Cell-free Hemolymph Research Articles

Naturally occurring agglutinins against trypanosomatid flagellates in the haemolymph of insects.

In vitro studies of the behaviour of the trypanosomatid flagellates Trypanosoma brucei and Leishmania hertigi in the presence of cell-free haemolymph of locusts, Schistocerca gregaria and cockroaches, Periplaneta americana revealed the presence of parasite agglutinins. The range of normal values of agglutination titres was 2(-4) to 2(-13). Physico-chemical treatment of haemolymph indicated that these agglutinins are protein or glycoprotein in nature and are only partially affected by heat treatment below 65 degrees C, at which temperature incubation of haemolymph for 30 min abrogated all agglutination. Agglutination was not dependent on the presence of Ca2+ or Mg2+. Prior injection of locusts and cockroaches with T. brucei and L. hertigi significantly increased agglutinin titres between Days 4 and 6 in cockroaches (P less than 0.05) and from Days 2 to 4 when L. hertigi was inoculated into locusts. The induced differences in titres observed in locusts infected with T. brucei were not significant. Lysozyme levels were significantly increased after inoculation of T. brucei into cockroaches compared with placebo-inoculated and uninoculated controls. L. hertigi inoculation produced significant increases in lysozyme levels compared with controls between Days 1 and 7 in locusts and 3 to 6 in cockroaches. These studies indicate that, at least in easily manipulated model systems, induced responses to intrahaemocoelic inoculation to trypanosomes and Leishmania can occur. As far as we are aware this is the first report of an induced response of an insect to such important parasites. The possibility that induced responses in natural vector to this parasites occurs requires investigation.

Schistosoma mansoni: Passive transfer of resistance by serum in the vector snail, Biomphalaria glabrata

Passive transfer of natural resistance to Schistosoma mansoni (PR-1 strain) has been successfully accomplished in the snail intermediate host, Biomphalaria glabrata (PR albino, M-line strain). Injection of serum (cell-free hemolymph) from a naturally schistosome-resistant strain of B. glabrata (10-R2) into PR albino snails induced a complete protection from a primary infection with the parasite in 29 of 48 snails (60.4%). In comparison, inoculation of homologous PR albino serum or heterologous proteins (fetal calf serum) had no effect. Moreover, this protection could be induced 24 hr prior to, or 24 hr after, exposure to the parasite, although heating of 10-R2 serum to 70 C for 30 min destroyed its protective ability. When in vitro transformed sporocysts were preincubated in 10-R2 or PR albino serum and then were injected into susceptible snails, a high level of infection (88.5 and 83.3%, respectively) was produced in both groups. Thus, the 10-R2 serum factor does not appear to be mediating specific parasite recognition by host hemocytes. Alternatively, our results suggest that 10-R2 serum possesses a heat-labile factor which specifically activate B. glabrata hemocytes to encapsulate and destroy sporocysts whereas PR albino serum lacks this factor.

The [formula omitted][formula omitted] generation of an antibacterial activity from the fat body and hemolymph of non-immunized larvae of [formula omitted][formula omitted

A state of immunity in Galleria mellonella against the pathogen Pseudomonas aeruginosa is known to be induced by the injection of lipopolysaccharide (LPS), isolated from the homologous organism. An in vitro mixture of the LPS and whole or cell-free hemolymph from non-immunized larvae is not antibacterial. In vitro mixtures of fat body and cell-free hemolymph from non-immunized larvae, incubated at 25°C for 20 hours generated a proteinaceous antibacterial activity. The generation of this activity was enhanced by the presence in the incubation mixture of LPS and/or hemocytes from non-immunized larvae. It is suggested that LPS causes the release of a hemocyte factor(s) which acts in conjunction with or directly on the fat body resulting in an enhanced production of antibacterial factors.

Lysosomal Enzyme Activities in Susceptible and Refractory Strains of Biomphalaria glabrata during the Course of Infection with Schistosoma mansoni

The distribution and abundance of the lysosomal enzyme markers, acid phosphatase (AP), peroxidase (PO), and nonspecific esterase (NE), within circulating blood cells (hemocytes) were examined in a schistosome-susceptible (PR albino M-line) and a resistant (10-R2) strain of Biomphalaria glabrata during the course of infection with Schistosoma mansoni. The dynamics of serum (cell-free hemolymph) AP activities and total hemocyte numbers in infected snails also were investigated. Hemocyte subpopulations, as determined by these enzyme markers, responded differently to parasite infection between snail strains. Generally, the hemocyte subpopulations within PR albino snails remained largely unchanged, whereas the same subpopulations in 10-R2 snails fluctuated considerably. The distribution of AP in the hemocytes of 10-R2 snails decreased by 1 hr postexposure (PE) to the parasite and remained low through 12 hr before increasing to control values at 24 hr and 2 wk PE. In comparison, PO activity increased by 1 hr PE and peaked at 12 hr before dropping to 0 hr values by 2 wk PE. The NE activity exhibited still another pattern with the percentage of NE-positive cells decreasing from 0 to 12 hr PE followed by a recovery to 0-hr values by 24 hr. The abundance of these hemocyte enzymes followed a similar pattern to that of their distribution, although some differences were observed. Serum AP values varied little in PR albino snails except for a significant increase at 2 wk PE, indicating a possible response to tissue damage resulting from migrating daughter sporocysts.(ABSTRACT TRUNCATED AT 250 WORDS)

Transfer of immunity against [formula omitted] P11-1 in [formula omitted] larvae

Larvae of Galleria mellonella may be immunized against Pseudomonas aeruginosa by the transfer of whole hemolymph, cell-free hemolymph or hemocytes from insects previously immunized with the lipopolysaccharide of the homologous organisms. Whole immune hemolymph injected as early as 3 hours after vaccination confers protection which persists as long as 40 hours. Hemocytes alone confer good protection when transferred as early as 30 minutes after, and up to about 4 hours after immunization. Transferred protection does not appear to be due to excess immunogen in the hemolymph.

Surface antigens of Biomphalaria glabrata (Gastropoda) hemocytes: Occurrence of membrane-associated hemolymph-like factors antigenically related to snail hemoglobin

A polyvalent antiserum (anti-H PR) generated in rabbits to cell-free hemolymph from a PR albino (M-line) stock of snail, Biomphalaria glabrata, was employed as a membrane probe to determine if antigens related to snail hemolymph were associated with the surface membranes of phosphate-buffered saline (PBS) washed hemocytes from a schistosome-susceptible (PR albino) and refractory (10-R2) stock of B. glabrata. Immunofluorescent and immunoelectron microscopical analyses revealed a strong cross-reactivity between anti-H PR antibodies and hemocytes from both PR albino and 10-R2 snails indicating the presence of surface-associated hemolymph or hemolymph-like antigens. Hemoglobin isolated from PR albino B. glabrata hemolymph competitively inhibited the binding of anti-H PR to hemocytes suggesting that cross-reactive membrane components were, at least in part, antigenically related to snail hemoglobin. Antigens reactive with antihemolymph antibodies also were resistant to protease treatment. No antigenic differences between PR albino and 10-R2 snail hemocytes could be detected due to the heterospecific nature of the probe antiserum, however, it is believed that the major cross-reactive membrane components, e.g., hemoglobin-like determinants, are shared in common by hemocytes of both snail stocks.

Inflammation in insects: The release of a plasmatocyte depletion factor following interaction between bacteria and haemocytes

Injection of a suspension of the Gram-positive bacterium Bacillus cereus into the haemocoel of Galleria mellonella larvae caused a rapid decrease in the number of circulating plasmatocytes. A similar phenomenon was observed following the injection of cell-free haemolymph collected from larvae previously challenged with bacteria, indicating that plasmatocyte depletion was mediated by a humoral factor released into the haemolymph. The release of plasmatocyte depletion factor could be effected in vitro by incubating adherent haemocytes with a suspension of B. cereus. The in vitro production of the factor did not require the presence of exogenous humoral components, was dose-dependent, was inhibited at low temperatures, and showed the same specificity with respect to bacterial species as observed in vivo. Activity of the plasmatocyte depletion factor destroyed by a 15-min incubation at 56°C. The release of the factor appears to be an early biochemical signal in the complex cellular response of this insect to bacterial infection.

Composition of the hemolymph of the tarantulaEurypelma californicum

1. Cell-free hemolymph of the tarantulaEurypelma californicum has been analysed for its inorganic and organic constituents (Tables 1, 2, and 3). 2. Free carbohydrates are 0.15 g/l, the main blood sugar being glucose. Protein-bound carbohydrates are 0.58 g/l. Protein concentration is 56.9 g/l. 3. The concentration of free amino acids is 0.52 mmol/l (Table 2), proline being the dominant free amino acid in the hemolymph. 4. The lipid content is 0.41 g/l (Table 3), the main fatty acids are palmitic and stearic acids. 5. The blood pH value was determined at 7.81. The osmolarity of 477 mosmol/l is fully accounted for by the concentrations of inorganic ions and other low molecular weight compounds, suggesting that essentially all blood components have been determined.

Changes in the circulating hemocyte population of Manduca sexta larvae following injection of bacteria

A technique for the collection of stable hemolymph from larvae of Manduca sexta has been developed. The method avoids the cell clumping and melanization reactions commonly encountered with insect hemolymph by minimizing contact between hemocytes and surfaces which provoke defensive or repair responses. The circulating hemocyte population of second-day, fifth-instar larvae (2dL5) of M. sexta consisted of 4.5 ± 2.5 × 10 6 cells/ml ( n = 15, range 2–7 × 10 6 cells/ml) and contained five cell types: prohemocytes, plasmatocytes, granulocytes, spherulocytes, and oenocytoids. Two strains of Pseudomonas aeruginosa which differ in pathogenicity (P11-1 and 9027) and Escherichia coli D31 grew well at 26°C in cell-free hemolymph prepared from naive (nonimmunized) 2dL5 M. sexta. When viable cells of any of the three bacteria were injected into M. sexta larvae, changes in both the total hemocyte count (THC) and differential hemocyte count were observed. Viable bacteria were not required to produce these changes since formalin-killed cells of P. aeruginosa 9027 produced a qualitatively and quantitatively similar response. Following injection of bacteria, the THC increased, reaching a maximal level at 1 hr postinjection, and remained elevated for at least 4 hr after injection. While prohemocytes, plasmatocytes, granulocytes, and spherulocytes all increased in number, 80% of the increased cell population at 1 hr postinjection of bacteria were the latter two cell types. Granulocytes and spherulocytes are cells with recognized defensive capabilities. The increased numbers of these cells in circulation soon after injection of bacteria may confer an advantage on M. sexta larvae in dealing with bacterial infections. This could explain in part the unusual resistance of M. sexta to certain bacterial pathogens.

Inducible Hemolytic Activity in Mercenaria Mercenaria Hemolymph

A naturally-occurring hemolysin directed against mammalian erythrocytes was found in the cell-free hemolymph, shell liquor, and hemocytes of the clam Mercenaria. The activity of the hemolysin showed some specificity as to erythrocyte type, was temperature-dependent, and required the presence of divalent cations. Experimental wounding was followed by a significant increment in hemolysin activity that persisted for at least 3 days. Injection of mammalian erythrocytes produced a further elevation of activity of shorter duration. No additional augmentation of activity resulted from multiple injections of erythrocytes. Presumably this humoral factor serves a protective function in the host, possibly related to antimicrobial activity. At this time there is no evidence to suggest any more than a superficial analogy between this hemolytic factor and vertebrate complement.

Macrophagelike Hemocytes of Resistant Biomphalaria glabrata Are Cytotoxic for Sporocysts of Schistosoma mansoni In vitro

When Schistosoma mansoni miracidia penetrate resistant individuals of the intermediate host snail Biomphalaria glabrata, the sporocyst is encapsulated by hemocytes (macrophagelike cells of the snail circulation) and killed. In our in vitro model the same fate requires only sporocysts and snail hemolymph. However, when cultured in plasma alone (cell-free hemolymph), sporocysts remain viable for more than 3 days, regardless of whether the plasma is from susceptible or resistant snails. When hemolymph is used from susceptible snails, the sporocysts retain a normal healthy appearance. Furthermore, the parasite appears to express an offensive response to the hemocytes of susceptible snails. Ultrastructural study reveals that resistant-strain hemocytes destroy the parasite tegument; within 24 hr the sporocyst is damaged severely throughout. This cell-mediated, internal, defensive response of an invertebrate host closely resembles antibody-dependent, cell-mediated damage to schistosomula in mammalian hosts.

Induction of specific humoral immunity to soluble proteins in the American cockroach ( Periplaneta americana). I. Nature of the primary response

Adult male American cockroaches ( Periplaneta americana) generated a specific protective response when injected with soluble protein toxins. This response developed over a period of time, peaking within 2 weeks and then gradually subsiding by the fifth week. Specificity of this reactivity was demonstrated by the fact that immunized animals were only protected against the original immunizing toxin, and not to a heterologous toxin. Passive transfer studies revealed that protection could be transferred to virgin animals with cell-free immune hemolymph. Thus, this advanced invertebrate is capable of generating a specific adaptive humoral immune response to these soluble proteins.

Immunological activation of phagocytic cells in Galleria mellonella

A phagocytosis-stimulating activity against the normally nonphagocytosable cells of Bacillus thuringiensis subtoxicus develops in the hemolymph of larvae of Galleria mellonella at different periods after injection with readily phagocytosable latex beads. The phagocytosis-stimulating factor can be transferred into new animals with the cell-free hemolymph of treated larvae. It is not detectable in the hemolymph of normal larva but is present in the supernatant of homogenates of the skin. The fractionation of activated hemolymph on Sephadex shows that the active substance has a low molecular weight. It appears to have a biological effect in cellular defense reactions as in the case of lymphokine in vertebrates.

Induction of lysozymelike activity in the hemolymph and hemocytes of an insect, Spodoptera eridania

Lysozymelike activity is present in the hemocytes and cell-free hemolymph of Spodoptera eridania. Its level remains essentially constant during larval development and can be induced by injection of various foreign materials. Serum bacteriolytic activity rises 24 hr after injection of saline, BSA, bacteria, bacterial endotoxin (LPS), latex particles, or sham injection. However, the magnitude and subsequent duration of the response depends on the nature of the injected material. The response is transient following sham injection or injection of soluble substances, such as saline and BSA, as compared to treatment with latex or bacteria. Both soluble and insoluble fractions of bacterial LPS preparations stimulated the lysozyme response. The response to a single injection of E. coli LPS was dose dependent and persisted for at least 5 days; however, additional injections had no effect on serum lysozyme level. The basal intracellular lysozyme level was significantly increased by E. coli LPS injection. Lysozyme release by hemocytes was proportional to intracellular concentration and did not increase after phagocytic stimulation of hemocytes.

Insect immunity: Early fate of bacteria injected in saturniid pupae

Saturniid pupae have previously been shown to synthesize a set of antibacterial proteins in response to an injection of viable nonpathogenic bacteria (Boman, H. G., Nilsson-Faye, I., Paul, K., and Rasmuson, T., Jr. 1974. Insect immunity. I. Characteristics of an inducible cell-free antibacterial reaction hemolymph of Samia cynthia pupae; Infec. Immun., 10, 136–145; Faye, I., Pye, A., Rasmuson, T., Boman, H. G., and Boman, I. A. 1975. Insect immunity. II. Simultaneous induction of antibacterial activity and selective synthesis of some hemolymph proteins in diapausing pupae of Hyalophora cecropia and Samia cynthia). Infec. Immun., 12, 1426–1438). It show here that two such injected bacteria, Enterobacter cloacae and Escherichia coli, were rapidly eliminated from the hemolymph. The distribution of the injected bacteria was studied by the use of radioactively labeled E. coli, which were traced by combustion of tissue samples and by radioautography. Both methods showed that the bacteria appeared most frequently in the upper distal ends of the pupae. In the radioautographic study this was expressed as a high number of silver grain-containing cells. These cells appeared singly or as two to five cells clumped together, preferentially attached to the fat body. No decisive effect was shown on either the elimination of bacteria from hemolymph or the appearance in the tissue when pupae were treated with actinomycin D or cycloheximide. Phagocytosis by adhesive hemocytes is discussed as an explanation of bacterial elimination from the hemolymph.

Bactericidal activity of the normal, cell-free hemolymph of silkworms (Bombyx mori)

Cell-free hemolymph from silkworm (Bombyx mori) larvae can kill Escherichia coli B/SM. The bactericidal principle can be resolved into at least two factors. One is a lysozyme-like enzyme that can be absorbed on crab shell chitin and on bentonite, and the other (cofactor) is an anionic factor that is of low molecular weight, can pass through the chitin column and a carboxymethyl-cellulose column, and can be eluted from a diethylaminoethyl-cellulose column at mu = 0.15 and pH 7.5. Egg-white lysozyme cannot replace the silkworm lysozyme-like enzyme for restoring the bactericidal activity when it is mixed with the cofactor, although it can enhance the bactericidal activity of the mixture of silkworm enzyme and cofactor. Mg2+ and Ca2+ can inhibit the bactericidal activity.

Biochemical and physiological studies of certain ticks (Ixodoidea): Specific gravity of hemolymph and coxal and gut fluids of Argas (Persicargas) persicus and Argas (Persicargas) arboreus (Argasidae)

Specific gravity (sp. gr.) of cell-free hemolymph and gut and coxal fluids was determined at different states of the gonotrophic cycle (unfed + 15 days, engorgement day before and after coxal fluid emission, engorgement + 1 day, oviposition day, and oviposition completion + 1 day) of female Argas (Persicargas) persicus and A. (P.) aboreus (Argasidae). The patterns of hemolymph and gut fluid sp. gr. change differed from each other during the gonotrophic cycle, but both patterns were similar in the 2 Argas species. Hemolymph sp. gr. decreased to a minimum one day after feeding (1.0085 and 1.0081 for persicus and arboreus, respectively), and increased through oviposition to a maximum on oviposition + 1 day (1.0187 and 1.0221). Minimum gut fluid sp. gr. occurred on engorgement day before coxal fluid emission (1.0565 and 1.0697). Afterward, gut fluid sp. gr. increased to a maximum on engorgement day + 1 for persicus (1.1089) and on oviposition day for arboreus (1.0973), and then decreased during oviposition in both species. In each tested state of each species, the sp. gr. was consistently higher in gut fluid than in hemolymph. In each species, coxal fluid and hemolymph sp. gr. were the same on engorgement day.

Dieldrin transport in the insect: an examination of Gerolt's hypothesis

[ 14C]-Dieldrin was detected in the hemolymph of adult male Periplaneta americana and P. brunnea 2.5 hr after an acetone solution of radiodieldrin (RD) was applied to the pronotum. Sephadex thin-layer gel filtration (TLG) of cell-free hemolymph (HL) from cockroaches to which RD was applied, and TLG of HL to which RD was added directly, indicated that dieldrin binds to a protein (s) of ca. 18,900 mol wt and two groups of proteins of mol wt ≥ 160,000. The capacity of Periplaneta HL for RD at 22°C was to at least 57 μ M, but the highest concentration of RD detected in HL after topical application was 29.9 μ M. The hemocytes contained about 37% of the dieldrin in whole cockroach hemolymph. When isolated cockroach legs were perfused with HL containing protein-bound RD, autoradiography of the dissected legs revealed 14C counts in the tissues. These findings are consistent with the translocation of topical dieldrin by hemolymph, but do not eliminate the possibility of translocation via the tracheal system.

A comparative study of the bactericidal activity of horseshoe crab ( Limulus polyphemus) hemolymph and vertebrate serum

This paper describes a partially heat-labile, naturally occurring bactericidal factor in cell-free hemolymph preparations obtained from Limulus polyphemus. This bactericidal activity has been shown to be directed against two Gram-negative bacteria, Escherichia coli and Klebsiella pneumoniae, whereas it had no effect on the Gram-positive bacteria tested, Micrococcus lysodeikticus and Staphylococcus aureus. Maximal bactericidal activity of this factor was observed at 30°C and pH 6.0. Since complement and antibody are required for antimicrobial activity in vertebrate sera, the activity of this factor in the presence of various complement inhibitors was assayed. The bactericidal activity of Limulus hemolymph is abolished by treatment with endotoxin; however, other anticomplementary substances were without effect. Limulus amebocyte lysate is known to contain protein which may be precipitated by endotoxin; it is possible that the reduction of bactericidal activity produced by endotoxin treatment may be caused by the denaturation of a bactericidal protein moiety produced by the hemocytes.

Antibacterial compounds in the cell-free haemolymph of Drosophila melanogaster

Third instar larvae, individuals in the white puparium stage, pharate pupae, and adults of Drosophila melanogaster were studied in order to detect the reason for the drop in the number of extracellular bacteria which occurs between the sixth and twenty-fourth hours after white puparium formation. Attempts were made to detect natural resistance in larvae and adults using unaltered haemolymph. Although larval haemolymph was free of bacteria, even after injection of bacteria, no isolatable antibacterial activity was found. After formation of the white puparium bacteria may be liberated from the alimentary tract by histolysis into the haemocoel. Other products of histolysis, antibacterial in nature, may also be present in the haemocoel. Several experiments were performed in order to detect such antibacterial compounds. Neutral extracts of whole larvae and pharate pupae and of fat bodies removed from larvae and from individuals in the white puparium stage proved ineffective in preventing bacterial growth. Significant antibacterial activity against Brevibacterium sp. and Bacillus sp. was found in acid extracts of haemolymph removed from pharate pupae, and its time of appearance corresponded to observed fluctuations in the numbers of gram-positive bacteria in vivo. A difference in the time of maximal antibacterial activity was found between two stocks; a difference in the response of the bacteria to the haemolymph extracts was also found, one of the bacteria, Bacillus sp., apparently being killed more efficiently by a lower concentration of haemolymph than a higher concentration. These results suggest that the growth of a bacterium must be adequately controlled if an association of Drosophila with bacteria is to be beneficial. Haemolymph of pharate pupae 24 hours after white puparium formation, when antibacterial activity against Brevibacterium sp. and Bacillus sp. is present, was tested against Escherichia coli and no antibacterial activity was found. This result agrees with the observation in vivo that numbers of E. coli did not drop at this time, and it is concluded that the material isolated in the acid is probably responsible for the fluctuations observed. Although the chemical nature of this basic material was not determined, indirect evidence indicates that it is a basic protein, either histone or lysosomal, and that variations in the amount or rate of release of such basic materials may be important in the association of an extracellular bacterium with a host Drosophila.