Changes in haemolymph parameters and insect ability to respond to immune challenge during overwintering

Overwintering is a challenging period in the life of temperate insects. A limited energy budget characteristic of this period can result in reduced investment in immune system. Here, we investigated selected physiological and immunological parameters in laboratory‐reared and field‐collected harlequin ladybirds (Harmonia axyridis). For laboratory‐reared beetles, we focused on the effects of winter temperature regime (cold, average, or warm winter) on total haemocyte concentration aiming to investigate potential effects of ongoing climate change on immune system in overwintering insects. We recorded strong reduction in haemocyte concentration during winter; however, there were only limited effects of winter temperature regime on changes in haemocyte concentration in the course of overwintering. For field‐collected beetles, we measured additional parameters, specifically: total protein concentration, antimicrobial activity against Escherichia coli, and haemocyte concentration before and after overwintering. The field experiment did not investigate effects of winter temperature, but focused on changes in inducibility of insect immune system during overwintering, that is, measured parameters were compared between naïve beetles and those challenged by Escherichia coli. Haemocyte concentration decreased during overwintering, but only in individuals challenged by Escherichia coli. Prior to overwintering, the challenged beetles had a significantly higher haemocyte concentration compared to naïve beetles, whereas no difference was observed after overwintering. A similar pattern was observed also for antimicrobial activity against Escherichia coli as challenged beetles outperformed naïve beetles before overwintering, but not after winter. In both sexes, total protein concentration increased in the course of overwintering, but females had a significantly higher total protein concentration in their hemolymph compared to males. In general, our results revealed that insect’s ability to respond to an immune challenge is significantly reduced in the course of overwintering.     

Insect haemolymph: Cooperation between humoral and cellular factors in Locusta migratoria

In Locusta migratoria, prophenoloxidase is present in the plasma and serum, but in reduced amounts relative to the haemocytes. This phenoloxidase activity cannot be induced by either heating or freezing and thawing and it is lost by heating at 70°C for 30 min. Both lipopolysaccharides and laminarin can elicit the prophenoloxidase activating system. These elicitors of prophenoloxidase activation are active in haemocyte lysate and in serum but never induce any phenoloxidase activity in plasma. In haemocyte lysate, the activating system is not heat resistant, and heating at 56°C for 30 min prior to incubation with laminarin or lipopolysaccharide precludes any phenoloxidase activity. Plasma contains a strong inhibitor of the prophenoloxidase activating system but serum does not. This inhibitor does not affect the phenoloxidase enzyme itself. The possible role of the activating system in immune recognition and the strategies evolved by parasites or pathogens to escape being recognized by their host are discussed.     

Steinernema carpocapsae DD136: metabolites limit the non-self adhesion responses of haemocytes of two lepidopteran larvae, Galleria mellonella (F. Pyralidae) and Malacosoma disstria (F. Lasiocampidae)

Live adult and juvenile entomopathogenic Steinernema carpocapsae DD136 (P. Nematoda) were not subjected to adhesion by haemocytes of lepidopteran insect larvae of Galleria mellonella or Malacosoma disstriain vitro or in vivo. In vitro freeze-killed nematodes exhibited haemocyte attachment, the intensity increasing with time. Accumulation of haemocytes on the dead nematodes was associated with host phenoloxidase activity; live nematodes and their exudates did not activate the enzyme whereas dead nematodes but not their exudate did activate phenoloxidase. Live-nematode exudate inhibited granular cell and some plasmatocyte adhesion to slides, increased granular cell but not plasmatocyte dissociation from preformed haemocyte monolayers and in vivo elevated total haemocyte counts and changed the floating haemocyte types while impairing bacterial removal from the haemolymph. Dead-nematode exudate did not affect these parameters thus immunosuppressant activity by live nematodes may represent the release of inhibitors not associated with their cuticle. The third stage juveniles released the inhibitors.     

Immunocompetence increases with larval body size in a phytophagous moth

Despite the obvious benefit of an immune system, its efficacy against pathogens and parasites may show great variation among individuals, populations and species. Understanding the causes of this variation is becoming a central theme in ecology. Many biotic and abiotic factors are known to influence immunocompetence (temperature, age, etc.). However, for a given age, size among individuals varies, probably as a result of accumulated resources. Thus, these variable resources could be allocated to immune defence and, consequently, body size may explain part of the variation in immune responsiveness. However, the influence of body size on immune defence is often overlooked. The present study investigates variations in haemocyte count and phenoloxidase activity in larvae of the phytophagous vine moth Eupoecilia ambiguella Hübner of the same age, although differing in body size. The measurements of immune function are made both when the insects are immunologically naïve and 24 h after a bacterial immune challenge. The base levels of these immune parameters do not covary with body size in naïve larvae. After the bacterial immune challenge, more haemocytes and phenoloxidase enzyme are mobilized, and the mobilization of these immune effectors is correlated positively with individual body size. Thus, larger larvae exhibit higher immunocompetence than smaller ones, suggesting that smaller larvae might be more vulnerable to infection. These results suggest that body size is probably an underestimated variable, which nevertheless modulates the insect immune system and should thus be considered as a covariate in insect immune system measurement. It is recommended therefore, that body size should be taken into account in ecological immunity studies with insects. © 2013 The Royal Entomological Society     

Cresolase, catecholase and laccase activities in haemocytes of the red swamp crayfish

Phenoloxidase activity in crayfish haemocyte lysates and extracts of haemocyte membranes were studied using native PAGE and SDS-PAGE gels and staining for cresolase, catecholase and laccase activities. The activation of the proenzyme, prophenoloxidase to phenoloxidase, in native PAGE was demonstrated following exposure to SDS. By staining samples separated in SDS-PAGE followed by renaturation, a high molecular mass phenoloxidase activity was identified in both the soluble and membrane fractions of haemocyte preparations. The membrane-associated activity appeared at only relatively high molecular mass (> 300 kDa), and could easily be eluted from membranes using detergents or NaCl. Further, this membrane-associated activity has a catecholase activity but not the cresolase activity seen in the soluble preparations. In addition, several other phenoloxidase enzymes were identified with different relative mobilities (250, 80, 72 and 10 kDa). Crayfish haemocytes also contained laccase activity, thought to be restricted to cuticle sclerotisation in the integument. Laccase activity in haemocytes might aid in the formation of capsule used to contain pathogens.     

Dim artificial light at night reduces the cellular immune response of the black field cricket,Teleogryllus commodus

A functioning immune system is crucial for protection against disease and illness, yet increasing evidence suggests that species living in urban areas could be suffering from immune suppression, due to the presence of artificial light at night (ALAN). This study examined the effects of ecologically relevant levels of ALAN on three key measures of immune function (haemocyte concentration, lytic activity, and phenoloxidase activity) using a model invertebrate species, the Australian black field cricket, Teleogryllus commodus. We reared crickets under an ecologically relevant daily light‐cycle consisting of 12 hr bright daylight (2600 lx) followed by either 12 h darkness (0 lx) or dim environmentally relevant ALAN (1, 10, 100 lx), and then assessed immune function at multiple time points throughout adult life using haemolymph samples. We found that the presence of ALAN had a clear negative effect on haemocytes, while the effects on lytic activity and phenoloxidase activity were more complex or largely unaffected by ALAN. Furthermore, the effects of lifelong exposure to ALAN of 1 lx were comparable to those of 10 and 100 lx. Our data suggest that the effects of ALAN could be large and widespread, and such reductions in the core immune response of individuals will likely have greater consequences for fitness and survival under more malign conditions, such as those of the natural environment.     

Costs of resistance: genetic correlations and potential trade-offs in an insect immune system

Theory predicts that natural selection will erode additive genetic variation in fitness-related traits. However, numerous studies have found considerable heritable variation in traits related to immune function, which should be closely linked to fitness. This could be due to trade-offs maintaining variation in these traits. We used the Egyptian cotton leafworm, Spodoptera littoralis, as a model system to examine the quantitative genetics of insect immune function. We estimated the heritabilities of several different measures of innate immunity and the genetic correlations between these immune traits and a number of life history traits. Our results provide the first evidence for a potential genetic trade-off within the insect immune system, with antibacterial activity (lysozyme-like) exhibiting a significant negative genetic correlation with haemocyte density, which itself is positively genetically correlated with both haemolymph phenoloxidase activity and cuticular melanization. We speculate on a potential trade-off between defence against parasites and predators, mediated by larval colour, and its role in maintaining genetic variation in traits under natural selection.     

Salinity effects on immune parameters of Ruditapes philippinarum challenged with Vibrio tapetis

The occurrence of brown ring disease (BRD) in farmed Manila clams Ruditapes philippinarum is seasonal. Development of the disease is believed to require the presence of the infective agent Vibrio tapetis and particular environmental conditions. This paper studies the effect of salinity (20 to 40 per thousand) on measurable immune parameters of Manila clams, and the progression of BRD in experimentally infected individuals. At 20 per thousand salinity, the total haemocyte count was reduced and disease prevalence was highest. At 40 per thousand salinity significantly fewer clams presented signs of BRD, and this was correlated with increases in the total haemocyte count, hyalinocyte count, phenoloxidase levels and phagocytic activity of haemocytes. Inoculation of clams with V. tapetis did not have a significant effect on the immune parameters measured. Thus, this laboratory-based study relates environmental stress to disease development.     

Dopamine depresses immunity in the tiger shrimp Penaeus monodon

The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, phagocytic activity and clearance efficiency to the pathogen Photobacterium damsela were measured when tiger shrimp Penaeus monodon (13.5+/-1.5 g) were individually injected with saline or dopamine at 10(-8), 10(-7), or 10(-6)mol shrimp(-1). Results showed that a transient period of immunosuppression occurred between 2 and 8h after injection of dopamine for all immune parameters except circulating haemocytes, and all immune parameters had returned to control values within 8-16 h after receiving dopamine. The injection of dopamine also significantly increased the mortality of P. monodon challenged with the pathogen Pho. damsela. These results suggest that stress-inducing dopamine suppresses the immune system, which in turn promotes the susceptibility of P. monodon to Pho. damsela.     

Indirect plant-mediated effects on insect immunity and disease resistance in a tritrophic system

Host plant quality can significantly influence the growth and condition of phytophagous insects, and consequently their susceptibility to pathogens. This study examined the relationship between host plant quality, insect condition, immune responsiveness and resistance to pathogens in the cabbage looper, Trichoplusia ni. Two baseline and induced immune parameters were estimated, haemocyte numbers and haemolymph phenoloxidase (PO) activity, for larvae on two host plants, broccoli and cucumber. Haemolymph protein concentration was assessed as an indication of insect condition, and the susceptibility of larvae to T. ni single nucleopolyhedrovirus (SNPV) was used as a measure of disease resistance. T. ni growth, survival and condition was much higher on broccoli than cucumber. Haemocyte numbers were significantly higher in broccoli-reared larvae, whereas PO activity was not. An immune challenge induced significantly elevated numbers of haemocytes for larvae reared on both host plants, but did not affect PO activity or protein concentrations. Susceptibility to T. ni SNPV was markedly higher in larvae reared on cucumber than on broccoli. These results clearly indicate that host plant quality can affect both immune response and disease resistance of T. ni larvae and that bottom-up effects could be important in interactions between insects and entomopathogens.     

Immune function responds to selection for cuticular colour in Tenebrio molitor

Cuticular colour in the mealworm beetle (Tenebrio molitor) is a quantitative trait, varying from tan to black. Population level variation in cuticular colour has been linked to pathogen resistance in this species and in several other insects: darker individuals are more resistant to pathogens. Given that cuticular colour has a heritable component, we have taken an experimental evolution approach: we selected 10 lines for black and 10 lines for tan adult cuticular phenotypes over at least six generations and measured the correlated responses to selection in a range of immune effector systems. Our results show that two immune parameters related to resistance (haemocyte density and pre-immune challenge activity of phenoloxidase (PO)) were significantly higher in selection lines of black beetles compared to tan lines. This may help to explain increased resistance to pathogens in darker individuals. Cuticular colour is dependent upon melanin production, which requires the enzyme PO that is present in its inactive form inside haemocytes. Thus, the observed correlated response to selection upon cuticular colour and immune variables probably results from these traits' shared dependence on melanin production.     

The antioxidants dimethylsulfoxide and dimethylthiourea affect the immediate adhesion responses of larval haemocytes from 3 lepidopteran insect species

Antioxidants, dimethylsulfoxide (DMSO) and dimethylthiourea (DMTU), at concentrations not affecting the viability of blood cells (haemocytes) from the larval stage of 3 lepidopteran insects - Galleria mellonella, Lymantria dispar, and Malacosoma disstria - differed in their influence on the innate binding of haemocytes to glass, bacteria to haemocytes, and on humoral responses to alien materials. In vitro DMSO had little effect, whereas DMTU substantially impaired the adhesion of the haemocyte types, the plasmatocytes and granular cells, to slides as well as the attachment of Bacillus subtilis to these haemocytes. Although both antioxidants increased lysozyme and phenoloxidase activities, there was no correlation of enzyme activity and haemocyte adhesion responses, possibly reflecting sequestered radicals. Nitric oxide and hydroxyl radicals offset the DMTU effect. In the absence of antioxidants, inactivate protein kinases A (PKA) and C (PKC) enhanced haemocyte aggregation. In general, DMSO, as opposed to DMTU, did not alter the effects of PKA and PKC activators and inhibitors on haemocyte aggregation or of PKC and PKA activities. High concentrations of DMSO and all levels of DMTU, although inhibiting PKA and PKC, inhibited haemocyte adhesion to slides. Comparable results occurred for DMTU-treated haemocytes incubated with B. subtilis. In vivo DMSO, unlike DMTU, did not impair plasmatocyte or granular cell responses to foreign materials, including bacterial removal from the haemolymph and nodulation.     

Immune responses of the scallop Chlamys farreri after air exposure to different temperatures

The present study examined the influence of air exposure at different temperatures: a common perturbation associated with aquaculture handling practices, on immune responses in zhikong scallop Chlamys farreri. Scallops were exposed to air for 2 h, 6 h, 12 h and 24 h at 5 °C, 17 °C and 25 °C respectively. Thereafter, a recovery period of 24 h at 17 °C was applied. Haemocyte mortality, phagocytosis and reactive oxygen species (ROS) production of haemocytes, acid phosphatase (ACP) and superoxide dismutase (SOD) activity in haemocyte lysates were chosen as immunomarkers of anoxic stress. The results showed that an increase of haemocyte mortality and a decrease of phagocytosis and ACP activity were observed after 2 h of air exposure for all temperatures tested. Moreover, a significant increase of ROS production occurred following 2 h of air exposure at 25 °C and 24 h of air exposure at 17 °C. Significant differences were also observed in haemocyte mortality, percentage of phagocytic cells and ACP and SOD activity depending on the temperature of air exposure. Finally, after 24 h of recovery at 17 °C, percentage of phagocytic haemocytes and ACP activity did not return to initial values. ROS production was significantly higher than before the recovery period and initial values for scallops subjected to air exposure at 5 °C. In our study, scallops showed a relative low anoxia tolerance under a high temperature. All the scallops air exposed to 25 °C died after the 6 h sampling. In conclusion, air exposure associated to aquaculture practices was demonstrated to strongly affect functional immune activities of scallop haemocytes, and high temperature air exposure caused reduced survival of scallops.     

Effect of water temperature on the immune response and infectivity pattern of white spot syndrome virus (WSSV) in freshwater crayfish

The susceptibility of two species of freshwater crayfish, Pacifastacus leniusculus and Astacus astacus, to white spot syndrome virus (WSSV) by intramuscular injection was compared and the results show that both species are susceptible to WSSV. The effect of water temperature on the development of white spot disease in crayfish was also studied. Crayfish were exposed to different temperatures after WSSV injection or oral exposure and the mortalities were recorded over a period of 45 days. No mortality was observed when crayfish were held at 4+/-2 degrees C or 12+/-2 degrees C and reached 100% when these crayfish transferred to 22+/-2 degrees C. The mortalities of nearly moribund crayfish at 22+/-2 degrees C with WSSV could be delayed after transfer to temperature below 16 degrees C. These results clearly show that low temperature affects the WSSV pathogenicity in crayfish. Moreover, haemocyte counts, phenoloxidase activity, mRNA levels of prophenoloxidase (proPO) and the lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) in crayfish exposed to various water temperatures were studied. Total haemocyte and granular cell counts of crayfish held at different temperatures were not significantly (P>0.05) different, except for the total haemocyte number at 18 degrees C was significantly (P<0.05) higher than in crayfish at 4 degrees C. The percentage of granular cells in crayfish held at 4 degrees C was the highest compared to crayfish maintained at other temperatures. The phenoloxidase activities in haemocyte lysate supernatant (HLS) of crayfish at all temperature groups remained similar. The amount of proPO-mRNAs in haemocytes was much higher than the amount of LGBP-m RNAs in all the experimental groups. However, there was no change in the level of pro PO-mRNA at the tested temperatures. Interestingly, the level of LGBP-mRNA of crayfish kept at 22 degrees C was much lower than in those held at lower temperatures. Proliferation of the haematopoietic tissues was higher at high temperatures which may support replication of WSSV, and explain the high mortality of crayfish with WSSV infection at high temperature. Based on these studies it is concluded that crayfish might act as a carrier of WSSV at low water temperature and could develop white spot disease if the water temperature is increased.     

In vivo dynamics of an immune response in the bumble bee Bombus terrestris

Concepts from evolutionary ecology have recently been applied to questions of immune defences. However, an important but often neglected aspect is the temporal dynamics of the simple immune measures used in ecological studies. Here, we present observations for workers of the bumble bee Bombus terrestris on the dynamics of the phenoloxidase (PO) system, antibacterial activity, and the total number of haemocytes following a challenge with immune elicitors (LPS, Laminarin), over a time-span ranging from 1min to 14 days. The dynamics of the PO measurement showed a complex pattern and was correlated with haemocyte counts. Antibacterial activity, on the other hand, increased sharply between 2 and 24h post-challenge followed by a slow decrease. Surprisingly, the effects of a challenge lasted up to 14 days.     

Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides

Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety (detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Rc treated cells (P相似文献   

Dopamine depresses the immune ability and increases susceptibility to Lactococcus garvieae in the freshwater giant prawn, Macrobrachium rosenbergii

The total haemocyte count (THC), phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase activity, and phagocytic activity and clearance efficiency to the pathogen Lactococcus garvieae were measured when freshwater giant prawns Macrobrachium rosenbergii (16.2 +/- 2.1 g) were individually injected with saline, or dopamine at 0.5, 5.0, or 50.0 pmol prawn(-1). The results show that a transient period of immunosuppression occurred between 2 and 8 h after injection of dopamine for all immune parameters except circulating haemocytes and all immune parameters returned to control values within 8-16 h after receiving dopamine. Injection of dopamine also significantly increased the mortality of M. rosenbergii challenged with the pathogen L. garvieae. These results suggest that stress-inducing dopamine suppresses the immune system, which in turn promotes the susceptibility to L. garvieae in M. rosenbergii.     

Effect of an entomopathogenic nematode,Steinernema carpocapsae on haemocyte profile and phenoloxidase activity of the Colorado potato beetle,Leptinotarsa decemlineata

The Colorado potato beetle (CPB), Leptinotarsa decemlineata Say is the most destructive insect pest of potato in many areas of the world. Little is known about the haemocyte types of the CPB and its plasma phenoloxidase (PO). In this regard, we investigated the haemocyte profile and PO of CPB and its immune response to the entomopathogenic nematode, Steinernema carpocapsae. Five types of haemocytes, the plasmatocytes (~67.4%), granulocytes (~23.5%), oenocytoids (~2.4%), spherulocytes (~0.25%) and prohaemocytes (~6.5%) were identified in fourth instar CPB larvae. Total haemocyte counts (THCs) were significantly reduced in nematode-injected insects compared with control groups (P < 0.05). Nematode cellular encapsulation observed in haemolymph of nematode-injected insects may partially explain decreased THCs. Plasma PO assay showed increased PO activity in nematode-injected insects compared with control groups (P < 0.05). Plasma PO assay on native polyacrylamide gel electrophoresis (PAGE) assay with L-3, 4-dihydroxyphenylalanine as substrate showed five bands (with molecular weights of approximately 200, 118, 68.5, 62.5 and 58.75 kDa).     

The role of haemocytes from the crab Carcinus aestuarii (Crustacea,Decapoda) in immune responses: A first survey

For the first time, a functional study of haemocytes from the crab Carcinus aestuarii was performed in order to evaluate their involvement in immune responses. Total haemocyte count (THC), phagocytosis, haemolymph opsonisation properties, hydrolytic and oxidative enzyme activities, and production of intracellular superoxide anion were evaluated. A great variability in THC was recorded among individuals, and haemocyte mean number was 6.4 (×10⁶) cells/ml haemolymph. Although only hyalinocytes were able to phagocytose yeast cells or Zymosan, phagocytic index was low (3%) and did not increase significantly (4%) after pre-incubation of yeast and Zymosan in cell-free haemolymph, suggesting that haemolymph did not have opsonising properties. All haemocyte types produced superoxide anion, whereas only granulocytes were positive to the hydrolytic enzymes assayed. In addition, only granulocytes were positive to phenoloxidase activity. Both Petri dish and spectrophotometric assays revealed a very low lysozyme-like activity in cell-free haemolymph (CFH) and haemocyte lysate (HL), although enzyme activity was higher in CFH than in HL. Interestingly, normalisation of data as to total protein content in CFH and HL resulted in an opposite situation, lysozyme-like activity being higher in HL than in CFH. This demonstrated that haemolymph of C. aestuarii has a high quantity of total proteins, functional properties of which need to be better investigated in future studies. Overall, the results obtained in the present study indicated that C. aestuarii haemocytes are not very active phagocytic cells, but they are more active in terms of both hydrolytic and oxidative enzyme activities and superoxide anion production.     

Effect of the insect pathogenic bacterium Photorhabdus on insect phagocytes

Photorhabdus are insect pathogenic bacteria that replicate within the insect haemocoel following release from their entomopathogenic nematode symbionts. To investigate how they escape the cellular immune response we examined the effects of two strains of Photorhabdus, W14 and K122, on Manduca sexta phagocytes (haemocytes), in vitro and in vivo. Following injection of Esherichia coli into Manduca larvae, these non-pathogenic bacteria are rapidly cleared from the haemolymph and the number of free haemocytes transiently increases. In contrast, following injection of either strain of pathogenic Photorhabdus, the bacteria grow rapidly while the number of haemocytes decreases dramatically. In vitro incubation of haemocytes with either Photorhabdus supernatant reduced haemocyte viability, and the W14 supernatant caused distinct changes in the actin cytoskeleton morphology of different haemocyte cell types. In phagocytosis assays both Photorhabdus strains can inhibit their own phagocytosis whether the bacterial cells are alive or dead. Further, the supernatant of W14 also contains a factor capable of inhibiting the phagocytosis of labelled E. coli. Together these results suggest that Photorhabdus evades the cellular immune response by killing haemocytes and suppressing phagocytosis by mechanisms that differ between strains.