Is cell surface calreticulin involved in phagocytosis by insect hemocytes?

The innate immune system of insects consists of humoral and cellular components involved in the recognition of and responses to intruding foreign micro- or macroorganisms. Several molecules have been identified so far that recognize molecular patterns present on microorganisms, such as lipopolysaccharides, peptidoglycans and lipoteichonic acid. These molecules, acting as opsonins, trigger immune responses such as phagocytosis, nodule formation, melanization and encapsulation. Here, we investigated the role of calreticulin (CRT) present on the surface of Pieris rapae hemocytes in phagocytosis. Comparative phagocytosis assays using yeast cells showed that hemocytes from different insects exhibit significant variation in their phagocytosing potential and relative CRT involvement.     

Cellular and humoral immune interactions between Drosophila and its parasitoids

The immune interactions occurring between parasitoids and their host insects, especially in Drosophila–wasp models, have long been the research focus of insect immunology and parasitology. Parasitoid infestation in Drosophila is counteracted by its multiple natural immune defense systems, which include cellular and humoral immunity. Occurring in the hemocoel, cellular immune responses involve the proliferation, differentiation, migration and spreading of host hemocytes and parasitoid encapsulation by them. Contrastingly, humoral immune responses rely more heavily on melanization and on the Toll, Imd and Jak/Stat immune pathways associated with antimicrobial peptides along with stress factors. On the wasps’ side, successful development is achieved by introducing various virulence factors to counteract immune responses of Drosophila. Some or all of these factors manipulate the host's immunity for successful parasitism. Here we review current knowledge of the cellular and humoral immune interactions between Drosophila and its parasitoids, focusing on the defense mechanisms used by Drosophila and the strategies evolved by parasitic wasps to outwit it.     

Insect hemocytes and their role in immunity

The innate immune system of insects is divided into humoral and cellular defense responses. Humoral defenses include antimicrobial peptides, the cascades that regulate coagulation and melanization of hemolymph, and the production of reactive intermediates of oxygen and nitrogen. Cellular defenses refer to hemocyte-mediated responses like phagocytosis and encapsulation. In this review, we discuss the cellular immune responses of insects with emphasis on studies in Lepidoptera and Diptera. Insect hemocytes originate from mesodermally derived stem cells that differentiate into specific lineages identified by morphology, function, and molecular markers. In Lepidoptera, most cellular defense responses involve granular cells and plasmatocytes, whereas in Drosophila they involve primarily plasmatocytes and lamellocytes. Insect hemocytes recognize a variety of foreign targets as well as alterations to self. Both humoral and cell surface receptors are involved in these recognition events. Once a target is recognized as foreign, hemocyte-mediated defense responses are regulated by signaling factors and effector molecules that control cell adhesion and cytotoxicity. Several lines of evidence indicate that humoral and cellular defense responses are well-coordinated with one another. Cross-talk between the immune and nervous system may also play a role in regulating inflammation-like responses in insects during infection.     

Regulators and signalling in insect haemocyte immunity

The innate immune system of insects relies on both humoral and cellular immune responses that are mediated via activation of several signalling pathways. Haemocytes are the primary mediators of cell-mediated immunity in insects, including phagocytosis, nodulation, encapsulation and melanization. The last years, research has focused on the mechanisms of microbial recognition and activation of haemocyte intracellular signalling molecules in response to invaders. The powerful tool, RNA interference gene silencing, helped several regulators involved in immune responses, to be identified. In this review, we summarize recent advances in understanding the role(s) of receptors and intracellular signalling molecules involved in immune responses.     

Characteristics of digestive enzymes of calanoid copepod species from different latitudes in relation to temperature, pH and food

In calanoid copepods it is poorly understood how enzymatic activities and patterns are affected by abiotic and biotic factors. Such knowledge, however, is crucial to assess metabolic functioning and performance of organisms in different habitats. Therefore, our study focuses on digestive enzyme activities in relation to temperature, pH and food in the Arctic species Calanus glacialis and in Centropages hamatus and Temora longicornis from the North Sea. Enzyme activities were measured over a range from 0 to 70 °C (lipases/esterases, proteinases) and pH 5 to 9 (proteinases). In all species, relative proteinases activity peaked at 40/50 °C and pH 6; relative lipases/esterases activity peaked at 30 °C. Between 0 and 20 °C, lipase activity of C. glacialis was higher (40-70% of maximum) than that of the boreal copepods (25-64%), which suggests thermal adaptation of the lipid metabolism in the polar species. Incubating C. glacialis with the diatom Thalassiosira weissflogii showed (i) that enzyme activities increased especially in the alkaline range and (ii) that enzyme patterns, revealed by gel electrophoresis, differed from that of starving individuals, indicating that feeding induced enzyme expression. Such studies, linking abiotic and biotic conditions to enzyme functioning, can help elucidating the capacity of copepods to respond to environmental changes.     

昆虫天然免疫相关基因研究进展

在长期进化过程中,昆虫形成了强大的天然免疫防御系统,即体液免疫和细胞免疫。体液免疫主要包括Toll、IMD和JAK/STAT 3条信号通路,通过信号转导及免疫途径调控免疫相关基因的表达,诱导产生抗菌肽和其他效应分子。细胞免疫由血细胞介导,主要完成对病原物的包裹、吞噬和集结等。近年来,昆虫基因组学快速发展,通过生物信息学等方法从昆虫基因组数据中已鉴定到大量免疫相关基因,对这些基因的研究加深了人们对昆虫天然免疫分子机制的认识和理解。根据基因功能,免疫相关基因分为识别、信号转导、调制器、效应分子、黑化反应、RNA干扰和其他基因等7类,这些基因通过互作来调控体液免疫和细胞免疫。本文对昆虫免疫相关基因的分类、功能及家族进化等方面的研究成果进行总结,并对今后昆虫免疫的研究重点进行了展望,以期为昆虫免疫分子机制的研究及开发新的害虫防治策略提供依据。     

Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti

Mosquitoes are vectors of many deadly and debilitating pathogens. In the current study, we used light and electron microscopies to study the immune response of Aedes aegypti hemocytes to bacterial inoculations, Plasmodium gallinaceum natural infections, and latex bead injections. After challenge, mosquitoes mounted strong phagocytic and melanization responses. Granulocytes phagocytosed bacteria singly or pooled them inside large membrane-delimited vesicles. Phagocytosis of bacteria, Plasmodium sporozoites, and latex beads was extensive; we estimated that individual granulocytes have the capacity to phagocytose hundreds of bacteria and thousands of latex particles. Oenocytoids were also seen to internalize bacteria and latex particles, although infrequently and with low capacity. Besides phagocytosis, mosquitoes cleared bacteria and sporozoites by melanization. Interestingly, the immune response toward 2 species of bacteria was different; most Escherichia coli were phagocytosed, but most Micrococcus luteus were melanized. Similar to E. coli, most Plasmodium sporozoites were phagocytosed. The immune response was rapid; phagocytosis and melanization of bacteria began as early as 5 min after inoculation. The magnitude and speed of the cellular response suggest that hemocytes, acting in concert with the humoral immune response, are the main force driving the battle against foreign invaders.     

Differential Effects of Serum Heat Treatment on Chemotaxis and Phagocytosis by Human Neutrophils

Neutrophils, in cooperation with serum, are vital gatekeepers of a host’s microbiome and frontline defenders against invading microbes. Yet because human neutrophils are not amenable to many biological techniques, the mechanisms governing their immunological functions remain poorly understood. We here combine state-of-the-art single-cell experiments with flow cytometry to examine how temperature-dependent heat treatment of serum affects human neutrophil interactions with “target” particles of the fungal model zymosan. Assessing separately both the chemotactic as well as the phagocytic neutrophil responses to zymosan, we find that serum heat treatment modulates these responses in a differential manner. Whereas serum treatment at 52°C impairs almost all chemotactic activity and reduces cell-target adhesion, neutrophils still readily engulf target particles that are maneuvered into contact with the cell surface under the same conditions. Higher serum-treatment temperatures gradually suppress phagocytosis even after enforced cell-target contact. Using fluorescent staining, we correlate the observed cell behavior with the amounts of C3b and IgG deposited on the zymosan surface in sera treated at the respective temperatures. This comparison not only affirms the critical role of complement in chemotactic and adhesive neutrophil interactions with fungal surfaces, but also unmasks an important participation of IgGs in the phagocytosis of yeast-like fungal particles. In summary, this study presents new insight into fundamental immune mechanisms, including the chemotactic recruitment of immune cells, the adhesive capacity of cell-surface receptors, the role of IgGs in fungal recognition, and the opsonin-dependent phagocytosis morphology of human neutrophils. Moreover, we show how, by fine-tuning the heat treatment of serum, one can selectively study chemotaxis or phagocytosis under otherwise identical conditions. These results not only refine our understanding of a widely used laboratory method, they also establish a basis for new applications of this method.     

Consistent pattern of local adaptation during an experimental heat wave in a pipefish-trematode host-parasite system

Extreme climate events such as heat waves are expected to increase in frequency under global change. As one indirect effect, they can alter magnitude and direction of species interactions, for example those between hosts and parasites. We simulated a summer heat wave to investigate how a changing environment affects the interaction between the broad-nosed pipefish (Syngnathus typhle) as a host and its digenean trematode parasite (Cryptocotyle lingua). In a fully reciprocal laboratory infection experiment, pipefish from three different coastal locations were exposed to sympatric and allopatric trematode cercariae. In order to examine whether an extreme climatic event disrupts patterns of locally adapted host-parasite combinations we measured the parasite's transmission success as well as the host's adaptive and innate immune defence under control and heat wave conditions. Independent of temperature, sympatric cercariae were always more successful than allopatric ones, indicating that parasites are locally adapted to their hosts. Hosts suffered from heat stress as suggested by fewer cells of the adaptive immune system (lymphocytes) compared to the same groups that were kept at 18°C. However, the proportion of the innate immune cells (monocytes) was higher in the 18°C water. Contrary to our expectations, no interaction between host immune defence, parasite infectivity and temperature stress were found, nor did the pattern of local adaptation change due to increased water temperature. Thus, in this host-parasite interaction, the sympatric parasite keeps ahead of the coevolutionary dynamics across sites, even under increasing temperatures as expected under marine global warming.     

Interactions between the cellular and humoral immune responses in Drosophila

Drosophila has highly efficient defenses against infection. These include both cellular immune responses, such as the phagocytosis of invading microorganisms, and humoral immune responses, such as the secretion of antimicrobial peptides into the hemolymph [1] [2]. These defense systems are thought to interact, but the nature and extent of these interactions is not known. Here we describe a method for inhibiting phagocytosis in Drosophila blood cells (hemocytes) by injecting polystyrene beads into the body cavity. This treatment does not in itself make a fly susceptible to Escherichia coli infection. However, when performed on flies carrying the mutation immune deficiency (imd), which affects the humoral immune response [3], the treatment results in a striking decrease in resistance to infection. We therefore carried out a sensitized genetic screen to identify immunocompromised mutants by co-injecting beads and E. coli. From this screen, we identified a new gene we have named red shirt and identified the caspase Dredd as a regulator of the Drosophila immune response. The observation that mutants with defects in the humoral immune response are further immunocompromised by blocking phagocytosis, and thus inhibiting the cellular immune response, shows that the Drosophila cellular and humoral immune responses act in concert to fight infection.     

Responses of disparate phenotypically-plastic,melanin-based traits to common cues: limits to the benefits of adaptive plasticity?

The evolution of perfect adaptive phenotypic plasticity of a given trait may be influenced by, among other things, phenotypic costs associated with the expression of a given trait value, relative to alternative trait values. One potential cause of such phenotypic costs is the allocation of limited resources to multiple traits. When multiple traits rely on the same resource, trait values for one adaptively plastic trait might be unavoidably associated with maladaptive trait values for other traits. I address this problem in three traits of Pieris rapae L. (the small cabbage white butterfly) that all rely on the pigment melanin and are adaptively plastic, but have very different functions: wing pattern, immune defense, and pupal color. Cool, short-day rearing conditions simultaneously increased total wing melanization and decreased a melanin-based immune response in females, consistent with predictions. However, cool, short days also reduced the melanin-based immune response in males, despite little effect on male wing melanization. Furthermore, contrary to predictions, these patterns were not altered by differences in dietary resources. Finally, dark-colored rearing backgrounds during pupation substantially increased pupal melanization in both sexes, but was not associated with differences in wing melanization. These results offer only mixed support for the hypothesis of melanin-based trade offs as a source of phenotypic costs to adaptive plasticity in these traits. However, patterns of sexual dimorphism for these traits suggest trade offs might be at work at another level: relative to males, females have consistently more heavily melanized wings but less heavily melanized pupae and immune responses. The reduced immune response under cool, short-day conditions may also have implications for the evolutionary ecology of these butterflies.     

The evolution of alternative morphs: density-dependent determination of larval colour dimorphism in a butterfly

Understanding the ultimate causes for the presence of polymorphisms within populations requires knowledge of how the expression of discrete morphs is regulated. In the present study, we explored the determination mechanism of a colour dimorphism in larvae of the butterfly Pararge xiphia (Satyrinae: Nymphalidae) with the ultimate aim of understanding its potential adaptive value. Last-instar larvae of P. xiphia develop into either a green or a brown morph, although all individuals are invariably green during the preceding three instars. A series of laboratory experiments reveal that morph development is strongly environmentally dependent and not the result of alternative alleles at one locus. Photoperiod, temperature, and in particular larval density, all influenced morph determination. The strong effect of a high larval density in inducing the brown morph parallels other known cases of density-dependent melanization in Lepidopteran larvae. Because melanization is often correlated with increased immune function, this type of determination mechanism is expected to be adaptive. However, the ecology and behaviour of P. xiphia larvae suggests that increased camouflage under high-density conditions may be an additional adaptive explanation. We conclude that the colour dimorphism of P. xiphia larvae is determined by a developmental threshold that is influenced both by heredity and by environmental conditions, and that selection for increased immune function and camouflage under high-density conditions may be responsible for maintaining the dimorphism.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 256–266.     

Mosquito phenoloxidase and defensin colocalize in melanization innate immune responses.

Mosquitoes mount strong humoral and cellular immune responses against foreign organisms. Two components of the mosquito immune response that have received much attention are the phenoloxidase cascade that leads to melanization and antimicrobial peptides. The purpose of the current study was to use immunocytochemistry and transmission electron microscopy to identify the location of the melanization rate-limiting enzyme phenoloxidase and the antimicrobial peptide defensin in innate immune reactions against Escherichia coli and Micrococcus luteus by the mosquito Aedes aegypti. Our results show that both phenoloxidase and defensin are present at the sites of melanin biosynthesis in immune reactions against bacteria. Furthermore, both proteins are often present inside the same melanotic capsules. When hemocytes were analyzed, phenoloxidase was present in the cytosol of oenocytoids, but no significant amounts of defensin were detected inside any hemocytes. In summary, these data show that phenoloxidase and defensin colocalize in melanization reactions against bacteria and argue for further studies into the potential role of defensin in phenoloxidase-based melanization innate immune responses in mosquitoes.     

The antibacterial innate immune response by the mosquito Aedes aegypti is mediated by hemocytes and independent of Gram type and pathogenicity

Previous mosquito studies showed that the hemocyte-mediated innate immune response against Gram- Escherichia coli is phagocytosis, but against Gram+ Micrococcus sp., is melanization. We examined the immune responses mounted by Aedes aegypti towards Gram- Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium, and Gram+ Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis. Using light microscopy, electron microscopy, and survival analysis, this investigation conclusively shows that the factors governing phagocytic vs. melanization responses are complex and independent of bacterial Gram type and pathogenicity. These data provide further evidence that hemocytes are central to the immune response against prokaryotes.     

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.     

The mean and variance of environmental temperature interact to determine physiological tolerance and fitness

Global climate change poses one of the greatest threats to biodiversity. Most analyses of the potential biological impacts have focused on changes in mean temperature, but changes in thermal variance will also impact organisms and populations. We assessed the combined effects of the mean and variance of temperature on thermal tolerances, organismal survival, and population growth in Drosophila melanogaster. Because the performance of ectotherms relates nonlinearly to temperature, we predicted that responses to thermal variation (±0° or ±5°C) would depend on the mean temperature (17° or 24°C). Consistent with our prediction, thermal variation enhanced the rate of population growth (r(max)) at a low mean temperature but depressed this rate at a high mean temperature. The interactive effect on fitness occurred despite the fact that flies improved their heat and cold tolerances through acclimation to thermal conditions. Flies exposed to a high mean and a high variance of temperature recovered from heat coma faster and survived heat exposure better than did flies that developed at other conditions. Relatively high survival following heat exposure was associated with low survival following cold exposure. Recovery from chill coma was affected primarily by the mean temperature; flies acclimated to a low mean temperature recovered much faster than did flies acclimated to a high mean temperature. To develop more realistic predictions about the biological impacts of climate change, one must consider the interactions between the mean environmental temperature and the variance of environmental temperature.     

Influence of elevated temperatures on the immune response of abalone, Haliotis rubra

Elevated water temperature can act as a stressor impacting the immune responses of molluscs, potentially increasing their susceptibility to microbial infections. Abalone are commercially important marine molluscs that have recently experienced disease outbreaks caused by a herpesvirus and Vibrio bacteria. Sampling of wild-caught Haliotis rubra showed a significant correlation between water temperature and both antiviral and antibacterial activity, with higher activity in summer than in winter months. However, antibacterial activity was compromised in favour of antiviral activity as the water temperatures peaked in summer. A controlled laboratory experiment was then used to investigate several immune responses of H. rubra, including total haemocyte count (THC), stimulated superoxide anion production (SO), antiviral activity against a model herpesvirus, herpes simplex virus type 1 and antibacterial activity against a representative pathogenic bacterium, Vibrio anguillarum, over one week after raising water temperature from 18 to 21 or 24 °C. THC and SO increased at day 1 and then dropped back to control levels by days 3 and 7. By comparison, the humoural immune parameters showed a delayed response with antibacterial and antiviral activity significantly increasing on days 3 and 7, respectively. Consistent with the field study, antibacterial activity became significantly depressed after prolonged exposure to elevated temperatures. A principal components analysis on the combined immune parameters showed a negative correlation between antiviral and antibacterial activity. SO was positively correlated to THC and neither of these cellular parameters were correlated to the humoural antimicrobial activity. Overall, this study indicates that abalone may have more resilience to viruses than bacterial pathogens under conditions of elevated temperature, such as those predicted under future climate change scenarios.     

Influence of temperature, pH and dissolved oxygen concentration on enhanced biological phosphorus removal under strictly aerobic conditions

Previous research has suggested that enhanced biological phosphorus removal (EBPR) from wastewater can be achieved under continuous aerobic conditions over the short term. However, little is known how environmental conditions might affect aerobic EBPR performance. Consequently we have investigated the impact of temperature, pH and dissolved oxygen (DO) concentrations on EBPR performance under strictly aerobic conditions. A sequencing batch reactor (SBR) was operated for 108 days on a six-hour cycle (four cycles a day). The SBR ran under alternating anaerobic-aerobic conditions as standard and then operated under strictly aerobic conditions for one cycle every three or four days. SBR operational temperature (10, 15, 20, 25 and 30°C), pH (6, 7, 8 and 9) and DO concentration (0.5, 2.0 and 3.5mg/L) were changed consecutively during the aerobic cycle. Recorded increases in mixed liquor phosphorus (P) concentrations during aerobic carbon source uptake (P release) were affected by the biomass P content rather than the imposed changes in the operational conditions. Thus, P release levels increased with biomass P content. By contrast, subsequent aerobic P assimilation (P uptake) levels were both affected by changes in operational temperature and pH, and peaked at 20-25°C and pH 7-8. Highest P uptake detected under these SBR operating conditions was 15.4 mg Pg-MLSS(-1) (at 25°C, pH 7 and DO 2.0mg/L). The ability of the community for linked aerobic P release and P uptake required the presence of acetate in the medium, a finding which differs from previous data, where these are reported to occur in the absence of any exogenous carbon source. Fluorescence in situ hybridization was performed on samples collected from the SBR, and Candidatus 'Accumulibacter phosphatis' cells were detected with PAOmix probes through the operational periods. Thus, Candidatus 'Accumulibacter phosphatis' seemed to perform P removal in the SBR as shown in previous studies on P removal under strictly aerobic conditions.