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.

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.

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.

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.

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.

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.

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.

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.

A new method for double immunolabelling with primary antibodies from identical species.

There are several double immunolabelling methods but each has its drawbacks. More often than not, antibodies with the required specificities are available in only one species and their use normally produces false labels due to cross-reactivity. We describe a new and reliable technique for staining with primary antibodies from the same species, that can even be employed on tissues of the donor species. The protocol avoids cross-reactivities without loss in sensitivity, uses commercially available reagents and takes advantage of enzymatic detection, although it can be adapted for fluorescent labelling. Briefly, tissue is incubated with one primary antibody, followed by a peroxidase-coupled secondary antibody which is detected using amino ethyl carbazol to give a red reaction product. Meanwhile, the next primary antibody is coupled in vitro to a biotinylated secondary antibody and excess binding sites quenched with normal immune serum from the same species as the primary antibody. This complex is applied to tissue and detected by the avidin-biotin/alkaline phosphatase technique using naphthol-AS-MX-phosphate/Fast Blue BB to produce a blue label. In addition to extensive controls, the reliability and broad applicability of this method has been confirmed in (1) murine skin cryostat sections to co-visualize antigen-presenting cells (MHC class II-immunoreactive; "-ir') with either antigen detecting T lymphocytes (CD4-ir) or Langerhans cells (NLDC-145-ir) and (2) locust (Insecta) abdominal ganglion paraffin sections, where it is known that immunoreactivities for octopamine and a FMRFamide-related peptide are colocalized in only one, uniquely identifiable neuron.

Hemolymph transfer as an assay for immunorecognition in the cockroach Periplaneta americana and the locust Schistocerca gregaria.

A quantitative assay for measuring the number of hemocytic nodules formed in response to foreign particles and soluble molecules has been used, in the locust Schistocerca and the cockroach Periplaneta, to investigate the response to transferred hemolymph. Xenogeneic test particles, rabbit neutrophil leukocytes, stimulate formation of nodules when injected into both insect species, compared with saline-injected controls. However, the number of nodules formed in the locust in response to cockroach hemolymph is significantly reduced compared with the response to other xenogeneic cells, and it is suggested that, in view of the strong reactivity of cockroach hemocytes to locust hemolymph and plasma, a graft-versus-host response might be occurring in the recipient locust. Whole hemolymph transferred allogeneically between Periplaneta, or xenogeneically from Blatta to Periplaneta, does not stimulate a response in the recipient. This corresponds well with results from other assays for immunorecognition of transplants and is further confirmation that allogeneic and, in some combinations xenogeneic, recognition is absent in insects.

Activation of the prophenoloxidase cascade and initiation of nodule formation in locusts by bacterial lipopolysaccharides.

The activation of the prophenoloxidase (proPO) system of the locusts, Schistocerca gregaria and Locusta migratoria, by several bacterial lipopolysaccharides (LPS) is described. Activation of proPO by LPS occurred only in the presence of whole blood homogenates and not with hemocyte lysate preparations alone. Levels of phenoloxidase generated by the different LPSs in vitro were also correlated with numbers of nodules formed in vivo by injection of these LPSs. This further strengthens the evidence for the involvement of proPO activation in the insect cellular defenses. Finally, the wisdom in using anticoagulants in order to stabilize fragile hemocytes in studies on the proPO system is discussed.

Serine proteinase inhibitors in arthropod immunity.

Arthropod hemolymph contains proteins with serine proteinase inhibitory activity. These inhibitors may exist in plasma or in hemocyte granules. Serine proteinase inhibitors from the Kazal, Kunitz, alpha-macroglobulin, and serpin families have been identified in arthropod hemolymph and have been characterized biochemically. Two new families of low molecular weight serine proteinase inhibitors have recently been discovered: one in silkworms (the Bombyx family) and another in locusts and a crayfish. The serine proteinase inhibitors in arthropod hemolymph are likely to function in protecting their hosts from infection by pathogens or parasites. Some may inhibit fungal or bacterial proteinases. Others probably have roles in regulating endogenous proteinases involved in coagulation, prophenol oxidase activation, or cytokine activation.

Changes in lipophorins are related to the activation of phenoloxidase in the haemolymph of Locusta migratoria in response to injection of immunogens.

In Locusta migratoria, activation of phenoloxidase in the haemolymph in response to injection of laminarin is age-dependent: being absent in fifth instar nymphs and newly emerged adults, and only becoming evident four days after the final moult. This pattern of change in phenoloxidase activation correlates with the pattern of change in the concentration of apolipophorin-III (apoLp-III) in the haemolymph. Injection of a conspecific adipokinetic hormone (Lom-AKH-I) has no effect on the phenoloxidase response in nymphs or newly emerged adults but, in adults older than four days, co-injection of the hormone with laminarin prolongs the activation of phenoloxidase in the haemolymph: a similar enhancement of the response to laminarin is observed in locusts that have been starved for 48 h but not injected with AKH-I. During most of the fifth stadium, injection of laminarin results in a decrease in the level of prophenoloxidase in the haemolymph; an effect that is not observed in adults of any age. Marked changes in the concentration of apoLp-III, and the formation of LDLp in the haemolymph, are observed after injection of laminarin (or LPS) and these are remarkably similar, at least qualitatively, to those that occur after injection of AKH-I. The involvement of lipophorins in the activation of locust prophenoloxidase in response to immunogens is discussed.

Selective groups of neuronal and mesodermal cells recognized early in grasshopper embryogenesis by a monoclonal antibody.

Our aim in generating monoclonal antibodies against the grasshopper nervous system is to identify molecules expressed early in neuronal development. A crude homogenate of the adult nervous system was used as the immunogen, and the hybridoma supernatants were screened on young grasshopper embryos. Here we report on the I-5 monoclonal antibody, which recognizes an antigen appearing in an interesting pattern of ectodermal and mesodermal cells early in the embryonic development of the grasshopper. Amongst the cells stained are the pioneer neurons in the central nervous system and the periphery, and the muscle pioneers.

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.