Morphology Of Hemocytes Research Articles

Characterising phagocytes and measuring phagocytosis from live Galleria mellonella larvae

ABSTRACT Over the last 20 years, the larva of the greater waxmoth, Galleria mellonella, has rapidly increased in popularity as an in vivo mammalian replacement model organism for the study of human pathogens. Experimental readouts of response to infection are most often limited to observing the melanization cascade and quantifying larval death and, whilst transcriptomic and proteomic approaches, and methods to determine microbial load are also used, a more comprehensive toolkit of profiling infection over time could transform the applicability of this model. As an invertebrate, Galleria harbour an innate immune system comprised of both humoral components and a repertoire of innate immune cells – termed haemocytes. Although information on subtypes of haemocytes exists, there are conflicting reports on their exact number and function. Flow cytometry has previously been used to assay Galleria haemocytes, but protocols include both centrifugation and fixation – physical methods which have the potential to affect haemocyte morphology prior to analysis. Here, we present a method for live haemocyte analysis by flow cytometry, revealing that Galleria haemocytes constitute only a single resolvable population, based on relative size or internal complexity. Using fluorescent zymosan particles, we extend our method to show that up to 80% of the Galleria haemocyte population display phagocytic capability. Finally, we demonstrate that the developed assay reliably replicates in vitro data, showing that cell wall β-1,3-glucan masking by Candida albicans subverts phagocytic responses. As such, our method provides a new tool with which to rapidly assess phagocytosis and understand live infection dynamics in Galleria.

Viability Analysis of Biomphalaria glabrata Hemocytes During Schistosoma mansoni Infection And Glyphosate-Based Herbicide Exposure

Pesticides are chemical agents that have a range of harmful effects on human health and the environment. They can cause drastic changes in natural communities, such as macroinvertebrates, plankton and fish. Snails, including Biomphalaria glabrata, are often present in aquatic communities and have multiple roles in limnic ecosystems. B. glabrata is an intermediate host of several species of helminths of medical and veterinary importance, such as Schistosoma mansoni, the etiological agent of schistosomiasis. The original Roundup® herbicide can affect snails and directly affects S. mansoni hemocytes, cells that act in the snails’ immune defense. Here we analyzed the effect of herbicide exposure on B. glabrata hemocytes divided into four groups: control group, infected-only group, treated-only group and infected+treated group. For this, flow cytometry and Neubauer-chamber counting were used to determine the morphology, viability and lectin expression profiles of hemocytes. We observed that the group infected by S. mansoni and treated with herbicide had a higher concentration of dead hemocytes in relation to the other groups. The treated group showed similar results to the control group, suggesting that the herbicide (Roundup™) alone does not interfere with the snails’ immune system. Regarding cellular-morphological-characterization analysis, hyalinocytes were the cells most commonly found in all groups studied. These findings suggest that S. mansoni infection and exposure to pesticides directly the immune system of the snails, stimulating the production of hemocytes, especially hyalinocytes, which have a high phagocytic power to quell the infection, but with toxic effects on the snail.

Hemocyte morphology of worker subcastes of the leaf-cutting ant Atta sexdens rubropilosa (Hymenoptera: Formicidae)

Hemocytes are cells present in the hemolymph of insects that play a role in combating invasive pathogens, ensuring defense by the immune system in these organisms. While the types of hemocytes are well known in some insect representatives, data on these cells in Hymenoptera are limited to certain bees and wasps, with little information available for ants. Among ants, the genus Atta has environmental and economic importance, forming highly organized colonies consisting of the queen and workers, with the latter subdivided into subcastes: gardeners, waste removers, foragers, and soldiers, which are exposed to different pathogens. This study describes the morphology of hemocytes in the worker subcastes of Atta sexdens rubropilosa. Hemolymph samples from the ant were submitted to light, confocal, and scanning electron microscopy analyses. Five types of hemocytes were identified in the hemolymph of all ant subcastes, including prohemocytes, oenocytoids, spherulocytes, plasmatocytes, and granulocytes. They exhibited nuclei with a predominance of decondensed chromatin. The granulocytes were the most abundant cell type in the subcastes, followed by prohemocytes, plasmatocytes, oenocytoids, and spherulocytes. Phagocytosis assays reveal that plasmatocytes and granulocytes are the main phagocytic cells in all castes evaluated. This study fills an important gap in understanding the immune response in this ant species.

Effect of hypersalinic stress on hemocyte morphology and hemolymph cellular composition of the ark clam (Anadarakagoshimensis).

Bivalve mollusks as typical osmoconformers are unable to maintain a constant level of internal osmolarity in conditions of salinity stress. Adaptation to fluctuations of environmental salinity is achieved through cellular osmoregulatory responses, which are accompanied with a substantial shift in functional state of cells. In the present work we investigated the effect of hypersalinity stress on hemolymph cellular composition and morphology of the ark clam (Anadara kagoshimensis) hemocytes. Ark clams were subjected to a gradual increase of environmental salinity from 18‰ to 35‰ and 45‰ and maintained at those conditions for two days. Exposure to hypersalinity 35‰ induced changes in erythrocyte morphology and led to a decrease of their diameter. At salinity 45‰ a substantial increase of hemocyte average diameter was observed, whereas the shape of cells did not change (18‰). Hyperosmotic stress was not associated with changes in hemocyte viability as well as changes in hemolymph cellular composition. The results of the present work demonstrate high tolerance of A. kagoshimensis to short-time exposure to hypersalinic conditions.

Cephalochordate Hemocytes: First Demonstration for Asymmetron lucayanum (Bahamas Lancelet) Plus Augmented Description for Branchiostoma floridae (Florida Amphioxus).

AbstractWithin phylum Chordata, the subphylum Cephalochordata (amphioxus and lancelets) has figured large in considerations of the evolutionary origin of the vertebrates. To date, these discussions have been predominantly based on knowledge of a single cephalochordate genus (Branchiostoma), almost to the exclusion of the other two genera (Asymmetron and Epigonichthys). This uneven pattern is illustrated by cephalochordate hematology, until now known entirely from work done on Branchiostoma. The main part of the present study is to describe hemocytes in the dorsal aorta of a species of Asymmetron by serial block-face scanning electron microscopy. This technique, which demonstrates three-dimensional fine structure, showed that the hemocytes have a relatively uniform morphology characterized by an oval shape and scanty cytoplasm. Ancillary information is also included for Branchiostoma hemocytes, known from previous studies to have relatively abundant cytoplasm; our serial block-face scanning electron microscopy provides more comprehensive views of the highly variable shapes of these cells, which typically extend one or several pseudopodium-like protrusions. The marked difference in hemocyte morphology found between Asymmetron and Branchiostoma was unexpected and directs attention to investigating comparable cells in the genus Epigonichthys. A broader knowledge of the hemocytes in all three cephalochordate genera would provide more balanced insights into the evolution of vertebrate hematopoiesis.

Effects of parasitism by the braconid wasp, Bracon hebetor (Hymenoptera: Braconidae), on the host hemolymph and phenoloxidase activation of the rice moth, Corcyra cephalonica larvae (Lepidoptera: Pyralidae)

BackgroundBiological control of insect pests is an economic, eco-friendly and harmless approach to integrated pest management strategies. Bracon hebetor (Say) (Hymenoptera: Braconidae) considers a polyphagous ectoparasitoid of various pests of the order Lepidoptera. The parasitized host insects' defense mechanisms are triggered as a result of the parasitic wasps' injury and penetration. Thus, induce the host cellular and humoral immune responses through a blend of secretions injected into the host body. The present study was designed to evaluate the efficacy of B. hebetor on the immune response of the parasitized full grown larval instar of the rice moth, Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) under natural envenomation.ResultsOn cellular level, the ultrastructure examination of the hemocytes displayed a considerable structural deformation in hemocyte morphology of the detected types of the hemocytes. Moreover, the effects of parasitism on both differential hemocyte counts (DHCs) and total hemocyte counts (THCs) were investigated. The number of prohemocytes (PR) (40.33 ± 5.61, 43 ± 8.33 and 26 ± 2.31) was statistically differed after (24, 48 and 72 h) of parasitism, respectively, compared to unparasitized (31.33 ± 6.49) larvae. Similar observations were recorded in plasmatocytes (PL) before and after the parasitism. However, Spherulocytes (SP) and Oenocytoids (OE) were recorded in the hemolymph with little abundance. On the other side, the quantitative analysis of total hemolymph proteins (THP) provoked a significant effect of considering parasitized and non-parasitized larvae. As the Phenoloxidase (PO) cascade plays a critical role in immune defenses, so the substantial activation of PO in the host's hemolymph following successive hours of parasitism compared to unparasitized larvae using L-DOPA as a substrate indicated the induction of larval immune system. The parasitized larvae showed a gradual increase in the PO activity (0.442 ± 0.103) after 24 h reached up to (1.482 ± 0.272) at the end of parasitism (72 h) in comparable to unparasitized larvae (0.177 ± 0.0.044).ConclusionThe present investigations clarified the efficacy of B. hebetor parasitism on the host immune mechanism, which will enable the progress of sustainable stored product protection approaches for the control of an important pest rice moth C. cephalonica.

Antioxidant and cellular immune response to acute hypoxia stress in the ark shell (Anadara broughtonii)

In this work we studied antioxidant status and cellular immunity of ark shells (A. broughtonii) to determine the range of the adaptive potential to hypoxia. For this, the effects of 24 h and 72 h periods of exposure to dissolved oxygen concentration 1.5–1.9 mg O2 L−1 were assessed. Hypoxia was not associated with changes in hemocyte morphology as well as with shifts in hemolymph cellular composition. Intracellular reactive oxygen species (ROS) production by hemocytes decreased following the 24 h exposure period but recovered after 72 h of hypoxia. Hypoxia contributed more to reduced phagocytosis and mitochondrial membrane potential of hemocytes. In gills, the activity of catalase and superoxide dismutase was suppressed upon exposure to hypoxia but the expression levels of the appropriate genes were stable. Metabolic depression state appeared to be the main driver for reduced cellular immune status and the activity of antioxidant enzymes in ark shells. Overall, ark shells demonstrated stability of functional state upon exposure to hypoxia and stabilization of cellular immune parameters under prolonged incubation to low dissolved oxygen concentrations.

Haemocyte characterization in invasive Carcinus maenas (Linnaeus, 1758) (Decapoda, Portunidae) from unpolluted vs polluted sites in Argentina

Abstract Pollution, biological invasions and climate change pose the greatest threats to biodiversity worldwide. Few studies explore the interlink between invasive species and local pollution sites. This study describes the type and cytochemical properties of haemocytes from invasive Carcinus maenas on the southwestern Atlantic coast (Argentina). Moreover, the morphology of haemocytes and the total haemocyte count (THC) as well as the differential haemocyte count (DHC) of crabs sampled at clean or unpolluted and polluted sites were compared, to determine whether environmental stresses were evident in their haemocytes. Our results revealed two groups or subpopulations of haemocytes: hyalinocytes, as well as granulocytes and semi-granulocytes. The type of haemocyte from crabs from both sites showed no differences in their morphology. For cytochemical assays, only neutral red dye showed a more intense staining of crab haemocytes at the polluted site. All haemocytes were positive to Periodic Acid-Schiff and Sudan Black. THC of crabs from the clean site were significantly higher, as well as having a significantly higher proportion of hyalinocytes and a lower proportion of granular cells. This study provides a baseline immunological assessment for C. maenas haemocytes, suggesting a differential haemocytic response to environmental stress.

EFFECT OF LEAD NITRATE ON HAEMOCYTE MORPHOLOGY OF FRESHWATER PRAWN, Macrobrachium dayanum (CRUSTACEA-DECAPODA)

Lead (Pb), a non-essential “Grey listed” heavy metal is a serious threat to aquatic flora and fauna. The haematological, nephrological, histopathological and neurological effects of lead are well known in vertebrates. Present study aims to investigate effect of lead on morphology of haemocytes, a important component of immune system of freshwater prawn, Macrobrachium dayanum. Prawns, collected at “Gulala-Ghat” from river Gomti, Lucknow, subjected to acute and sub-acute concentrations of lead nitrate (116.46 mg/l, 96h LC50; 29.12 mg/l, 25% of 96h LC50) showed morphological changes in haemocytes after 24, 48, 72 and 96h in acute exposure and after 10, 20 and 30 days in sub-acute exposure. The chief morphological changes like surface blebbing, vacuolization, cytoplasmic darkening, fragmentation of nuclear material, cone formation, nuclear pycnosis and breaking of plasma membrane were noticed after acute exposure (116.46 mg/litre). Black granular depositions, nuclear pycnosis and fragmentation of nuclear material; pseudopodial projections, blackening of the plasma membrane, achromatia were observed after sub-acute exposure (29.12 mg/litre). These changes were pronounced in prohaemocytes, granulocytes, plasmatocytes and spindle cells. The severity of morphological changes was dose- and duration-dependent. The present study found a positive correlation between heavy metal lead exposure and morphological changes in haemocyte morphology leading to severe immune dysfunction in freshwater prawn M. dayanum. This parameter can be used as bio-marker in health monitoring of prawns as well as environmental monitoring without sacrificing the animal.

Identification and Functional Characterization of Toxoneuron nigriceps Ovarian Proteins Involved in the Early Suppression of Host Immune Response.

Simple SummaryThe endoparasitoid of the tobacco budworm Heliothis virescens, Toxoneuron nigriceps, has several strategies to survive, including venom and calyx fluid. This latter contains a Polydnavirus and Ovarian Proteins (OPs). They are injected into the host body together with the egg. Although much research has focused on venom protein components, little is known about OPs. OPs can disrupt the cellular immune response of the host, acting on host hemocytes, the immune cells. In this study we investigated the action of HPLC fractions derived from OPs. Two fractions caused multiple and significant changes in hemocytes, including cellular oxidative stress and actin cytoskeleton disruption, which might explain the high incidence of hemocyte death and loss of function. Furthermore, using a transcriptome and proteomic approach, we identify the proteins of the two fractions that may be involved in the observed host hemocyte alterations. Our results will help to better understand the OP components and their involvement in parasitization strategies.The endophagous parasitoid Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) of the larval stages of the tobacco budworm Heliothis virescens (Fabricius) (Lepidoptera, Noctuidae) injects the egg, the venom, the calyx fluid, which includes a Polydnavirus (T. nigriceps BracoVirus: TnBV) and the Ovarian Proteins (OPs) into the host body during oviposition. The host metabolism and immune system are disrupted prematurely shortly after parasitization by the combined action of the TnBV, venom, and OPs. OPs are involved in the early suppression of host immune response, before TnBV infects and expresses its genes in the host tissues. In this work, we evaluated the effect of HPLC fractions deriving from in toto OPs. Two fractions caused a reduction in hemocyte viability and were subsequently tested to detect changes in hemocyte morphology and functionality. The two fractions provoked severe oxidative stress and actin cytoskeleton disruption, which might explain the high rate of hemocyte mortality, loss of hemocyte functioning, and hence the host’s reduced hemocyte encapsulation ability. Moreover, through a transcriptome and proteomic approach we identify the proteins of the two fractions: eight proteins were identified that might be involved in the observed host hemocyte changes. Our findings will contribute to a better understanding of the secreted ovarian components and their role in parasitoid wasp strategy for evading host immune responses.

Shift in functional and morphological parameters of the Pacific oyster hemocytes after exposure to hypoxia

Bivalve mollusks are widely spread in shallow areas where they are subjected to fluctuations of dissolved oxygen concentration. The rapidly occurring hypoxia can represent a significant impact on bivalve immunity and functional status of the organism. The Pacific oyster (Crassostrea gigas) is an intensively cultivated introduced species in the Black Sea, which is poorly studied as a region for aquaculture. In this work the effect of 72 h hypoxia (2.1 mg O2L−1) on hemocyte functional parameters as well as immune markers of C. gigas were investigated. Hemolymph cellular composition, hemocyte proliferation level, mortality, and mitochondrial membrane potential, phagocytic capacity and spontaneous reactive oxygen species (ROS) production were evaluated using flow cytometry and microscopy. Exposure to hypoxia significantly affected hemolymph cellular composition by increasing the number of agranulocytes. Granulocyte and hyalinocyte number did not change. After 72 h exposure period changes in hemocyte morphology and massive cell aggregation were observed on slides. Cellular and nuclear diameter of agranulocytes decreased upon exposure to hypoxia The non-stimulated production of ROS significantly decreased in all hemocyte types. Phagocytic activity, hemocyte proliferation and mortality level were not significantly influenced by oxygen deficiency. The results of the present work indicate a pronounced negative impact of 72 h hypoxia on physiological parameters of hemocytes, which, however, was not associated with a decrease of their immune capacity.

First Evidence of In Vitro Effects of C6O4-A Substitute of PFOA-On Haemocytes of the Clam Ruditapes philippinarum.

Alternative chemicals to per- and poly-fluoroalkyl substances have recently been introduced in various industrial processes. C6O4 (difluoro{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetic acid) is a new surfactant and emulsifier used as a replacement for perfluorooctanoic acid (PFOA). From an ecotoxicological point of view, in vitro assays are useful tools for assessing the negative effects and understanding the mechanisms of action of chemicals at the cellular level. Here, we present the results of an in vitro study in which the effects of C6O4 were evaluated—for the first time—on haemocytes of the clam Ruditapes philippinarum. Cells were exposed to three concentrations of C6O4 (0.05, 0.5, 5 μg/mL) and the effects on haemocyte viability, haemocyte morphology, differential haemocyte count, lysosomal membrane stability, superoxide anion production, acid phosphatase, and β-glucuronidase activities, as well as on the percentage of micronuclei and chromosomal aberrations were evaluated. The results demonstrated that C6O4 significantly affected haemocyte morphology, lysosomal membrane stability, hydrolytic enzyme activity, and superoxide anion production, and promoted chromosomal aberrations. To the best of our knowledge, this is the first study revealing the in vitro effects of C6O4, a substitute for PFOA, on haemocytes from a bivalve species.

Effect of Short-Term Desiccation, Recovery Time, and CAPA-PVK Neuropeptide on the Immune System of the Burying Beetle Nicrophorus vespilloides.

Environmental conditions, especially related to winter, are crucial for shaping activity of insect immune system. However, our previous research clearly indicates differences in the immune system functioning when the cold stress was induced in the laboratory conditions and when the beetles were collected from natural environment during winter. This is probably related to the multiplication of observed effects by simultaneous presence of different stress factors characteristic of winter, including desiccation. For these reasons, our next step was analysis of the effects of short-term desiccation and recovery time on the functioning of immune system of burying beetle Nicrophorus vespilloides. Also, the effect of Tenmo–PVK-2 (tenebrionid periviscerokinin), member of the CAPA–PVK neuropeptide family, was investigated to better understand observed changes. Short-term desiccation decreases the phagocytic activity of burying beetle haemocytes, which is correlated with a reduction in their adhesive ability. On the other hand, there was a significant increase in phenoloxidase (PO) activity and the level of proPO expression, which may suggest sealing the cuticula by melanin deposition and prevention of water loss. Additionally, the elevated level of defensin expression may be associated with the cross-talk between mechanisms, which participate in insect response to environmental stress, including pathogen infection. After 1 h of recovery time, the activity of tested cellular and humoral mechanisms was mostly back to the control level. However, inhibition of the activity of PO and down-regulation of proPO were noted. These results also indicate importance of melanin deposition during water loss. Moreover, it suggests that some changes in immune system functioning during stress conditions do not have an immune function. Interestingly, part of the effects characteristic of recovery time were also observed after the application of Tenmo–PVK-2, mainly related to haemocyte morphology. These results indicate that CAPA–PVK neuropeptides may also influence on activity of burying beetle immune system. It should be also highlighted that, because of the study of the effects of CAPA–PVK neuropeptides, homologs of vertebrate neuromedin U, the results may be interesting for search evolutionary similarities in the functioning of the neuroendocrine system of insects and vertebrates.

Flow cytometric analysis of morphologic and immunological characterisation of the tiger shrimp Penaeus monodon haemocytes

Morphology and immune functions of Penaeus monodon haemocytes were analysed by flow cytometry (FCM). Basing on the relative size and granularity, there are three types of haemocyte identified by FCM, including hyaline cells (HC), semigranular cells (SGC) and granular cells (GC). GC and SGC were significantly larger than HC. GC contained more granules in the cytoplasm than the two other types. SGC were the most abundant cells, while HC were the least. The mean total apoptotic cell ratio was 5.09 % in the fresh shrimp haemocytes. SGC contributed most to the phagocytic activity, and HC contributed least, probably due to the great difference of percentage. GC had most mass of mitochondria and lysosome, and HC contained least. Defined by different characteristic of lysosomal mass, three subsets of haemocytes were observed. GC were most active in non-induced and zymosan-induced reactive oxygen species (ROS) production, while HC showed lowest oxidative activity. Both non-induced and zymosan-induced esterase activity (EA) in GC were higher than those in SGC and HC. The present flow cytometric analysis clearly demonstrated the variation in morphologic and immunological characterisation among the three haemocyte subpopulations. The facts that SGC account for most of the number of circulating haemocyte, while GC contain most organelles in relation to energy supply and immune defence, and have highest immune activites, suggesting that GC and SGC are the main haemocyte types involved in the cellular defence of P. monodon.

Seasonal abundance of Cotesia ruficrus (Hymenoptera: Braconidae) and its host tea looper, Hyposidra talaca (Lepidoptera: Geometridae) in tea ecosystem

Investigations were undertaken for a better understanding of seasonality, interaction with tea ecosystem, and parasitic impact of an endoparasitoid Cotesia ruficrus (Haliday) (Hymenoptera: Braconidae) on the destructive tea looper Hyposidra talaca (Walker) (Lepidoptera: Geometridae). The population abundance of C. ruficrus was noticed as high in the months of pre-winter and spring seasons which was coincided with the population increase of H. talaca. C. ruficrus found to foraging and collecting nectar from the flowers of Lantana camera L., Moringa oleifera Lam., and Clerodendron infortunatum L. associated with the tea ecosystem. The life table parameters including intrinsic rate of natural increase (rm ) and net reproductive rate (Ro) of C. ruficrus were 0.203 per day and 41.11 female offspring/female, respectively, with the gross reproductive rate (∑mx) 50.09 eggs/female. The parasitic impact of C. ruficrus has significantly affected the food consumption and development of H. talaca. Resultantly, a drastic reduction in body weight, changes in differential hemocyte count (DHC), and morphology of hemocytes were observed in parasitized host larvae. Toxic effect of quinalphos and thiamethoxam on C. ruficrus showed 100% mortality followed by bifenthrin > deltamethrin > emamectin benzoate > flubendiamide > fenpyroxymate after 72 h of the application.

Biocidal efficacy of tutin and its influence on immune cells and expression of growth-blocking and neuroglian peptides in Mythimna separata (Lepidoptera: Noctuidae).

Mythimna separata Walker (Lepidoptera: Noctuidae) is one of the major pests that can cause severe damage to grain crops. The development of low-toxicity and high-performance botanical insecticides is becoming the focus of new pesticide research to control M. separata. Tutin, a sesquiterpene lactone compound obtained from Coriaria sinica Maxim, a native Chinese poisonous plant, has antifeedant, absorption, and stomach poisoning against a variety of pests. To understand the toxic effect of tutin on M. separata larvae, we set out to determine their antifeedant, mortality, paralysis, weight change, and to examine the spreading of M. separata hemocytes under different concentrations of tutin treatment. Tissue distribution of the immune-associated gene growth-blocking peptide (GBP) and neuroglian peptide (Nrg) was detected by reverse transcription polymerase chain reaction (PCR). Furthermore, real-time quantitative PCR was carried out to determine the expression profiles of GBP and Nrg after different concentrations of tutin stimulation. Our results revealed that tutin exhibited significant antifeedant and insecticidal activities, paralysis, weight loss to M. separata. Besides, tutin significantly influenced on the morphology of hemocytes and enhanced the expression of GBP and Nrg in M. separata.

Pendimethalin-based herbicide impairs cellular immune response and haemocyte morphology in a beneficial ground beetle

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.

IMPACT OF LOW SALINITY ON HEMOCYTES MORPHOLOGY AND FUNCTIONAL ASPECTS IN INVASIVE CLAM ANADARA KAGOSHIMENSIS (TOKUNAGA, 1906)

Impact of low salinity on morphology and function of hemocytes in ark clam species Anadara kagoshimensis was investigated using light microscopy and flow cytometry. In control group the water salinity was adjusted to 19.6‰, and experimental group was maintained at 14.8‰ and 8.8‰. Two cell types, amebocytes and erythrocytes, were identified in control group of ark clams. Erythrocytes constituted the main type of the cells, and amounted to 92.3±3.9 %. Hyposalinity changed that proportion: the number of amebocytes decreased 2.7 times and number of erythrocytes increased 7.6 times. Morphometric characteristics of hemocytes didn’t show statistically significant changes. As far as salinity decreased, the number of erythrocyte shades in hemolymph increased (3.5 times at salinity 8.8‰) and, in hemocytes, the reactive oxygen species (ROS) production grew (3.5. times at salinity 8.8‰).

Functional and Morphological Changes Induced in Mytilus Hemocytes by Selected Nanoparticles.

Nanoparticles (NPs) show various properties depending on their composition, size, and surface coating, which shape their interactions with biological systems. In particular, NPs have been shown to interact with immune cells, that represent a sensitive surveillance system of external and internal stimuli. In this light, in vitro models represent useful tools for investigating nano-bio-interactions in immune cells of different organisms, including invertebrates. In this work, the effects of selected types of NPs with different core composition, size and functionalization (custom-made PVP-AuNP and commercial nanopolystyrenes PS-NH2 and PS-COOH) were investigated in the hemocytes of the marine bivalve Mytilus galloprovincialis. The role of exposure medium was evaluated using either artificial seawater (ASW) or hemolymph serum (HS). Hemocyte morphology was investigated by scanning electron microscopy (SEM) and different functional parameters (lysosomal membrane stability, phagocytosis, and lysozyme release) were evaluated. The results show distinct morphological and functional changes induced in mussel hemocytes depending on the NP type and exposure medium. Mussel hemocytes may represent a powerful alternative in vitro model for a rapid pre-screening strategy for NPs, whose utilization will contribute to the understanding of the possible impact of environmental exposure to NPs in marine invertebrates.

Plants impact cellular immunity of caterpillars to an entomovirus.

Tri-trophic interactions among plants, insect herbivores and entomopathogens are one of the hot topics in ecology. Although plants have been shown to impact the interactions between herbivores and entomopathogens, it is still unclear how plants affect the cellular immunity of herbivores to entomopathogens. The number of hemocytes and the proportion of two main cell types (granular hemocytes and plasmatocytes), plasmatocyte-spreading rate, apoptosis rate, two Spodoptera exigua caspase (SeCasp-1, SeCasp-5) activities and gene expressions were all higher and the activities and gene expression of S. exigua inhibitor of apoptosis protein (SeIAP) were lower in nucleopolyhedrovirus (NPV)-infected caterpillars fed Ipomoea aquatica than those fed other plants or artificial diet. Scanning electron microscopy images were consistent with molecular patterns of immune responses. This study suggests that host plants affect the immune responses of herbivores to entomopathogens by manipulating the composition, morphology and apoptosis of herbivore hemocytes, which sheds light on the mechanisms that allow host plants to influence multi-trophic interactions. © 2021 Society of Chemical Industry.