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Herbicides have become the most commonly applied agrochemicals in agroecosystems. Thus, basic knowledge of their physiological effects on insects is needed, especially for understanding their impact on beneficial insect species. In this study, we evaluated the effect of a pendimethalin-based herbicide (PND) on the cellular immune response of the carabid beetle Harpalus (Pseudoophonus) rufipes (De Geer 1774) (Coleoptera, Carabidae), acting as biocontrol agent in agroecosystems. Total and differential haemocyte counts and phagocytosis assay, performed by injecting in vivo carboxylate-modified polystyrene latex beads, were measured in beetles exposed to a recommended field dose (4L per ha) of PND to evaluate the exposure effects over the time. The pattern of haemocyte subpopulations and the decrease of the phagocytic index after the exposure to PND suggested a lowering of P. rufipes ability to face an infection performing a cell-mediated response. PND was also found to cause cytotoxic effects on the haemocyte ultrastructure. Ultrastructural alterations such as irregular shape, large vacuolization of the cytoplasm, and condensation of marginated chromatin were recorded from 2d of exposure. The loss of RER, Golgi apparatus, mitochondria integrity and the swelling of the outer nuclear membrane found in some haemocytes suggested an interference of PND with the membrane permeability. Results indicated that the exposure to PND impairs the distribution, morphology and physiological functions of haemocytes causing a decrease of P. rufipes immunocompetence. Moreover, the sensitivity to herbicide exposure makes this species a suitable model and a useful bioindicator for monitoring exposure effects on non-target species. This study provides useful information to protect and preserve biodiversity of insects in agroecosystems., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The aim of this study was to evaluate the metabolic condition of Steatoda grossa (Theridiidae) spider, from their hemocytes, after a short-term (four-week) exposure to cadmium and copper in sublethal doses by administering them into the body of the preys. The ultrastructure of the dominant types of hemocytes, such as granulocytes, plasmatocytes and prohemocytes, was evaluated using transmission electron microscope (TEM). Quantitative evaluation of apoptotic and necrotic cells, as well as the ones with depolarized mitochondria in hemolymph, was performed using flow cytometry, while ATP concentration and ADP/ATP ratio in hemocytes were measured by luminescent methods. Cadmium, unlike copper, demonstrated proapoptotic and pronecrotic activity. Low ATP levels and high ADP/ATP ratio in hemocytes indicate a disturbance in the energy metabolism of cells and may account for their qualitative and quantitative degenerative changes. The intensification of death processes in hemocytes after an exposure to cadmium-contaminated food may impair the ability of these cells to fight infectious diseases. Copper at the applied dosage was safe for the spiders without causing visible changes in the hemocyte ultrastructure and in the level of analyzed cell death indices., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Toxicity of Cd was tested with the hemocytes of the freshwater crab, Sinopotamon henanense, which were exposed to concentrations of 0, 0.725, 1.450, and 2.900mgL -1 Cd for 7, 14 and 21 d. We investigated the effects of Cd on the total antioxidant capacity (TAC), and oxidative damage of biomarkers, such as malondialdehyde (MDA), protein carbonyl derivates (PCO), and DNA-protein crosslink (DPC). Transmission electron microscopy (TEM) was applied to assess ultrastructural changes of hemocytes. The mRNA expression levels of prophenoloxidase (proPO), lysozyme (LSZ), metallothionein (MT), and the activity of phenoloxidase (PO) were also determined. Our results showed that TAC was inhibited by Cd, resulting in an increase of MDA contents, PCO contents, and DPC levels in hemocytes, respectively. Ultrastructural observations revealed that chromatin condensation, nucleus deformation, mitochondrial dilation, rough endoplasmatic reticulum (rER) degranulation and secondary or tertiary lysosomes were observed in hemocytes of crabs exposed to Cd. Meanwhile, the expression levels of proPO were down-regulated, while the activity of PO was up-regulated in hemocytes. The expression levels of LSZ and MT were up-regulated to some extent. Our findings suggest these parameters could be used as biomarkers in the monitoring of heavy metal pollution and quantitative risk assessments of pollutant exposure., (Copyright © 2016. Published by Elsevier Inc.)

Individuals of colonial animals (e.g. zooids) are in continuous turnover. In ascidians colonial or solitary species have evolved by convergence multiple times. Colonial Botryllus and Botrylloides are well-studied genera that exhibit colony-wide developmental mechanisms that regulate synchronous and orchestrated cycles of budding and turnover of zooids. The origins of modular developmental mechanisms that facilitated the evolution of coloniality in this group remain unclear. To reconstruct ancestral states of coloniality we studied Symplegma brakenhielmi, a sister taxon of the botryllids. S. brakenhielmi zooids are embedded in a common tunic and present a similar vascular system as the botrylloides, however development and turnover of zooids occurs asynchronously and in a more independent manner. We generated a table of common stages of budding in Symplegma and Botryllus for comparative studies of asexual development. We tested dependent processes of budding among individuals of the colony by systemic bud or zooid removals. Although our results showed a higher degree of independence in bud development in S. brakenhielmi, we found a subtle colony-wide regulatory mechanism of modular development, i.e. new buds expedited development after the removal of all buds in the colony. Next, we characterized external morphology, ultrastructure, and abundance of circulatory blood cells in the vascular system of S. brakenhielmi. Macrophage-like cells (MLCs) are involved in zooid resorption and turnover. Proportions of MLCs in the blood of S. brakenhielmi corresponded to the peak of occurrence of this cell type during the budding cycle of B. schlosseri. We found several new blood cell types in S. brakenhielmi, including two cell types that resemble circulatory progenitor stem cells of other botryllid colonial ascidians. These cells showed features of undifferentiated cells and expressed mitotic marker Phospho-histone H3. Comparative studies of S. brakenhielmi and B. schlosseri allow us to discuss possible changes in the regulation of modular development (i.e. regulation of life and death in the colony), and a possible contribution of circulatory blood cells in budding processes. We propose that the higher degree of developmental independence in S. brakenhielmi budding is a result of its ancestral solitary mode of development., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Toxic metals, such as mercury, contribute substantially to anthropogenic pollution in many estuarine environments. Animals living in those environments, particularly invertebrate filter feeders like tunicates, can be used as bioindicators. In an attempt to identify cellular markers for revealing pollution, this study examined in vitro the effects of different concentrations of methyl mercury on Styela plicata hemocytes. The harvested hemocytes from S. plicata that were exposed to the metal had a significant mortality, cellular count and morphometric alterations. These findings provided evidence of MeHg immunotoxic effects on S. plicata, resulting in hemocyte death and morphological changes induced by cytoskeleton alterations. Thus, a morphometric cellular parameter, such as spreading ability, was used as a complementary method for differentiation between hemocytes treated with a marine solution (as a negative control) and hemocytes incubated with methylmercury and/or Sicilian seawater samples., (Copyright © 2016 Elsevier Inc. All rights reserved.)

When lepidopteran larvae are infected by a large quantity of pathogens or parasitized by nonadaptive parasitoids, hemocytes in the hemocoel will encapsulate these foreign invaders. Cellular encapsulation requires hemocytes, particularly plasmatocytes, to change their states from nonadhesive, spherical cells into adhesive, spreading cells. However, it is unclear how the changes of plasmatocytes are regulated. Here we report that the integrin β1 subunit from hemocytes of Ostrinia furnacalis (Ofint β1) plays an important role in regulating the spreading of plasmatocytes. The full length cDNA sequence (4477 bp) of Ofint β1 was cloned from hemocytes. Phylogenetic analysis showed that Ofint β1 belonged to the integrin βPS family of Drosophila melanogaster with highest sequence identity (78.7%) to the β-integrin of Pseudoplusia includens. Structural analysis of the deduced amino acid sequence indicated that Ofint β1 had similar functional domains to known β-integrins in other lepidopteran insects. RT-PCR, Northern blotting, Western blotting and immunohistochemical analyses showed that OfINT β1 was expressed mainly in hemocytes, especially in plasmatocytes, and weakly in fat body, Malpighian tubes and epidermis. After hemocytes had spread onto slides, fewer antibodies to OfINT β1 bound to the surface of plasmatocytes. Furthermore, anti-OfINT β1 serum clearly inhibited the spreading of plasmatocytes. Together these results indicate that OfINT β1 may play an important role in regulating the spreading of plasmatocytes., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Several bivalve species, including mussels (Mytilus spp.) and clams (Mya spp.), are susceptible to a leukemia-like disease called haemic neoplasia that has been known to decimate whole populations. Previous studies of molecular processes associated with late stages of this disease have implicated analogs of the p53 tumour suppressor protein family in disease etiology. We detected synonymous single nucleotide polymorphisms (SNPs) in the coding region sequence of p53-like cDNA from Mytilus trossulus (bay mussel) that differ between normal and neoplastic haemolymph. SNPs were located at positions 182, 392 and 821 bp. Most (94%) of the late leukemic animals sampled from cages in Burrard Inlet (British Columbia, Canada) had the same p53-like genotype, C182T G392G C821T, whereas 75% of the healthy animals were homozygous at positions C182C and T821T, independent of the genotype at the 392 bp position. As well, we detected an increased number of allelic variants in the leukemic animals that may arise from separate somatic mutation events in haemocyte precursors or from additional p53-like gene copies in polyploidy. Therefore, detection of these SNPs may provide a useful genetic biomarker for efficient monitoring of mussel population health., (2010 Elsevier B.V. All rights reserved.)

Classification of insect larvae circulating haemocytes is the subject of controversy, and the terminology used to designate each cellular type is often different from one species to another. However, a survey of the literature on insect haemocytes suggests that there are resemblances for most of the cell types and functions, in different insect species. In this review paper, we compare the structure and functions of circulating haemocytes in those insect species that are, by far, the most often used species for insect physiology studies, i.e. lepidopteran species and Drosophila. We show that there is high degree of homology of haemocyte types and suggest possible synonymies in terminology among species from these taxa.

The Diachasmimorpha longicaudata entomopoxvirus (DlEPV), the first reported symbiotic entomopoxvirus, occurs in the venom apparatus of D. longicaudata female wasps and is introduced into Anastrepha suspensa larvae during parasitism. The DlEPV 250-300 kb double stranded DNA genome encodes putative proteins having 30 to >60% amino acid identity with poxvirus homologs such as DNA helicase, DNA-dependent RNA polymerase, and the poxvirus-specific rifampicin resistance protein. Although the molecular characterization of DlEPV is progressing, little is known about its morphogenesis in and effects on host haemocytes. This paper describes (1) haemocytes of third instar A. suspensa, (2) DlEPV infection and morphogenesis, and (3) DlEPV-induced changes in haemocytes. A. suspensa third instars have 3-4 haemocyte morphotypes. Dot blots of DNA from infected haemocytes hybridized with a digoxigenin-labeled DlEPV genomic probe as early as 4 h post-parasitism (hpp) and the intensity of the signal increased with time through 40 hpp. Immunofluorescence microscopy localized DlEPV proteins in cytoplasmic (but not nuclear) sites of infected haemocytes, within 24-36 hpp. Electron microscopy confirmed the presence of viral envelopes, immature spheroids with centric nucleoids, budding virus, and extracellular enveloped virus in three haemocyte types, 24-84 hpp and later. Infected haemocytes exhibited blebbing, DNA concatenation, and inability to encapsulate sephadex beads in vitro. These data indicate that DlEPV disrupts the normal function of host haemocytes, thereby insuring the successful development of D. longicaudata offspring and as such should be regarded as a symbiont of the wasp.

We have discovered a new type of haemocyte in the larval stage of the tobacco hornworm moth Manduca sexta that has extreme phagocytic ability; each cell can engulf up to 500 bacteria. This level of phagocytosis may be unprecedented among animal cells. Although these hyperphagocytic cells (HP) only represent about 1% of the circulating haemocytes, they are responsible for sequestering the majority of the bacteria by circulating haemocytes when non-pathogenic, heat-killed Escherichia coli are injected into the haemolymph. Extreme phagocytosis by HP is not limited to Gram-negative bacteria since heat-killed Staphylococcus aureus as well as positively and negatively charged microspheres are also highly phagocytosed. Evidence is presented to show that phagocytosis by HP is involved in the early stages of nodule formation in infected insects. In addition, HP are also present in non-infected insects, characterised by their distinctive spreading morphology, which becomes impaired following hyperphagocytosis of bacteria. This is the first time that a dedicated "professional" phagocytic class of haemocyte has been reported for an invertebrate. The importance of these specialised cell types in the M. sexta immune response and their role in nodule formation is discussed.

Hemopoiesis in orthopteran insects occurs in a hemopoietic organ that is located bilaterally along the aorta. This organ is also known as a reticulo-hemopoietic organ because of the rich presence of reticular cells. This study was performed to further elucidate hemopoiesis in the reticulo-hemopoietic organ of an orthopteran, Euprepocnemis shirakii. We focused on the question why reticular cells are so abundant (35% of cells in hemopoietic organ). Interestingly, 21% of these reticular cells surrounded hemocytes with their reticular cytoplasm. The surrounded hemocytes were distinguished by their different size and darkly stained nucleus. These cells were characterized by immunostaining using antibodies against several types of hemocytes: 45% of the surrounded hemocytes were CD34+, and these positive cells were double stained (over 85%) when immunostained by another hemopoietic pluripotent cell marker, Sca-1. Transmission electron microscopic analysis showed that reticular cells surrounded hemocytes containing large nuclei and poorly developed cytoplasmic organelles. This strongly suggests that the reticular cells surround hemopoietic stem cells. Additionally, surrounded hemopoietic progenitor cells are undergoing apoptosis as indicated by the TUNEL assay. The enclosed apoptotic cells are engulfed and then phagocytosed by reticular cells. Our results suggest that reticular cells are related to the differentiation and apoptosis of hemopoietic stem cells.

Although the lepidopteran larva Pseudaletia separata is attacked by the gregarious ectoparasitoid Euplectrus separatae, it continues to feed and grow. Lipid concentration in the hemolymph of the parasitized host was higher than that of the nonparasitized host from 3 to 8 days after parasitization. Artificial injection of parasitoid venom also elevated lipid concentration in the host hemolymph. One day after venom injection the host's fat body contained many lipid particles, but most of the lipid particles disappeared 7 days later. Light microscopy and transmission electron microscopy showed the lipid particles leaving the fat body cells as a result of the lysis of the fat body cells. These results suggest that the venom elevated the lipid concentration in the host hemolymph by provoking the release of lipid particles from the fat body. Though most of the lipid particles were freely floating in the host hemolymph, a portion of the released lipid particles were phagocytized by hemocytes. The amount of lipid that was loaded to lipophorin in the hemolymph of the venom-injected host was measured, but it was not sufficient to explain the high lipid titer in the hemolymph of parasitized and venom-injected host larvae. The fact that parasitoid larva consumed many hemocytes as evidenced by their presence in the midgut supported the hypothesis that the parasitoid larvae fed on the host hemolymph containing the free lipid particles, the hemocytes phagocytizing the lipid particles, and the lipid-loaded lipophorin. The possibility of the venom contribution to the disruption of the intercellular matrix was examined. The venom showed high activity of matrix metalloproteinase (MMP), especially when it was mixed with the hemolymph of non-parasitized 5th instar larvae. We suggest that the MMP in the venom was activated by some components of the host hemolymph. On the other hand, the venom mixed with hemolymph could not decompose gelatin on zymography, suggesting that the venom-MMP is a different type from gelatinase. Activity of phospholipases A(2), B, C and hyaluronidase were measured with agar plates. High activities of phospholipase B and hyaluronidase were detected. These results suggest that the venom-MMP initially attacked the specific site of the intercellular-matrix of the fat body, and then the hyaluronidase and the phospholipase B cause lysis of the fat body cell, allowing lipid particles to be released into the host hemolymph.

The hemocytes of Rhodnius prolixus were analyzed during the course of infection with the protozoan Trypanosoma rangeli. The following cell types were identified: prohemocyte, plasmatocyte, adipocyte, granular cell and oenocytoid. The number of these cells changes during the infection course thus indicating a cell response to infection of R. prolixus by T. rangeli. Transmission electron microscopy showed that plasmatocytes were able to ingest epimastigote forms of the parasite, which were then found within a parasitophorous vacuole. Amorphous material was seen within the vacuole suggesting that fusion of host cell lysosomes with the vacuole took place. Intravacuolar parasites in process of digestion were observed. In addition, reaction product indicative of the presence of acid phosphatase was observed in parasite-containing vacuoles. No dividing parasites were seen within the vacuole in contrast to what was observed outside the host cells.

Phagocytosis is a complex and apparently evolutionarily conserved process that plays a central role in the immune response to infection. By ultrastructural and functional criteria, Drosophila hemocyte (macrophage) phagocytosis resembles mammalian phagocytosis. Using a non-saturated forward genetic screen for larval hemocyte phagocytosis mutants, D-SCAR and profilin were identified as important regulators of phagocytosis in Drosophila. In both hemocytes ex vivo and the macrophage-like S2 cell line, lack of D-SCAR significantly decreased phagocytosis of Escherichia coli and Staphylococcus aureus. In contrast, profilin mutant hemocytes exhibited increased phagocytic activity. Analysis of double mutants suggests that D-SCAR and profilin interact during phagocytosis. Finally, RNA interference studies in S2 cells indicated that the D-SCAR homolog D-WASp also participates in phagocytosis. This study demonstrates that Drosophila provides a viable model system in which to dissect the complex interactions that regulate phagocytosis.

We have investigated the blood cell types present in Drosophila at postembryonic stages and have analysed their modifications during development and under immune conditions. The anterior lobes of the larval hematopoietic organ or lymph gland contain numerous active secretory cells, plasmatocytes, few crystal cells, and a number of undifferentiated prohemocytes. The posterior lobes contain essentially prohemocytes. The blood cell population in larval hemolymph differs and consists mainly of plasmatocytes which are phagocytes, and of a low percentage of crystal cells which reportedly play a role in humoral melanisation. We show that the cells in the lymph gland can differentiate into a given blood cell lineage when solicited. Under normal nonimmune conditions, we observe a massive differentiation into active macrophages at the onset of metamorphosis in all lobes. Simultaneously, circulating plasmatocytes modify their adhesion and phagocytic properties to become pupal macrophages. All phagocytic cells participate in metamorphosis by ingesting doomed larval tissues. The most dramatic effect on larval hematopoiesis was observed following infestation by a parasitoid wasp. Cells within all lymph gland lobes, including prohemocytes from posterior lobes, massively differentiate into a new cell type specifically devoted to encapsulation, the lamellocyte., (Copyright 2001 Academic Press.)

The frequency of micronuclei (MN) induced by pentachlorophenol (PCP) in haemocytes of zebra mussel, Dreissena polymorpha Pall. and great ramshorn snail, Planorbarius corneus L. was determined over a 14 days of exposure (sampling after 4, 7 and 14 days) under laboratory conditions. PCP doses for zebra mussel ranged from 10 to 150 microg/l, and for ramshorn snail from 10 to 450 microg/l. Micronuclei were detected after bisbenzimide fluorescent staining. Positive responses were observed in both species. The mean MN frequencies in treated mussels ranged between 0.69 and 7.50 per thousand, and between 2.07 and 13.80 per thousand in treated snails. The spontaneous MN levels in mussels averaged from 0.5 to 2.75 per thousand, and in snails from 1.56 to 2.00 per thousand. Our results suggest that haemolymph of both species represent an appropriate test tissue in environmental genotoxicity assessment.

Caged zebra mussels, Dreissena polymorpha, were transplanted to 6 monitoring sites receiving industrial effluents suspected of containing genotoxic chemicals. After a residence time of 2 months, the induction of micronuclei (MN) in haemocytes was determined as a criterion for genetic damage. The mean MN frequencies observed in mussels exposed to effluents ranged between 5.0 and 8.8/1000. These rates were significantly higher than the baseline level of 2.0/1000 recorded in a concurrent control mussel group reintroduced at the reference location. Biological fitness descriptors (mortality, attachment biotest, condition index, gonadic index) revealed no relationship between the general well-being of the mussels exposed under contrasted environmental conditions and the frequency of MN induced in their haemocytes. The biological feasibility of the transfer technique of zebra mussels, together with a moderate, but significant, inducibility of MN, are major features towards the development of a first tool for in situ monitoring of genotoxicity in freshwater environments using a common indicator species.

The micronucleus (MN) test was performed in vivo and in vitro on the oyster Crassostrea gigas to evaluate the genotoxic effect of the marine environment. In vitro tests were carried out on adult and young (spat) specimens exposed to benzo[a]pyrene (BaP: 0.5, 5, 500 and 1000 micrograms.l-1) and an effluent (5, 50, 75 and 100%) of Seine Bay, one of the most highly contaminated sites in France. MN frequency observed after 48 h exposure to the two pollutants was much greater in adults than spats. A preliminary test of the genotoxic effect of BaP (0.05, 0.5, 1 and 500 micrograms.l-1), cupric sulfate (10, 25, 50 and 100 micrograms.l-1) and a paper mill effluent (1, 3, 10 and 30 mg.l-1) was performed in C. gigas heart cells cultured for 6 days. Comparison of the MN assay with the C. gigas larva test showed the clastogenic action of BaP and the toxic effect of cupric sulfate on culture cells as well as the slighter toxic effect of paper mill effluent on spats. An in vivo study was conducted in an oyster-farming area contaminated by cadmium and copper. MN frequency was not very sensitive to a pollution gradient but showed high interindividual variability. The absence of precise criteria for MN identification in mollusks and the identification of highly basophilic spherical inclusions in the cytoplasm of gill tissue hemocytes in oysters during viral infection are handicap for application of the micronuclei assay in the marine environment. Another limitation of the assay is the particularly onerous requirement for manual observation. Optimization of the assay by automated analysis is necessary but can only be achieved if cytologic preparations are of good quality.

Mytilus edulis has been the subject of recent studies to determine whether the relationship between the immune and nervous systems seen in vertebrates also exists in invertebrates. In the present study the effects of experimentally induced "stressful" stimuli on immunoactive hemocytes were studied in this mollusc. This subpopulation of invertebrate blood cells, resembling vertebrate granulocytes, has been previously shown to produce and react to opioid peptides. Their activation, like that of vertebrate immunocytes, expresses itself in distinctive conformational changes preceding cellular mobilization. The cellular response to "stress" observed is the same as that to the administration of exogenous mammalian opioid peptides. This strongly suggests that under the conditions of stressful stimuli, the immune/defense system can be altered by endogenous neuropeptides. The involvement of opioids in neuroimmunoregulatory phenomena appears to have a long evolutionary history.