadily for the first three weeks following dealation. VG levels declined slightly near eclosion of the first workers (= nanitics) and dropped sharply after nanitic emergence at five weeks following dealation. Oocyte maturation peaked at days 15-25 following dealation, but otherwise remained low but steady. These studies provide the basis for future investigations into endocrine regulations of vitellogenesis in S. invicta queens.     

The use of critical power as a determinant for establishing the onset of blood lactate accumulation

Eight highly trained male kayakers were studied to determine the relationship between critical power (CP) and the onset of blood lactate accumulation (OBLA). Four exercise sessions of 90 s, 240 s, 600 s, and 1200 s were used to identify the CP of each kayaker. Each individual CP was obtained from the line of best fit (LBF_CP) obtained from the progressive work output/time relationships. The OBLA was identified by the 4 mmol·l^–1 blood lactate concentration and the work output at this level was determined using a lactate curve test. This consisted of paddling at 50 W for 5 min after which a 1-min rest was taken during which a 25-l blood sample was taken to analyse for lactate. Exercise was increased by 50 W every 5 min until exhaustion, with the blood sample being taken in the 1-min rest period. The exercise intensity at the OBLA for each subject was then calculated and this was compared to the exercise intensity at the LBF_CP. The intensity at LBF_CP was found to be significantly higher (t=2.115, P<0.05) than that at the OBLA of 4 mmol·1^–1. These results were further confirmed by significant differences being obtained in blood lactate concentration (t=8.063, P<0.05) and heart rate values (t=2.90, P<0.05) obtained from the exercise intensity at LBF_CP over a 20-min period and that of the anaerobic threshold (Th_an) parameters obtained from the lactate/heart rate curve. These differences suggest that CP and Th_an are different physiological events and that athletes have utilised either one or the other methods for monitoring training and its effects.     

Purification and characterization of laccase from Monocillium indicum Saxena

Summary An ascomycete Monocillium indicum Saxena producing extracellular laccase was isolated. The culture filtrate on native polyacrylamide gel electrophoresis (PAGE) revealed four bands of activity, one of which was a major one. The major laccase band, a glycoprotein, was purified and characterized. Gel filtration chromatography showed that the relative molecular weight (M_r) of laccase was 100 000. On sodium dodecyl sulphate (SDS)-PAGE the major laccase band further resolved into three proteins of M_r 72 000, 56 000 and 24 000. The enzyme had a pH optimum of 3.0 and was active on a number of o-phenols and aromatic acids. The 72 000 M_r protein was found to share common immunological properties with laccases of Coriolus versicolor, Agaricus bisporus and lignin peroxidase of Phanerochaete chrysosporium. Correspondence to: K. Koteswara Rao     

Experimental Parameterisation of Principal Physics in Buoyancy Variations of Marine Teleost Eggs

It is generally accepted that the high buoyancy of pelagic marine eggs is due to substantial influx of water across the cell membrane just before ovulation. Here we further develop the theoretical basis by applying laboratory observations of the various components of the fertilized egg in first-principle equations for egg specific gravity (ρ_egg) followed by statistical validation. We selected Atlantic cod as a model animal due to the affluent amount of literature on this species, but also undertook additional dedicated experimental works. We found that specific gravity of yolk plus embryo is central in influencing ρ_egg and thereby the buoyancy. However, our established framework documents the effect on ρ_egg of the initial deposition of the heavy chorion material in the gonad prior to spawning. Thereafter, we describe the temporal changes in ρ_egg during incubation: Generally, the eggs showed a slight rise in ρ_egg from fertilization to mid-gastrulation followed by a gradual decrease until full development of main embryonic organs just before hatching. Ontogenetic changes in ρ_egg were significantly associated with volume and mass changes of yolk plus embryo. The initial ρ_egg at fertilization appeared significantly influenced by the chorion volume fraction which is determined by the combination of the final chorion volume of the oocyte and of the degree of swelling (hydrolyzation) prior to spawning. The outlined principles and algorithms are universal in nature and should therefore be applicable to fish eggs in general.     

Vitellogenin and egg production in the moth,Heliothis virescens

The characteristics of vitellogenin (Vg) and the relationship between Vg production and egg production in the tobacco budworm, Heliothis virescens, were studied. The relationship between Vg production and juvenile hormone (JH) and the impact of mating on Vg and egg production were also investigated. Vg appears in the hemolymph of H. virescens about 6 h after moth eclosion. Vg may be separated into two apoproteins (ApoVg-I and ApoVg-II) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights were calculated to be 156,065 ± 800 for ApoVg-I and 39,887 ± 323 for ApoVg-II. SDS-PAGE analysis revealed that the female hemolymph Vg polypeptides appear to be identical to those from eggs but are absent in male hemolymph. Vg concentration was significantly higher in mated females than in virgin females of the same age at 48 h after emergence. Rates of egg production increased as Vg production increased; rates of egg production in mated females were significantly higher than those of virgin females at 48, 72, 96, and 120 h postemergence. Vg production is dependent on JH, because hemolymph from decapitated females lacked Vg while that of decapitated females treated with synthetic JH had Vg at levels comparable to similarly aged, normal H. virescens females. Hemolymph JH titers in mated females were significantly higher compared with those in virgin females at all sampling periods. The high JH level in mated females may explain the high Vg and egg production in mated H. virescens. Arch. Insect Biochem. Physiol. 34:287–300, 1997. © 1997 Wiley-Liss, Inc.     

Identification of Contaminations Hiding Beneath the α- and β-Subunits of Partially Purified Nitrogenase MoFe Protein on the Sodium Dodecyl Sulfate Gel

To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, the partially purified MoFe protein （Avl） preparation was obtained from Azotobacter vinelandii Lipmann OP by chroma- tography on DEAE-cellulose （DE52） and Sephacryl S-200 columns and analyzed by PAGE and matrix- assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometry. The Av 1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Avl on the SDS gel. However, on the anoxic native PAGE, in addition to the Avl band, the preparation was shown to have three other main bands that migrated slower than Av 1. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Avl, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase （PGI）. In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av 1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av 1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaC1 concentration gradient. Thus, Avl with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.     

Purification of β-N-Acetylhexosaminidase from Egg White and the Microsomal and Lysosomal Fractions of Hen Oviduct

β-N-Acetylhexosaminidase (NAHase) was purified from egg white and the lysosomal and microsomal fractions of hen oviduct. The purification procedure included affinity chromatography using Sepharose 4B coupled with IgG specific for NAHase of hen oviduct. The isoelectric points of the three enzymes were different, but their antigen determinants were identical. In sodium dodecyl sulfate polyacrylamide gel electrophoresis, both the egg white and lysosomal enzyme gave only one protein band each, corresponding to a MW of 68000 and 53000, respectively, but the microsomal enzyme gave two protein bands, corresponding to those of the lysosomal and egg white enzymes.     

Identification,characterization, and developmental profile of a high molecular weight,juvenile hormone-binding protein in the hemolymph of the migratory grasshopper,Melanoplus sanguinipes

In the hemolymph of Melanoplus sanguinipes, a high molecular weight juvenile hormone binding protein (JHBP) was identified by photoaffinity labelling and found to have a M_r of 480,000. The JHBP, purified using native gel electrophoresis followed by electroelution, has an equilibrium dissociation constant for JH III of 2.1 nM and preferentially binds JH III over JH I. Antibody raised against JHBP recognized only the 480,000 band. Under denaturing conditions the native JHBP gave a single band with a M_r 78,000. The antibody against native JHBP recognized only the 78,000 protein in SDS-treated hemolymph samples, indicating that JHBP is a hexamer in this species. The concentration of JHBP fluctuates in both the sexes during nymphal and adult development in parallel with total protein content of hemolymph. © 1995 Wiley-Liss, Inc.     

Effects of precocene II on female-specific hemolymph polypeptides in Oncopeltus fasciatus

Using polyacrylamide gel electrophoresis (PAGE) under denaturing conditions, two major polypeptides of 200,000 and 170,000 daltons were detected in the hemolymph of mature female Oncopeltus fasciatus, but they were not found in the hemolymph of males or newly emerged females. Those polypeptides constituted the two major bands of early vitellogenic oocytes; however, they were absent from the yolk of mature eggs. The slower-migrating band (200,000 daltons) appears to correspond to a vitellogenic protein already identified in O. fasciatus, whose synthesis has been suggested to be independent of juvenile hormone (JH). Treatment of newly emerged adult females with the corpus allatum cytotoxin precocene II prevented the appearance of the female-specific bands and induced an important accumulation of other proteins in the hemolymph. Yolk deposition was also inhibited in those animals. Topical application of JH to precocene-treated females restored the appearance of the 200,000 and 170,000 dalton polypeptides in the hemolymph. These results suggest that JH is required for the synthesis of female-specific polypeptides in O. fasciatus.     

Effect of age on acetylcholinesterase and other hemolymph proteins in Aplysia

Summary Hemolymph was examined in young (ca. 86 days old), mature (ca. 163 days old), and old (ca. 294 days old) Aplysia for age-related changes in constituent proteins. In young, mature, and old animals protein concentrations were 1.6±0.27, 1.41±0.53, and 1.45±0.43 mg·ml^-1, respectively. The copper-containing respiratory protein, hemocyanin, measured by determining the copper concentration, was found to increase significantly from young (0.98±0.51 g·ml^-1) to mature (2.02±0.95 g·ml^-1) Aplysia, with little change between mature and old (1.92±0.43 g·ml^-1) animals. These findings were consistent with the results obtained when hemocyanin was directly measured by spectrophotometric absorption at 340 nm. Acetylcholinesterase (AChE) was present in the hemolymph of Aplysia. Its activity was highest in mature animals (3121±1627 units·mg^-1) and least in old animals (1463±599 units·mg^-1). Young animals had intermediate levels (2080±762 units·mg^-1). SDS-PAGE revealed a distinct pattern of protein bands for hemolymph from each age group; hemolymph from the young group contained six prominent protein bands with molecular weights (MW) from 13 to 300 kDa. Hemolymph of mature and old animals exhibited four and three prominent protein bands, respectively, with MW between 45 and 300 kDa. A prominent band at 97 kDa was present in samples from the mature group, but was faint in samples from the old group and absent in samples from the young group. Under non-denaturing conditions the hemolymph protein band patterns for each group differed from the others, thereby demonstrating that the age-dependent differences in the protein profiles are intrinsic to hemolymph in vivo. Isoelectric focusing of the hemolymph samples revealed that the proteins were all acidic (pI ca. 3.0–6.5). The hemolymph from the young differed from the other two groups in having an additional acidic protein (pI ca. 4.0). A possible link between age-related changes in hemolymph proteins and age-related changes in the nervous system is proposed.Abbreviations 2-ME 2-mercaptoethanol - AChE acetylcholinesterase - FMRFamide amidated tetrapeptide containing phenylalanine, methionine, arginine and phenylalanine - MW molecular weight - PAS periodic acid-Schiff - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TRIS tris-(hydroxymethyl)-aminomethane