Response of the insect immune system to three different immune challenges

Insects rely on an innate immune system to effectively respond to pathogenic challenges. Most studies on the insect immune system describe changes in only one or two immune parameters following a single immune challenge. In addition, a variety of insect models, often at different developmental stages, have been used, making it difficult to compare results across studies. In this study, we used adult male Acheta domesticus crickets to characterize the response of the insect innate immune system to three different immune challenges: injection of bacterial lipopolysaccharides (LPS); injection of live Serratia marcescens bacteria; or insertion of a nylon filament into the abdomen. For each challenge, we measured and compared hemolymph phenoloxidase (PO) and lysozyme-like enzyme activities; the number of circulating hemocytes; and the nodulation responses of challenged and un-challenged crickets. We found that injection of an LD₅₀ dose of LPS from Escherichia coli elicited a more rapid response than an LD₅₀ dose of LPS from S. marcescens. LPS injection could cause a rapid decrease 2 hpi, followed by an increase by 7 dpi, in the number of circulating hemocytes. In contrast, injection of live S. marcescens produced a rapid increase and then decrease in hemocyte number. This was followed by an increase in the number of hemocytes at 7 dpi, similar to that observed following LPS injection. Both LPS and live bacteria decreased hemolymph PO activity, but the timing of this effect was dependent on the challenge. Live bacteria, but not LPS, induced an increase in lysozyme-like activity in the hemolymph. Insertion of a nylon filament induced a decrease in hemolymph PO activity 2 h after insertion of the filament, but had no effect on hemocyte number or lytic activity. Our results indicate that the innate immune system’s response to each type of challenge can vary greatly in both magnitude and timing, so it is important to assess multiple parameters at multiple time points in order to obtain a comprehensive view of such responses.     

中华绒螯蟹血细胞原代培养条件的优化

比较了几种常见血细胞培养基(L-15、2×L-15、3×L-15、M199和RMPI-1640)对中华绒螯蟹(Eriocheir sinensis)血细胞原代培养中细胞形态以及存活率的影响,以及在筛选获得的最佳培养基中添加不同比例胎牛血清(FBS)(0%、5%、10%和15%),进一步观察了血清对中华绒螯蟹血细胞培养效果的比较。结果表明,3×L-15培养基培养效果较好,所培养的细胞形态相对完整,数量较多,培养至96 h时血细胞存活率仍大于60%;而其他4种培养基效果较差,培养12 h存活率均低于50%,且细胞形态结构变化明显。以3×L-15培养基为基础,添加不同比例胎牛血清后发现,对细胞存活有显著影响,存活率明显降低。因此,不添加血清的3×L-15培养基对中华绒螯蟹血细胞的生长较为适宜。     

Specificity of anti-Vibrio immune response through p38 MAPK and PKC activation in the hemocytes of the mussel Mytilusgalloprovincialis

In mussel (Mytilus sp.) hemocytes, differential functional responses to injection with different types of live and heat-killed Vibrio species have been recently demonstrated.In this work, responses of Mytilus hemocytes to heat-killed Vibrio splendidus LGP32 and the mechanisms involved were investigated in vitro and the results were compared with those obtained with Vibrio anguillarum (ATCC 19264). Adhesion of hemocytes after incubation with bacteria was evaluated by flow cytometry: both total hemocyte counts (THC) and percentage of hemocyte sub-populations were determined in non-adherent cells. Functional parameters such as lysosomal membrane stability, lysozyme release, extracellular ROS production and NO production were evaluated, as well as the phosphorylation state of the stress-activated p38 MAPK and PKC. Neither Vibrio affected total hemocyte adhesion, while both induced similar lysosomal destabilization and NO production. However, V. splendidus decreased adhesion of large granulocytes, induced rapid and persistent lysozyme release and stimulated extracellular ROS production: these effects were associated with persistent activation of p38 MAPK and PKC. In contrast, V. anguillarum decreased adhesion of large semigranular hemocytes and increased that of hyalinocytes, had no effect on the extracellular ROS production, and induced significantly lower lysozyme release and phosphorylation of p-38 MAPK and PKC than V. splendidus. These data reinforced the existence of specific interactions between mussel hemocytes and V. splendidus LGP32 and suggest that this Vibrio strain affects bivalve hemocytes through disregulation of immune signaling. The results support the hypothesis that responses of bivalve hemocytes to different bacterial stimuli may depend not only on the nature of the stimulus, but also on the cell subtype, thus leading to differential activation of signaling components.     

In vitro bacteridical capacity of Blaberus craniifer hemocytes

Blaberus craniifer hemocytes, maintained in short-term culture, are capable of phagocytosing and destroying Staphylococcus aureus, Staphylococcus albus, Streptococcus faecalis, Serratia marcescens, and Proteus mirabilis. The observed bactericidal activity of the hemocyte suspensions was entirely a function of the phagocytes; the medium, the hemolymph, and cell products elaborated during incubations were not bactericidal. No humoral opsonic factors were required for, or facilitated, bacterial phagocytosis in vitro. Washed hemocyte monolayers bathed by hemolymph-free medium were capable of phagocytosing bacteria. The addition of hemolymph concentrated by ultrafiltration did not increase the bactericidal capacity of the hemocytes. Bacteria opsonized with concentrated hemolymph were not killed more efficiently than were untreated bacteria.A partial blockage of bactericidal capacity was induced by prior exposure of the hemocytes to bacteria or to latex particles. The functional blockade was more complete with bacteria than with latex particles.Pseudomonas aeruginosa, Escherichia coli, Salmonella typhosa, and Diplococcus pneumoniae were phagocytosed but not killed by the hemocytes. This lack of bactericidal activity suggests that roaches may encounter difficulty in eliminating these organisms from the hemocoel. However, deficient bactericidal capacity probably does not entirely correlate with pathogenicity since the known insect pathogens, Staphylococcus albus, Serratia marcescens, and Proteus mirabilis, are killed by the hemocytes. Pathogenicity seems to depend on a complex of factors including bacterial strain, dose received, and intracellular survival of ingested bacteria. A possible connection between the lack of hemocytic bactericidal capacity and the role of roaches as potential disease vectors warrants further investigation.     

Histopathology of Aerococcus viridans var. homari infection (Gaffkemia) in the lobster, Homarus americanus, and a comparison with histological reactions to a gram-negative species, Pseudomonas perolens

Histological response of lobsters to injection of Aerococcus viridans var. homari, cause of gaffkemia, was followed over a 14-day period. Salient features in infected lobsters, Homarus americanus, were: aggregations of hemocytes occurring in hemal spaces throughout the tissues and increasing in number and size with time; the early phagocytosis of bacteria by the system of fixed phagocytes (FPs) present in hemal spaces of the hepatopancreas; and premature release of differentiating hemocytes from the hemopoietic tissue, so that by 14 days that tissue consisted mainly of large stem cells. Mass release of differentiating hemocytes presumably occurred to replace hemocytes lost from the circulation by their incorporation into aggregations or by lysis of individual cells ruptured through the pressure of phagocytized bacteria that were multiplying in them. Bacteria and their remains were present in FPs at 2 days but not visible in single or aggregated hemocytes until 6 days, when free bacteria were also present in the hemolymph. By 6 days, all bacteria, whether phagocytized or free, appeared normal and were surrounded by nonstaining halos that extended well beyond the stainable capsular material. As predicted earlier in physiological studies, gaffkemia is a nontoxic, noninvasive bacteremia. There was hemal stasis and consequent injury in the antennal gland due to free and aggregated hemocytes that occluded hemal spaces of that organ, but other tissues and organs appeared normal except for depletion of glycogen. Aggregations of hemocytes were present in lobsters 2 and 12 days after injection of a nonpathogenic, Gram-negative bacterium, Pseudomonas perolens. Unlike the case with gaffkemia, necrotic hemocytes were common in the aggregations, presumably in response to damage by endotoxin. A further difference was that aggregations were common in the heart of P. perolens-injected lobsters but rare in the heart of gaffkemic lobsters. Bacteria were not seen in hemolymph, hemocytes, or other cells of P. perolens-injected lobsters.     

An improved method of cell culture system from eye stalk,hepatopancreas, muscle,ovary, and hemocytes of <Emphasis Type="Italic">Penaeus vannamei</Emphasis>

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace’s insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO₂. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO₂ supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.     

Hemocytes and hemocytopoiesis in Silkworms.

A brief review is presented of the current state of ultrastructure, cytochemistry, and physiology of the hemocytes and meso- and metathoracic peri-imaginal-wing organs in silkworms. According to the accepted morphological classification, five circulating types of hemocytes are recognized in Bombyx mori as well as in Antheraea pernyi. They are prophemocytes or stem cells, plasmatocytes or pre-differentiated cells and three specialized cells, granulocytes, spherule cells and oenocytoids. During post-embryonic development the last four types are the most common in the circulating hemolymph. Plasmatocytes are considered to be pluripotent cells from which granulocytes, spherule cells and oenocytoids are derived. Contrary to the situation in most insects the plasmatocytes are not phagocytic in Antheraea. The granulocytes are efficient phagocytes. Both plasmatocytes and granulocytes are involved in pinocytosis. Another possible function of the granulocytes is hemolymph coagulation. The function of the spherule cells which contain a paracrystalline material (muco- or glycoproteins) is by no means clear. The phenoloxidase activity found within the cytosol of oenocytoids appears effective against the natural monophenol and diphenol substrates. The involvement of oenocytoids in the complex metabolism of phenols and particularly in the production of plasma phenolases has been reported. The mitotic division of five circulating hemocyte types is well known and was long regarded as the only mechanism of postembryonic hemocyte production. We present for silkworms, experimental evidence of the hemocytopoietic function of the meso- and metathoracic organs surrounding the imaginal wing discs. Ablation experiments demonstrate that the mitotic activity of free hemocytes is unable to maintain the normal hemocytogram in the absence of the two paris of organs. These organs are typically divided into cell islets ensheathed by a connective tissue membrane. Two types of islets may be classified by the disposition of the cells : the compact islets or aggregations of stem cells and the reticulate islets which are mainly composed of hemocytes at different steps of differentiation. The relative number of prohemocytes in the total hemocyte population ranges from 84 to 97 p. cent in organs of Antheraea pernyi. This well-defined cell type appears to be the major hemocyte type in hemocytopoietic organs. In Antheraea, the mitotic index (the relative number of mitotic hemocytes in the total cell population) varies from 0.5 to about 3 p. cent. Finally, our data direct attention to cyclic functional changes such as mitotic divisions and hemocyte differentiation which run parallel to the molting cycle.     

Effects of pH, temperature, and osmolarity on the morphology and survival rate of primary hemocyte cultures from the Mitten Crab, Eriocheir sinensis

Hemocytes are the main immune defense cells in crustacean, and its in vitro culture can be a useful tool for the study of host and pathogen interaction. In the present study, the primary hemocyte culture of Chinese mitten crab (Eriocheir sinensis), including mixed and single hemocyte, was set up for the first time. In this study, different pH (6.4, 6.8, 7.2, 7.6, and 8.0), temperature (26, 28, and 30°C), and osmolarity (500, 700, 900, 1,100, and 1,300 mOsm kg^?1) values were tested. Moreover, the effects of two types of medium (1× L-15 and 3× L-15) with the same osmolarity on hemocyte culture were evaluated. After incubation at different culture conditions, the morphological changes (degranulation, lysis, shrinkage, and detachment) and survival rate of hemocytes were taken into account in order to evaluate the culture condition effect. Our results showed that the total hemocyte counts of Chinese mitten crab were about 2.5?×?10⁷ cells ml^?1, and three subpopulations of hemocytes were distinguished as granulocytes (43.46?±?4.98%), semigranulocytes (31.04?±1.95%), and hyalinocytes (25.50?±4.89%). The optimal culture condition for primary hemocytes of Chinese mitten crab was 3× L-15 medium, 1,100 mOsm kg^?1, pH 6.8 at 28°C. Hemocytes at optimal culture condition could retain a better morphology and higher survival rate: hemocytes retained a survival rate >60% after 5 d and >40% after 7 d. Furthermore, the hemocyte subpopulations were isolated by Percoll step gradient centrifugation and cultured in optimized hemocyte culture conditions. The results showed that hyalinocytes and semigranulocytes could maintain a survival rate of >50% after 15 d, while granulocytes only retained a survival rate of 26% after 5 d.     

In vivo insect hemocyte destruction by UV-irradiated Chilo iridescent virus

Large inoculum of infective or UV-irradiated Chilo iridescent virus (CIV) caused an early drastic cytopathy of the larval hemocytes of the greater wax moth, Galleria mellonella. Cytopathy of the hemocyte could be detected 2 hr after virus injection at 25°C and most hemocytes were completely destroyed within 8 hr after virus injection. Heat-treated CIV did not produce any cytopathy of Galleria hemocytes. Antiserum to CIV neutralized this cytopathic effect, while that to Tipula iridescent virus did not demonstrate any inhibitory effect. Actinomycin D, mitomycin C, and puromycin did not inhibit hemocyte destruction by UV-irradiated CIV. Galleria larva injected with a large dose of UV-irradiated CIV progressively became stunted with a decrease of body weight and did not regain its hemocyte numbers. Hemocytes infected with CIV were not destroyed by any additive injection of a large dose of UV-irradiated CIV.     

Serratia marcescens Suppresses Host Cellular Immunity via the Production of an Adhesion-inhibitory Factor against Immunosurveillance Cells

Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis.     

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 × 10⁶ cells/ml (n = 15, range 2–7 × 10⁶ 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.     

Characterization of cell clusters in larval hemolymph of the cabbage armyworm Mamestra brassicae and their role in maintenance of hemocyte populations

Maintenance of hemocyte populations is critical for both development and immune responses. In insects, the maintenance of hemocyte populations is regulated by mitotic division of circulating hemocytes and by discharge from hematopoietic organs. We found cell clusters in the hemolymph of Mamestra brassicae larvae that are composed of small, spherical cells. Microscopic observations revealed that the cells in these clusters are similar to immature or precursor cells present in hematopoietic organs. The results of bromodeoxyuridine (BrdU) incorporation experiments demonstrate that these cells are mitotically active. Furthermore, these cells maintain their immature state and proliferate until late in the last larval instar. The results of in vitro experiments showed that most of the cells changed their morphology to one consistent with plasmatocytes or granulocytes, and that the change was promoted by addition of larval hemolymph to the culture medium, in particular when hemolymph was collected at a prepupal stage. Taken together, our results suggested that cells in clusters may be an additional source of hemocytes during larval development.     

Chemotactic attraction between hemocytes of the oyster,Crassostrea virginica,and bacteria

Experiments were conducted to ascertain whether there is chemotactic attraction by Bacillus megaterium and Micrococcus varians, both Gram-positive species, and Escherichia coli and Vibrio parahaemolyticus, both Gram-negative species, for hemocytes of the American oyster, Crassostrea virginica. It was ascertained quantitatively that oyster hemocytes are attracted to live E. coli, B. megaterium, and M. varians but not to heat-killed bacteria. Furthermore, oyster cells are not attracted to either live or heat-killed V. parahaemolyticus. It is concluded that the chemoattractant is some molecule emitted by living vegetative cells of certain Gram-positive as well as Gramnegative bacteria.     

Investigations on cellular defense reactions with Galleria mellonella against Bacillus thuringiensis

In contrast to a great number of foreign particles (bacteria and inorganic materials), cells of some strains of Bacillus thuringiensis are not phagocytosed in the first hours after injection into the hemocoel of Galleria mellonella. This phenomenon is not caused by the production of β-exotoxin or exoenzymes, because heat-killed cells are not phagocytosed and the phagocytosis of latex particles is not prevented by the presence of living B. thuringiensis. The phagocytosis of heat-killed B. thuringiensis subtoxicus can be encouraged by treatment of the cells and by simultaneous injection of latex particles. A factor stimulating the phagocytosis is discussed. It is induced by the injection of phagocytosable latex particles into the hemocoel but not by injection of living or killed B. thuringiensis subtoxicus.     

Hemocytes of Biomphalaria glabrata: Factors affecting variability

Variability in the hemocyte number of two geographic strains of Biomphalaria glabrata was studied. In each strain a logarithmic increase in hemocyte number associated with increasing shell size was observed. A two fold increase in circulating hemocytes occurred 2 hr following the exposure of a susceptible strain of B. glabrata to miracidia of Schistosoma mansoni. The hemocyte number was dependent on the temperatures at which the snails were maintained.     

Pathological changes in the nephrocytes of the shore crab, Carcinus maenas, following injection of bacteria

The gills of Carcinus maenas were examined by light and electron microscopy following injection of either sterile saline or the bacteria Bacillus cereus and Moraxella sp., to determine any role(s) for the nephrocytes in the host defense reactions. The results showed that although intact bacteria were not sequestered to the nephrocytes, these cells were active in the removal of large quantities of cell debris from the hemolymph. Much of this material was derived from the breakdown of the hemocytes in response to the presence of bacteria and it's accumulation in the central vacuoles of the nephrocytes resulted in the degradation of these cells. It is proposed that while nephrocytes do not phagocytose intact bacteria, they augment the host defenses by clearing much of the hemocyte and associated bacterial debris from the gills, thus preventing blockage of the lamellar sinuses and subsequent impairment of respiration.     

Physiological functions of hemocytes newly emerged from the cultured hematopoietic organs in the silkworm, Bombyx mori

Abstract  Cellular immunity is a very important part of insect innate immunity. It is not clear if hemocytes entering the hemolymph require a maturation process to become competent. The establishment of a tissue culture system for the insect hematopoietic organs would enable physiological function assays with hemocytes newly emerged from hematopoietic organs. To this end, we established a hematopoietic organ culture system for the purebred silkworm pnd pS and then studied the physiological functions of the newly emerged hemocytes. We found that Grace's medium supplemented with 10% heated silkworm larval plasma was better for culturing the hematopoietic organs of pnd pS . Newly emerged hemocytes phagocytosed propidium iodide-labeled bacteria and encapsulated the Iml-2 coated nickel beads as well as pupal tissue debris. This culture system is therefore capable of generating physiologically functional hemocytes. These hemocytes can be used to study the mechanisms of the hemocyte immune response among others.     

Lysozyme gene expression and hemocyte behaviour in the Mediterranean mussel, Mytilus galloprovincialis, after injection of various bacteria or temperature stresses

The aim of the present study was to evaluate the expression of the Mytilus galloprovincialis lysozyme gene in different in vivo stress situations, including injection of bacteria Vibrio splendidus LGP32, Vibrio anguillarum or Micrococcus lysodeikticus, as well as heat shock at 30 degrees C and cold stress at 5 degrees C. Injection of V. splendidus LGP32 resulted in: (i) a general down-regulation of lysozyme gene expression, as quantified by Q-PCR; (ii) reduction in the number of circulating hemocytes; (iii) decrease in the percentage of circulating hemocytes expressing lysozyme mRNA which was now restricted to only small cells, as observed by ISH; and (iv) accumulation of hemocytes expressing lysozyme in the muscle sinus where injection took place. Injection of V. anguillarum or M. lysodeikticus induced significant up-regulation of lysozyme gene expression, but only 2-3days post-injection, with no change in the total hemocyte counts but an increased percentage of hemocytes expressing lysozyme mRNA. Neither the control injection of PBS-NaCl nor temperature stress modified the lysozyme expression pattern. Consequently, the hemocyte population appears to be capable of discriminating between stress factors, and even between 2 Vibrio species.