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.     

Unaltered hematocrit values and differential hemocyte counts in acanthocephalan-infected Armadillidium vulgare

Three cell types were present in Armadillidium vulgare hemolymph. Hyaline cells, 8.2 to 12.0 μm in size with a few fine granules in the cytoplasm, comprised 42% of the cells. Twelve percent of the cells were nonexplosive granulocytes, 8.8 to 15.0 μm, with many cytoplasmic granules of medium size. A third cell type, an explosive 7.2- to 12.0-μm granulocyte with coarse cytoplasmic granules, comprised 46% of the cells. Within 48 hr after adult specimens of A. vulgare were fed eggs of the acanthocephalan, Plagiorhynchus cylindraceus, isopod hemocytes aggregated in the intestinal epithelium surrounding penetrating acanthors. Encapsulation and death of the parasite routinely followed. No significant difference in hematocrit values or in differential hemocyte counts occurred between infected and uninfected control isopods.     

STUDIES ON MORPHOLOGY AND DEVELOPMENT OF OEDMEUS ASIATICUS ENTOMOPOXVIRUS PROPAGATED IN OEDALEUS INFERNALIS*

Abstract The morphological characteristics and development of Oedaleus asiaticus entomopoxvirus propagated in Oeddeus infernalis are reported. This virus mainly infected host's fat bodies and hemocytes. Three kinds of spheroids were observed during propagation: big spheroid (30. 41 μm × 25. 40 μm), ellipsoid (6. 58 μm × 4. 78 μm) and small spheroid (3. 35 μm × 2. 60 μm). The virions embeded in them were oval, measuring 230 nm × 176 nm. The typical characteristic of poxviruses as spherical units with the mulberry-like surface could be seen under transmission electron microscope. The lateral body was cylinder-shaped. The rope-like substances in the core folded back only once; therefore two spots in transverse sections were seen. The development of the virions included four stages: the appearance of viro-plasm, the formation of spherical particles, the differentiation of core and capsid. The grasshoppers only in the same genus could be infected by this virus.     

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。     

The structure of the hemocytes of Galleria mellonella (Lepidoptera)

Four hemocyte types have been identified in the late last larval instar of Galleria mellonella. Plasmatocytoids are round to spindle shaped cells, 10–20 μ long and 5–10 μ wide. The cytoplasm contains no distinguishing inclusions. Golgi complexes, rough endoplasmic reticulum and free ribosomes are abundant. Granular hemocytes are oval shaped cells, 10–20 μ long and 5–10 μ wide. The granules, their most characteristic feature, have a diameter of 0.2 μ, a microtubular sub-structure, and are made up of acidic mucosubstances. Lipid droplets may be present in these cells at some stage of development. These cells appear to be phagocytic. Spherule cells are oval shaped, 15–20 μ long and 5–10 μ wide. The spherules, approximately 2 μ in diameter, have a highly ordered substructure and are made up of acidic mucosubstances. Oenocytoids are the largest cells, 20 by 40 μ. The cytoplasm contains mostly free ribosomes and microtubules.     

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.     

Coccidian Parasites (Apicomplexa: Eimeriidae) from Insectivores. VI. Six New Species from the Eastern Mole,Scalopus aquaticus1

ABSTRACT Thirteen eastern moles, Scalopus aquaticus, collected in West Texas were examined for coccidian oocysts; 11 (85%) were infected and eight (73%) of these had multiple infections representing two or more species. One cyclosporan, three eimerians, and two isosporans were studied and all are described as new species. Sporulated oocysts of Cyclospora megacephali n. sp. were subspheroidal, 18.5 × 15.7 (14–21 × 12–18) μm; they had sporocysts pointed at one end with Stieda bodies nearly as wide as the sporocysts themselves, and were 15.0 × 7.2 (11–17 × 6–9) μm; C. megacephali was found in four (31%) hosts. Sporulated oocysts of Eimeria scalopi n. sp. were spheroidal to subspheroidal, 13.6 × 12.6 (11–17 × 11–15) μm with sporocysts lemon-shaped, 8.7 × 5.5 (7–10 × 4–7) μm; it was found in six (46%) hosts. Sporulated oocysts of Eimeria aquatici n. sp. were asymmetrically ellipsoidal, 17.0 × 10.6 (14–20 × 9–14) μm, with sporocysts elongately ovoidal, 9.0 × 5.2 (8–11 × 4–6) μm; it was found in two (15%) hosts. Sporulated oocysts of Eimeria motleiensis n. sp. were subspheroidal, 17.0 × 15.3 (15–20 × 13–18) μm with sporocysts ovoidal, 10.7 × 6.8 (10–13 × 6–8) μm; it was found in seven (54%) hosts. Sporulated oocysts of Isospora motleiensis n. sp. were spheroidal to subspheroidal, 13.6 × 12.0 (10–17 × 8–15) μm with sporocysts broadly ovoidal, 9.5 × 6.7 (7–11 × 4–8) μm; it was found in nine (69%) hosts. Sporulated oocysts of Isospora aquatici n. sp. were subspheroidal, 20.9 × 18.4 (15–24 × 13–21) μm with sporocysts ellipsoidal, 11.8 × 9.0 (9–14 × 7–11) μm; it was found in two (15%) hosts.     

Parasitism of Pieris rapae (Lepidoptera: Pieridae) by the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae): Effects of parasitism on differential hemocyte counts,micro‐ and ultra‐structures of host hemocytes

Abstract Parasitism by the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae) by using only its associated venom, can suppress the immunal responses of Pieris rapae (Lepidoptera: Pieridae). However, up to now, current knowledge of the mechanisms has been limited. The response of host hemocytes to parasitism was investigated using a combination of light and transmission electron microscopy (TEM). Five hemocyte types, prohemocytes (PRs), granulocytes (GRs), plasmatocytes (PLs), oenocytoids (OEs) and coagulocytes (COs), were observed and characterized from both unparasitized and parasitized Pieris rapae pupae. Light microscopy showed that both GRs and PLs became more round and spread abnormally after parasitism, whereas the shape of other types of hemocytes remained unaffected. In addition, the size of PRs and PLs became larger while OEs became smaller. The proportion of PRs significantly increased after parasitism and that of PLs decreased by 43.9%, but there was no significant increase of GRs and OEs. TEM showed that all types of hemocytes except COs were damaged to various degrees after parasitism, especially resulting in electron opaque cytoplasm and nucleus, fewer cell organelles of rough endoplasmic reticulum, mitochondria and vesicles. Our results indicate that parasitism by P. puparum affects differential hemocyte counts and structures of host hemocytes, particularly for GRs and PLs, which may be the main cause of the parasitoid suppressing host cellular immune responses.     

Bacterial challenge and eicosanoids act in plasmatocyte spreading

We report on experiments designed to more thoroughly document the roles of eicosanoids as crucial elements in cell spreading and on experiments designed to test the hypothesis that in vivo bacterial infections influence cell spreading on glass surfaces. We used hemocytes prepared from tobacco hornworms, Manduca sexta (L.) (Lepidoptera: Sphingidae) and four species of bacteria (Serratia marcescens, Escherichia coli, Bacillus subtilis, and Micrococcus luteus) in each experiment. Our protocols yielded several important points: (i) hemocytes prepared from hornworms at 15 and 60 min following infection with, separately, each of the four bacterial species were fundamentally altered in size (all less than the 15‐µm counting cut‐off) and none of the hemocytes exhibited cell‐spreading behavior; (ii) the influence of bacterial challenge on cell spreading declined with incubation time post‐challenge; (iii) conditioned medium (CM) prepared by exposing hemocytes to bacterial cells in vitro exerted a strong dose‐dependent influence on cell spreading. Specifically, plasmatocytes increased in length from about 38 µm with 2.5% CM to a maximum of about 54 µm at 100% CM; and (iv) the retarding influence of dexamethasone (an eicosanoid biosynthesis inhibitor) on cell spreading was reversed by arachidonic acid, prostaglandin H₂, and CM. Taken together, these findings indicate that both bacterial infection and eicosanoids influence hemocyte‐spreading processes.     

A systematic study on hemocyte identification and plasma prophenoloxidase from Culex pipiens quinquefasciatus at different developmental stages

Culexpipiens quinquefasciatus (C. quinquefasciatus) is an important vector that can transmit human diseases such as West Nile virus, lymphatic filariasis, Japanese encephalitis and St. Louis encephalitis. However, very limited research concerning the humoral and cellular immune defenses of C. quinquefasciatus has been done. Here we present the research on hemocyte identification and plasma including hemocyte prophenoloxidase from C. quinquefasciatus at all developmental stages in order to obtain a complete picture of C. quinquefasciatus innate immunity. We identified hemocytes into four types: prohemocytes, oenocytoids, plasmatocytes and granulocytes. Prophenoloxidase (PPO) is an essential enzyme to induce melanization after encapsulation. PPO-positive hemocytes and plasma PPO were observed at all developmental stages. As for specific hemocyte types, prophenoloxidase was found in the plasmatocytes at larval stage alone and in the smallest prohemocytes during almost all developmental stages. Moreover, the granulocytes were PPO-positive from blood-fed female mosquitoes and oenocytoids were observed PPO-positive in pupae and in adult females after blood-feeding. As for plasma, there were different patterns of PPO in C. quinquefasciatus at different developmental stages. These results are forming a basis for further studies on the function of C. quinquefasciatus hemocytes and prophenoloxidase as well as their involvement in fighting against mosquito-borne pathogens.     

Coccidia of Brazilian Mammals: Eimeria corticulata N. Sp. (Apicomplexa: Eimeriidae) from the Anteater Tamandua tetradactyla (Xenarthra: Myrmecophagidae) and Eimeria zygodontomyis N. Sp. from the Cane Mouse Zygodontomys lasiurus (Rodentia: Cricetidae)

Feces from a specimen of Tamandua tetradactyla (Linn.) from Portel, Para State, north Brazil, contained two different coccidial oocysts; one identified as Eimeria tamanduae Lainson 1968, and the other as a new species, described here as Eimeria corticulata n. sp. Oocysts of E. corticulata are ellipsoidal, 37.4 × 30.4 (31.2–43.7 × 23.7–35.0) μm, shape index (length/width) 1.2 (1.0–1.5). Oocyst wall 2.5–3.7 μm thick and composed of two layers; an outer thick, brown-yellow one with radial striations, and a thin inner smooth one: no visible micropyle. Oocyst residuum a large globule of about 10.7 × 10.3 μm, usually accompanied by a number of smaller attached globules. Sporocysts ellipsoidal, 21.0 × 11.0 (20.0–22.5 × 10.0–12.5) μm, with a conspicuous Stieda body: shape index 1.9 (1.6–2.2). Sporocyst residuum a small number of scattered granules: sporozoites 18.7 × 5.0 μm, with a large posterior refractile body. Eimeria zygodontomyis n. sp. is described in feces from Zygodontomys lasiurus (Lund) from the Serra dos Carajas, Para. Oocysts ellipsoidal to cylindrical, 16.5 × 12.0 (13.7–18.7 × 11.2–12.3) μm, shape index 1.4 (1.2–1.5). Wall colorless, smooth, single-layered and about 0.6 μm thick: no micropyle. No oocyst residuum, but a polar granule of about 1.8 × 1.0 μm is sometimes present. Sporocysts ellipsoidal, 8.4 × 5.5(7.5–8.7 × 5.0–6.2) μm, shape index 1.5 (1.4–1.7), with a thin colorless wall and a delicate Stieda body. Sporozoites enclose a compact residuum of about 2.5 × 3.7 μm.     

A comparative ultrastructural study of blood cells from nine insect orders

Summary An ultrastructual study of hemocytes from 9 different insect orders has led to the identification of 8 cell types: (1) Plasmatocytes, whose cytoplasm is filled with small dense lysosomes and large heterogeneous structures, are phagocytic cells. (2) Granulocytes, filled with uniformly electron dense granules, are involved in capsule formation. (3) Coagulocytes, which contain granules and structured globules and which possess a well developed RER, are involved in phagocytosis. (4) Spherule cells are filled with large spherical inclusions. (5) Oenocytoids are large cells with few cytoplasmic organelles. These 5 hemocyte types represent the majority of insect blood cells. (6) Prohemocytes, blastic cells which are one of the stem cells of hemocytes, are very few in number in each species investigated. (7) Thrombocytoids and (8) Prodocytes are restricted to a small number of insect species.The ultrastructural characteristics of these hemocyte types are discussed.     

Phenoloxidases in ascidian hemocytes: characterization of the pro-phenoloxidase activating system

The phenoloxidase (PO) activity of the hemocytes lysate supernatant from three ascidians species, assayed by means of 3-methyl-2-benzothiazolinone hydrazone hydrochloride, have been compared. PO-containing hemocytes were identified by a cytochemical reaction and the enzymatic activity measured by a spectrophotometric assay of lysate supernatant from hemocyte populations separated on a discontinuous Percoll density gradient. In Styela plicata, the enzyme appeared to be contained in morula cells only. In Ciona intestinalis, PO activity was shown in univacuolar refractile granulocyte and granular hemocyte. In Phallusia mammillata both compartment cell and granular hemocytes were positive. Enzymatic assay following electrophoretic analysis on polyacrylamide gel electrophoresis (PAGE) or SDS-PAGE indicated that hemocyte lysate presented orthodiphenoloxidase (catecholase) activity. The enzymes from the three species differed in molecular size, activating substances and trypsin sensitivity.     

Effects of the endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae) parasitism,venom, and calyx fluid on cellular and humoral immunity of its host Chilo suppressalis (Lepidoptera: Crambidae) larvae

The larval endoparasitoid Cotesia chilonis injects venom and bracoviruses into its host Chilo suppressalis during oviposition. Here we study the effects of the polydnavirus (PDV)-carrying endoparasitoid C. chilonis (Hymenoptera: Braconidae) parasitism, venom and calyx fluid on host cellular and humoral immunity, specifically hemocyte composition, cellular spreading, encapsulation and melanization. Total hemocyte counts (THCs) were higher in parasitized larvae than in unparasitized larvae in the late stages following parasitization. While both plasmatocyte and granulocyte fractions and hemocyte mortality did not differ between parasitized and unparasitized hosts, in vitro spreading behavior of hemocytes was inhibited significantly by parasitism throughout the course of parasitoid development. C. chilonis parasitism suppressed the encapsulation response and melanization in the early stages. Venom alone did not alter cellular immune responses, including effects on THCs, mortality, hemocyte composition, cell spreading and encapsulation, but venom did inhibit humoral immunity by reducing melanization within 6 h after injection. In contrast to venom, calyx fluid had a significant effect on cell spreading, encapsulation and melanization from 6 h after injection. Dose–response injection studies indicated the effects of venom and calyx fluid synergized, showing a stronger and more persistent reduction in immune system responses than the effect of either injected alone.     

Hemocytes of the palaemonids Macrobrachium rosenbergiiand M. acanthurus,and of the Penaeid Penaeus paulensis

The hemocytes of two palaemonids and one penaeid were characterized using light and transmission electron microscopy (TEM). The blood cells in all three species were classified as hyaline hemocytes (HH), small granule hemocytes (SGH), and large granule hemocytes (LGH). The HH are unstable hemocytes with a characteristic high nucleo-cytoplasmic ratio. Their cytoplasm appears particularly dense and has from few to numerous granules that often exhibit a typical striated substructure. In both palaemonids, the great majority of the HH contain numerous granules, whereas in Penaeus paulensis, a small number of these cells have few or no granules. The cytoplasm of some HH of the penaeid exhibits typical electron-dense deposits. The granulocytes, LGH and SGH, contain abundant electron-dense granules that are usually smaller in the SGH. In both hemocyte types, the cytosol, but not the granules, is rich in carbohydrates (PAS positive) and numerous vesicles contain acid phosphatase (Gomori reactive). In all studied shrimps, the SGH and LGH were actively phagocytic when examined on blood cell monolayers incubated with the yeast Saccharomyces cerevisiae. A few mitotic figures (less than 1%) were observed in the granulocytes of P. paulensis, but not in the palaemonids. SGH is the main circulating blood cell type in both palaemonids, whereas HH is predominant in the penaeid. Based on morphological and functional features, it appears that the hyaline and the granular hemocytes of the three shrimp species represent different cell lineages. J. Morphol. 236:209–221, 1998. © 1998 Wiley-Liss, Inc.     

VENOM FROM THE ECTOPARASITIC WASP Habrobracon hebetor ACTIVATES CALCIUM‐DEPENDENT DEGRADATION OF Galleria mellonella LARVAL HEMOCYTES

Ectoparasitoids inject venom into hemolymph during oviposition. We determined the influence of envenomation by the parasitoid, Habrobracon hebetor, on the hemocytes of its larval host, Galleria mellonella. An increase in both intracellular Са²⁺ content and phospholipase C activity of the host hemocytes was recorded during 2 days following envenomation by the parasitoid. The decreased hemocyte viability was detected 1, 2, and 24 h after the envenomation. Injecting of the crude venom (final protein concentration 3 μg/ml) into the G. mellonella larvae led to the reduced hemocyte adhesion. The larval envenomation caused a decrease in transmembrane potential of the hemocytes. These findings document the suppression of hemocytic immune effectors in the parasitized host larvae.     

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.     

华北大黑鳃金龟幼虫血细胞的超微结构观察

应用光学和电子显微镜技术检查了华北大黑鳃金龟3龄幼虫血淋巴内的血细胞,识别出5种类型的血细胞(原血胞、浆血胞、颗粒血胞、珠血胞和凝血胞)并对每一种血细胞的超微结构特点进行描述。     

Ultrastructural studies of the hemocytes of Panstrongylus megistus (Hemiptera: Reduviidae)

Ultrastructural analyses revealed the presence of six hemocyte types in the hemolymph of Panstrongylus megistus, partially confirming our previous results obtained through light microscopy. Prohemocytes: small, round hemocytes with a thin cytoplasm layer, especially rich in free ribosomes and poor in membranous systems. Plasmatocytes: polymorphic cells, whose cytoplasm contains many lysosomes and a well developed rough endoplasmic reticulum (RER). They are extremely phagocytic. Sometimes, they show a large vacuolation. Granulocytes: granular hemocytes whose granules show different degrees of electrodensity. Most of them, have an internal structuration. Coagulocytes: oval or elongated hemocytes, which show pronounced perinuclear cisternae as normally observed in coagulocytes. The cytoplasm is usually electrodense, poor in membranous systems and contains many labile granules. Oenocytoids: large and very stable hemocytes, whose homogeneous cytoplasm is rich in loose ribosomes and poor in membranous systems. Adipohemocytes: large cells, containing several characteristic lipid droplets. The cytoplasm is also rich in glycogen, RER and large mitochondria. The total and differential hemocyte count (THC and DHC) were also calculated for this reduviid. THC increases from 2,900 hemocytes/mm3 of hemolymph in the 4th instar to 4,350 in the 5th and then, decreases to 1,950 in the adults. Plasmatocytes and coagulocytes are the predominant hemocyte types.     

Characterisation of Mytilus edulis hemocyte subpopulations by single cell time-lapse motility imaging

In bivalve molluscs, defence against pathogens mainly relies on fast tissue infiltration by immunocompetent hemocytes that migrate from circulating hemolymph to sites of infection, in order to deliver, in situ, an effective immune response. In the present work, we have investigated dynamics of hemocyte subpopulations motility by combining flow cytometry coupled to Coulter-type cell volume determination, Hoffman modulation contrast microscopy, time-lapse imaging and off-line analysis of cell shape changes. Our results revealed fast modifications of hemocyte aspect in vitro, with bidirectional transitions from spread outlines to condensed cell body morphologies, in the minute range. Amoeboid or non-amoeboid types of locomotion were observed, depending on the cell shapes and on the cell subtypes, with velocities reaching up to 30 μm min^?1. Correlations between motion profiles, Hemacolor staining and flow cytometry analysis on living cells help to propose a functional mussel hemocyte classification including the motile properties of these cells. In particular, basophils were shown to be involved in dynamic hemocyte–hemocyte interactions and in the constitution of aggregation cores. Physiological implications, in terms of immune response in organisms devoid of endothelium-closed vascular system, and potential applications of hemocyte motility studies for the development and the interpretation of experiments involving hemocytes in the field of marine ecotoxicology are discussed.