Variation of TNF modulates cellular immunity of gregarious and solitary locusts against fungal pathogen Metarhizium anisopliae.

Changes in population density lead to phenotypic differentiation of solitary and gregarious locusts, which display different resistance to fungal pathogens; however, how to regulate their cellular immune strategies remains unknown. Here, our stochastic simulation of pathogen proliferation suggested that humoral defense always enhanced resistance to fungal pathogens, while phagocytosis sometimes reduced defense against pathogens. Further experimental data proved that gregarious locusts had significantly decreased phagocytosis of hemocytes compared to solitary locusts. Additionally, transcriptional analysis showed that gregarious locusts promoted immune effector expression (gnbp1 and dfp) and reduced phagocytic gene expression (eater) and the cytokine tumor necrosis factor (TNF). Interestingly, higher expression of the cytokine TNF in solitary locusts simultaneously promoted eater expression and inhibited gnbp1 and dfp expression. Moreover, inhibition of TNF increased the survival of solitary locusts, and injection of TNF decreased the survival of gregarious locusts after fungal infection. Therefore, our results indicate that the alerted expression of TNF regulated the immune strategy of locusts to adapt to environmental changes.

MaPacC, a pH-responsive transcription factor, negatively regulates thermotolerance and contributes to conidiation and virulence in Metarhizium acridum.

PacC is a pH-responsive transcription factor gene highly expressed at alkaline pH and plays distinct roles in environmental fitness, conidiation and virulence of different fungi. Here, we show biological functions of orthologous MaPacC in the locust-specific fungal pathogen Metarhizium acridum. Disruption of MapacC slowed down the fungal growth only under alkaline conditions. Intriguingly, the fungal thermotolerance was enhanced by the MapacC deletion, accompanied by transcriptional upregulation of some heat shock-responsive genes. The disruptant suffered a reduction in conidial yield and a change in conidial surface structure, but showed little change in cell wall integrity. The virulence of the disruptant against a locust species was markedly attenuated due to delayed appressorium formation, repressed expression of some insect cuticle hydrolases and slowed growth in locust hemolymph. The phenoloxidase activity and nodules of the locusts infected by the disruptant were also boosted. All of these phenotypic changes were restored by targeted gene complementation. Our results indicate that MaPacC acts a negative regulator of thermotolerance and contributes to the virulence of M. acridum by an involvement in hyphal penetration through insect cuticle and evasion from insect immunity.

Spatial and temporal transcriptomic analyses reveal locust initiation of immune responses to Metarhizium acridum at the pre-penetration stage.

Insect hemocyte and fat body tissues play critical functional roles in insect immunity. Little, however, is known concerning the dynamic responses of these tissues to fungal infection. Here, we report on a time course of locust hemocyte and fat body transcriptomic responses to infection by the acridid specific fungal pathogen, Metarhizium acridum. Fat body responses were more pronounced at all infection stages as compared to hemocytes. Immune and other related genes were induced far earlier than previously considered including at pre-penetration stages. Differential expression in hemocyte and fat body tissues persisted throughout the course of infection up until host death. Our data indicate selective pressure on the host to recognize the infection as early as possible in order to limit its spread. Overall, fat body and hemocyte tissues launch a robust multi-tiered response to combat the fungal pathogen, with our data providing potential host targets for exploitation in pest control.

Condition-dependence and sexual ornamentation: Effects of immune challenges on a highly sexually dimorphic grasshopper.

Sexual ornaments contribute substantially to phenotypic diversity and it is particularly relevant to understand their evolution. Ornaments can assume the function of signals-of-quality that the choosy sex uses to evaluate potential mating partners. Often there are no obvious direct benefits and investment into mate choice is primarily rewarded by beneficial alleles that are inherited to the offspring. Inter-sexual communication via sexual ornaments requires honesty of the sexual signal, yet the question of what maintains honesty remains only partially solved. One solution is that honesty is maintained by trait expression being dependent on individual condition, since condition-dependent trait expression offers an effectively inexhaustible source of genetic variability. Here we test in the highly sexually dimorphic club-legged grasshopper Gomphocerus sibiricus if putative sexual ornaments, in particular the striking front-leg clubs, are more strongly affected by a lipopolysaccharide (LPS) immune challenge than putatively not sexually selected traits. Our results show overall little condition-dependent expression of morphological and song traits, with sexually selected traits exhibiting effects comparable to nonsexually selected traits (with the possible exception of stridulatory file length and syllable-to-pause ratio in advertisement songs). Interestingly, field observations of individuals of lethally parasitized individuals suggest that a very strong environmental challenge can specifically affect the expression of the front-leg clubs. The presence of 1% of males in natural populations with missing or heavily deformed clubs plus 5% with minor club deformations furthermore indicate that there are risks associated with club development during final ecdysis and this might act as a filter against deleterious alleles.

Molecular characterization of a c-type lysozyme from the desert locust, Schistocerca gregaria (Orthoptera: Acrididae).

Lysozymes are bacteriolytic peptides that are implicated in the insect nonspecific innate immune responses. In this study, a full-length cDNA encoding a c-type lysozyme from Schistocerca gregaria (SgLys) has been cloned and characterized from the fat body of immune-challenged 5(th) instar. The deduced mature lysozyme is 119 amino acid residues in length, has a calculated molecular mass of 13.4 kDa and an isoelectric point (Ip) of 9.2. SgLys showed high identities with other insect lysozymes, ranging from 41.5% to 93.3% by BLASTp search in NCBI. Eukaryotic in vitro expression of the SgLys ORF (rSgLys) with an apparent molecular mass of ∼16 kDa under SDS-PAGE is close to the calculated molecular weight of the full-length protein. rSgLys displayed growth inhibitory activity against Gram-negative and Gram-positive bacteria. 3D structure modeling of SgLys, based on comparison with that of silkworm lysozyme, and sequence comparison with the helix-loop-helix (α-hairpin) structure of hen egg white lysozyme (HEWL) were employed to interpret the antibacterial potencies. Phylogenetic alignments indicate that SgLys aligns well with insect c-type lysozymes that expressed principally in fat body and hemocytes and whose role has been defined as immune-related. Western blot analysis showed that SgLys expression was highest at 6-12 h post-bacterial challenge and subsequently decreased with time. Transcriptional profiles of SgLys were determined by semi-quantitative RT-PCR analysis. SgLys transcript was upregulated at the highest level in fat body, hemocytes, salivary gland, thoracic muscles, and epidermal tissue. It was expressed in all developmental stages from egg to adult. These data indicate that SgLys is a predominant acute-phase protein that is expressed and upregulated upon immune challenge.

Body condition constrains immune function in field populations of female Australian plague locust Chortoicetes terminifera.

The insect innate immune system comprises both humoral and cellular defence responses. In the laboratory, the insect immune system is well characterized. In the field, however, little is known about the role of constitutive insect immune function and how it varies within and between populations. Laboratory studies suggest that host nutrition has significant impact upon insect immune function. Thus, the rationale for this study was to sample natural populations of the Australian Plague Locust Chortoicetes terminifera to establish whether locust body condition (as determined by protein and lipid content) impacted their constitutive immune system and, as a result, has the potential to impact on their capacity to respond to a pathogenic challenge. We found that body condition varied greatly between individual female locusts within sites and that haemolymph protein levels, but not body lipid content, varied between sites. Moreover, our measures of immune function were correlated with the haemolymph levels of protein (in the case of haemocyte density), lipid (prophenoloxidase activity) or both (lysozyme-like antimicrobial activity). We discuss the implications of these findings for the role of biological pesticides in the control of locust populations.

Locusts increase carbohydrate consumption to protect against a fungal biopesticide.

There is growing evidence to suggest that hosts can alter their dietary intake to recoup the specific resources involved in mounting effective resistance against parasites and pathogens. We examined macronutrient ingestion and disease-resistance in the Australian plague locust (Chortoicetes terminifera), challenged with a fungal pathogen (Metarhizium acridum) under dietary regimes varying in their relative amounts of protein and digestible carbohydrate. Dietary protein influenced constitutive immune function to a greater extent than did carbohydrate, indicating higher protein costs of mounting an immune defence than carbohydrate or overall energy costs. However, it appears that increased immune function, as a result of greater protein ingestion, was not sufficient to protect locusts from fungal disease. We found that locusts restricted to diets high in protein (P) and low in carbohydrate (C) were more likely to die of a fungal infection than those restricted to diets with a low P:C ratio. We hypothesise that the fungus is more efficient at exploiting protein in the insect's haemolymph than the host is at producing immune effectors, tipping the balance in favour of the pathogen on high-protein diets. When allowed free-choice, survivors of a fungus-challenge chose a less-protein-rich diet than those succumbing to infection and those not challenged with fungus locusts. These results are contrary to previous studies on caterpillars in the genus Spodoptera challenged with bacterial and baculoviral pathogens, indicating that nutrient ingestion and pathogen resistance may be a complex interaction specific to different host species and disease agents.

Lipophorins can adhere to dsRNA, bacteria and fungi present in the hemolymph of the desert locust: a role as general scavenger for pathogens in the open body cavity.

Desert locusts are characterized by a highly sensitive and effective RNA interference (RNAi) response. Moreover, delivery of dsRNA into the open body cavity will elicit potent silencing effects throughout the body. On the other hand, many other insect species, such as Bombyx mori and Drosophila melanogaster, lack the ability to efficiently spread the RNAi-signal. In this study, we demonstrated that, in the serum of the desert locust, lipophorins adhere to dsRNA-fragments. Lipophorins can be subdivided into high density and low density lipophorins (HDLp and LDLp), according to their buoyant density, and we showed that both types of lipophorins can interact with dsRNA fragments. Furthermore, in the presence of (gram-positive) bacteria or fungi, LDLp induce the formation of pathogen aggregates, while no clear aggregation effects were detected in the presence of HDLp.

Gene cloning and expression patterns of two prophenoloxidases from Catantops pinguis (Orthoptera: Catantopidae).

In insect, fat body plays major roles in insect innate immunity. Phenoloxidase (PO) is an important component in insect innate immunity and is necessary for acclimatization. In our study, two prophenoloxidase (PPO) subunits were obtained from fat body of Catantops pinguis (Stål). The full-length cDNA sequence of one PPO (CpPPO1) consisted of 2347 bp with an open reading frame (ORF) of 2187 bp encoding 728 amino acids, while the other subunit (CpPPO2) had a full length of 2445 bp, encoding 691 amino acids. Both the PPO gene products are predicted to possess all the structural features of other PPO members, including two putative tyrosinase copper-binding motifs with six highly conserved histidine residues and a thiolester-like motif. Tissue distribution analysis showed that both PPO mRNAs were abundantly expressed in the fat body among 11 tissues examined, and they were transiently up-regulated after Escherichia coli infection, consistent with them being immune-responsive genes. Total levels of CpPPO1 and CpPPO2 mRNA transcripts were much higher in first instar larvae and adults. A much higher transcript level of CpPPO1 was detected in several months, while there were extremely high mRNA expression levels of CpPPO2 in January, July, October, and December. The above results suggested that PPO from fat body might also bring significant function during the processes of development and acclimatization for C. pinguis.

Altered immunity in crowded locust reduced fungal (Metarhizium anisopliae) pathogenesis.

The stress of living conditions, similar to infections, alters animal immunity. High population density is empirically considered to induce prophylactic immunity to reduce the infection risk, which was challenged by a model of low connectivity between infectious and susceptible individuals in crowded animals. The migratory locust, which exhibits polyphenism through gregarious and solitary phases in response to population density and displays different resistance to fungal biopesticide (Metarhizium anisopliae), was used to observe the prophylactic immunity of crowded animals. We applied an RNA-sequencing assay to investigate differential expression in fat body samples of gregarious and solitary locusts before and after infection. Solitary locusts devoted at least twice the number of genes for combating M. anisopliae infection than gregarious locusts. The transcription of immune molecules such as pattern recognition proteins, protease inhibitors, and anti-oxidation proteins, was increased in prophylactic immunity of gregarious locusts. The differentially expressed transcripts reducing gregarious locust susceptibility to M. anisopliae were confirmed at the transcriptional and translational level. Further investigation revealed that locust GNBP3 was susceptible to proteolysis while GNBP1, induced by M. anisopliae infection, resisted proteolysis. Silencing of gnbp3 by RNAi significantly shortened the life span of gregarious locusts but not solitary locusts. By contrast, gnbp1 silencing did not affect the life span of both gregarious and solitary locusts after M. anisopliae infection. Thus, the GNBP3-dependent immune responses were involved in the phenotypic resistance of gregarious locusts to fungal infection, but were redundant in solitary locusts. Our results indicated that gregarious locusts prophylactically activated upstream modulators of immune cascades rather than downstream effectors, preferring to quarantine rather than eliminate pathogens to conserve energy meanwhile increasing the "distance" of infectious and target individuals. Our study has obvious implications for bio-pesticides management of crowded pests, and for understanding disease epidemics and adaptiveness of pathogens.

[Roles of phosphatases in pathogen infection: a review].

Phosphatases play a key role not only in cell physiological functions of an organism, but also in host-pathogen interactions. Many studies demonstrated that some Gram-negative pathogenic bacteria could evade host immunity and promote pathogenicity by injecting phosphatases into host cells through type III secretion system. However, there were few reports about pathogenic fungi evading the immunity of hosts. Our researches indicated that the entomogenic fungus Metarhizium anisopliae could dephosphorylate the signal transduction substance of locust humoral immunity specifically in vitro by secreting extracellular protein tyrosine phosphatase, which implied that the fungus might interfere with the immune defense of locust. To provide reference for further studies of the functions of phosphatases, we reviewed the types of phosphatases and their roles in pathogen infection.

The inhibitory effect of the fungal toxin, destruxin A, on behavioural fever in the desert locust.

During an infection locusts behaviourally fever by seeking out higher environmental temperatures. This behaviour places the pathogen at sub-optimal growth temperatures while improving the efficiency of the immune system, thereby prolonging the lifespan of the host. It is therefore in the interest of the pathogen to either adapt to fever-like temperatures or to evolve mechanisms to interfere with, or inhibit fever. We investigated the behavioural fever response of desert locusts to two fungal pathogens. A prolonged fever was observed in locusts infected with Metarhizium acridum. However, fever was comparatively short-lived during infection with Metarhizium robertsii. In both cases restriction of thermoregulation reduced lifespan. Destruxin A (dtx A) produced by M. robertsii, but not M. acridum has previously been associated with the inhibition of the insect immune system. Injection of dtx A during infection with the fever-causing M. acridum inhibited fever and was particularly effective when administered early on in infection. Furthermore, locusts injected with dtx A were more susceptible to M. acridum infection. Therefore engineering M. acridum isolates currently used for locust biocontrol, to express dtx A may improve efficiency of control by interfering with fever.

Crowded locusts produce hatchlings vulnerable to fungal attack.

Transgenerational effects of parental experience on offspring immunity are well documented in the vertebrate literature (where antibodies play an obligatory role), but have only recently been described in invertebrates. We have assessed the impact of parental rearing density upon offspring disease resistance by challenging day-old locust hatchlings (Schistocerca gregaria) from either crowd- or solitary-reared parents with the fungal pathogen Metarhizium anisopliae var. acridum. When immersed in standardized conidia suspensions, hatchlings from gregarious parents suffered greater pathogen-induced mortality than hatchlings from solitary-reared parents. This observation contradicts the basic theory of positive density-dependent prophylaxis and demonstrates that crowding has a transgenerational influence upon locust disease resistance.

Adipokinetic hormone and the immune responses of locusts to infection.

Injections of Bacillus, or of blastospores from the entomopathogenic fungus, Metarhizium anisopliae, activate the prophenoloxidase (PPO) cascade, and coinjection of adipokinetic hormone-I (AKH) enhances and prolongs these responses. When injected concurrently with an immunizing dose of live bacteria, AKH suppresses the appearance of antimicrobial activity and, after a short delay, increases the growth of bacteria within the hemocoel. Injections of live Escherichia coli or Pseudomonas aeruginosa into locusts fail to activate PPO in the hemolymph, even when coinjected with AKH. The coinjection of bacteria and hormone is rarely lethal to the locust. However, if locusts are injected with AKH when they are infected with Metarhizium, they die more rapidly than if no AKH is administered.

Induced hyperlipaemia and immune challenge in locusts.

Injections of immunogens, such as beta-1,3-glucan or lipopolysaccharide (LPS), bring about a marked hyperlipaemia with associated changes in lipophorins and apolipophorin-III in the haemolymph of Locusta migratoria. These changes are similar to those observed after injection of adipokinetic hormone (AKH). The possibility that endogenous AKH is released as part of the response to these immunogens is investigated using passive immunisation against AKH-I, and measurement of AKH-I titre in the haemolymph after injection of immunogens. The data presented show that, despite the similarity of the changes brought about by the presence of immunogens in the haemolymph to those brought about by AKH, there is no release of endogenous AKH after injection of laminarin or LPS. A direct effect of the immunogens on release of neutral lipids by the fat body cannot be demonstrated in vitro, and the mechanism by which hyperlipaemia is induced during immune challenge remains uncertain.

Molecular evidence for the expression of angiotensin converting enzyme in hemocytes of Locusta migratoria: stimulation by bacterial lipopolysaccharide challenge.

The presence of angiotensin converting enzyme (ACE) in insects has been reported many times, but numerous questions about the functional role of this enzyme in insects remain. Here we show by RT-PCR experiments that ACE has a wide tissue distribution in Locusta migratoria, suggesting diverse roles for this enzyme in the locust. Immune challenge through injection of bacterial lipopolysaccharides resulted in a tenfold increase of ACE gene transcripts in the hemocytes and is suggestive for a role of ACE in the cellular defense of the locust. However, phenotypic knockout experiments with the ACE inhibitor captopril showed that ACE is not essential for the efficient clearance of injected E. coli bacteria.

Adipokinetic hormone enhances nodule formation and phenoloxidase activation in adult locusts injected with bacterial lipopolysaccharide.

Interactions between the locust endocrine and immune systems have been studied in vivo in relation to nodule formation and activation of the prophenoloxidase cascade in the haemolymph. Injection of bacterial lipopolysaccharide (LPS) extracted from Escherichia coli induces nodule formation in larval and adult locusts but does not increase phenoloxidase activity in the haemolymph. Nodule formation starts rapidly after injection of LPS and is virtually complete within 8 h, nodules occurring mainly associated with the dorsal diaphragm on either side of the heart, but sometimes with smaller numbers associated with the ventral diaphragm on either side of the nerve cord. Co-injection of adipokinetic hormone-I (Lom-AKH-I) with LPS stimulates greater numbers of nodules to be formed in larval and adult locusts, and activates phenoloxidase in the haemolymph of mature adults but not of nymphs. The effect of co-injection of Lom-AKH-I with LPS on nodule formation is seen at low doses of hormone; only 0.4 pmol of Lom-AKH-I per adult locust is needed to produce a 50% increase in the number of nodules formed. When different components of LPS from the E. coli Rd mutant are tested, the mono- and the diphosphoryl Lipid A components have similar effects to the intact LPS. Remarkably, detoxified LPS activates phenoloxidase in the absence of Lom-AKH-I, although co-injection with hormone does enhance this response. Both diphosphoryl Lipid A and detoxified LPS induce a level of nodule formation that is enhanced by co-injection of Lom-AKH-I, but monophosphoryl Lipid A does not initiate nodule formation even when injected with hormone. Co-injection of a water-soluble inhibitor of eicosanoid synthesis, diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid), reduces nodule formation in response to injections of LPS (both in the absence and presence of hormone) in a dose-dependent manner, but does not prevent activation of phenoloxidase in adult locusts. It is shown that nodule formation and activation of the prophenoloxidase in locust haemolymph can both be enhanced by Lom-AKH-I, but it is argued that these processes involve distinct mechanisms in which eicosanoid synthesis is important for nodule formation, but not for the increased phenoloxidase activity.