Insect immunity - adaptive mechanisms and survival strategies / Odpornosc owadów - mechanizmy adaptacyjne i strategie przetrwania.

Host-pathogen interactions play an important role in understanding the dynamics of the insect immune system. The analysis of environmental modulators, both biotic and abiotic, directs our attention to the impact of the surroundings on the effectiveness of immunological responses. This knowledge is essential for the comprehensive understanding of insect's immune reaction after infection with a pathogen. This article discusses the role of the immune system in insect, with a special emphasis on greater wax moth Galleria mellonella and highlights its adaptive capabilities. The processes are not only extremely interesting area of scientific research but also indicate potential practical applications in the context of plan protection, pest population control and medicine.

Juvenile responses to immune challenges are not carried through to subsequent life stages in an insect.

Environmental variability can significantly impact individual survival and reproduction. Meanwhile, high population densities can lead to resource scarcity and increased exposure to parasites and pathogens. Studies with insects can offer valuable insights into eco-immunology, allowing us to explore the connections between these variables. Here we use the moth Anticarsia gemmatalis to examine how increases in population density and immunological challenge during the larval stage shape its investment in immune defence and reproduction. Larvae reared at a high population density exhibited greater lytic activity against bacteria compared to those reared at low density, whilst bacterial challenge (i.e. bacteria-immersed needles) also increased lytic activity. There was no interaction between the variables population density and bacterial challenge, indicating that these are independent. Surprisingly, neither increase in lytic activity carried through to activity in prepupal haemolymph. Rearing of larvae at a high density delayed pupation and decreased pupal weight. The immunological stimulus did not significantly influence pupal development. Lower population density as a larva resulted in greater adult weight, but did not significantly influence lytic activity in the eggs or the number of eggs laid. Negative correlations were found between lytic activity in the eggs and the number of eggs, as well as between adult weight and the number of eggs. Overall, this study demonstrates that high population density and immune challenge trigger increased lytic activity in caterpillars, but this effect is transient, not persisting into later stages. The trade-offs observed, such as delayed pupation and reduced prepupal weights under high density, suggest a balancing act between immune investment and developmental aspects. The findings hint at a short-term adaptive response rather than a sustained strategy. The implications of delayed pupation and smaller adult moths could influence the moth's life history strategy, impacting its role in the ecosystem. Further research tracking larval immune investment and subsequent reproductive success will unveil the evolutionary dynamics of this relationship in changing environments.

Targeting Design of Human Anti-idiotypic Genetically Engineered Antibody for Simulating the Structure and Insecticidal Function of Bt Cry1C Toxin.

The ß-type anti-Id (Ab2ß) is considered to have potential for simulating the structure and function of the antigen. In this study, a ß-type anti-Id (3A7 anti-I-GEAb) of the Cry1C toxin was captured from a GEAb library. Subsequently, a higher activity of mutant (3A7 mutant 8) was obtained from the mutagenesis library based on 3A7 anti-I-GEAb. The LD50 values of 3A7 anti-I-GEAb and 3A7 mutant 8 reach up to 38.9% and 46.8% of Cry1C toxin for P. xylostella and reach up to 32.9% and 37.4% of Cry1C toxin for H. armigera. Additionally, an IC-ELISA was established based on 3A7 mutant 8 (as the coated "antigen"), with an LOD value of 0.35 ng/mL, exhibiting good accuracy and stability for detecting Cry1C toxin in spiked samples. The present ß-type anti-I-GEAb not only exhibits insecticidal activity similar to Cry1C toxin, offering potential for environmentally friendly pest management, but it can also replace the Cry1C toxin structure to establish a highly sensitive and specific IC-ELISA for monitoring Cry1C toxin.

Comparative transcriptome analysis reveals disruption of Plutella xylostella immune system by fungal peptide cyclosporin C.

The diamondback moth (Plutella xylostella), a major threat to crucifers across the globe, has developed resistance against the majority of insecticides enhancing the need for alternate control measures against this pest. Recently cyclosporin C, a secondary metabolite produced by the insect pathogenic fungus Purpeocillium lilacinum, has been reported to induce lethal and sub-lethal effects against P. xylostella. To date, little is known about the molecular mechanisms of interaction between cyclosporin C and P. xylostella immune systems. This study reports the transcriptome-based immune response of P. xylostella to cyclosprin C treatment. Our results showed differential expression of 322, 97, and 504 differentially expressed genes (DEGS) in P. xylostella treated with cyclosporin C compared to control 24, 48, and 72 h post-treatment, respectively. Thirteen DEGs were commonly expressed at different time intervals in P. xylostella larvae treated with cyclosporin C compared to control. Cyclosporin C treatment induced the down-regulated expression of majority of immune-related genes related to pattern recognition responses, signal modulation, Toll and IMD pathways, antimicrobial peptides and antioxidant responses confirming the ability to suppress immune response of P. xylostella. These results will further improve our knowledge of the infection mechanism and complex biochemical processes involved in interaction between cyclosporin C and insect immune systems.

De novo transcriptome assembly of the oak processionary moth Thaumetopoea processionea.

OBJECTIVES: The oak processionary moth (OPM) (Thaumetopoea processionea) is a species of moth (order: Lepidoptera) native to parts of central Europe. However, in recent years, it has become an invasive species in various countries, particularly in the United Kingdom and the Netherlands. The larvae of the OPM are covered with urticating barbed hairs (setae) causing irritating and allergic reactions at the three last larval stages (L3-L5). The aim of our study was to generate a de novo transcriptomic assembly for OPM larvae by including one non-allergenic stage (L2) and two allergenic stages (L4 and L5). A transcriptomic assembly will help identify potential allergenic peptides produced by OPM larvae, providing valuable information for developing novel therapeutic strategies and allergic immunodiagnostic assays. DATA: Transcriptomes of three larval stages of the OPM were de novo assembled and annotated using Trinity and Trinotate, respectively. A total of 145,251 transcripts from 99,868 genes were identified. Bench-marking universal single-copy orthologues analysis indicated high completeness of the assembly. About 19,600 genes are differentially expressed between the non-allergenic and allergenic larval stages. The data provided here contribute to the characterization of OPM, which is both an invasive species and a health hazard.

Development of a Fully Protective Pandemic Avian Influenza Subunit Vaccine in Insect Pupae.

In this study, we pioneered an alternative technology for manufacturing subunit influenza hemagglutinin (HA)-based vaccines. This innovative method involves harnessing the pupae of the Lepidoptera Trichoplusia ni (T. ni) as natural biofactories in combination with baculovirus vectors (using CrisBio® technology). We engineered recombinant baculoviruses encoding two versions of the HA protein (trimeric or monomeric) derived from a pandemic avian H7N1 virus A strain (A/chicken/Italy/5093/99). These were then used to infect T. ni pupae, resulting in the production of the desired recombinant antigens. The obtained HA proteins were purified using affinity chromatography, consistently yielding approximately 75 mg/L of insect extract. The vaccine antigen effectively immunized poultry, which were subsequently challenged with a virulent H7N1 avian influenza virus. Following infection, all vaccinated animals survived without displaying any clinical symptoms, while none of the mock-vaccinated control animals survived. The CrisBio®-derived antigens induced high titers of HA-specific antibodies in the vaccinated poultry, demonstrating hemagglutination inhibition activity against avian H7N1 and human H7N9 viruses. These results suggest that the CrisBio® technology platform has the potential to address major industry challenges associated with producing recombinant influenza subunit vaccines, such as enhancing production yields, scalability, and the speed of development, facilitating the global deployment of highly effective influenza vaccines.

The role of a novel secretory peptidoglycan recognition protein with antibacterial ability from the Chinese Oak Silkworm Antheraea pernyi in humoral immunity.

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors that play a critical role in the immune response of invertebrates and vertebrates. Herein, the short ApPGRP-D gene was cloned from the model lepidopteran Antheraea pernyi. Quantitative PCR (qPCR) confirmed that ApPGRP-D is an immune-related protein and that the expression of ApPGRP-D can be induced by microorganisms. ApPGRP-D is a broad-spectrum pattern recognition protein that activates the prophenoloxidase cascade activation system and promotes the agglutination of microbial cells. Likely due to its amidase activity, ApPGRP-D can inhibit the growth of E. coli and S. aureus. In addition, we demonstrated for the first time that zinc ions, as important metal coenzymes, could promote multiple functions of ApPGRP-D but not its amidase activity.

Expression of a viral ecdysteroid UDP-glucosyltransferase enhanced the insecticidal activity of the insect pathogenic fungus Beauveria bassiana.

BACKGROUND: Entomopathogenic fungi, such as Beauveria bassiana, hold promise as biological control agents against insect pests. However, the efficacy of these fungi can be hindered by insect immune responses. One strategy to enhance fungal virulence is to manipulate host immune by targeting key regulatory molecules like 20-hydroxyecdysone (20E). RESULTS: In this study, we engineered B. bassiana strains to constitutively express the enzyme ecdysteroid UDP-glucosyltransferase (EGT), which inactivates 20E, a crucial insect molting hormone. The engineered strain Bb::EGT-1 exhibited robust expression of EGT, leading to a significant reduction in insect 20E levels upon infection. Moreover, infection with Bb::EGT-1 resulted in accelerated larval mortality. Immune responses analysis revealed repression of insect immune response genes and decreased phenoloxidase (PO) activity in larvae infected with Bb::EGT-1. Microbiome analysis indicated alterations in bacterial composition within infected insects, with increased abundance observed during infection with Bb::EGT-1. Additionally, the presence of bacteria hindered hyphal emergence from insect cadavers, suggesting a role for microbial competition in fungal dissemination. CONCLUSIONS: Constitutive expression of EGT in B. bassiana enhances fungal virulence by reducing insect 20E levels, suppressing immune responses, and altering the insect microbiome. These findings highlighted the potential of engineered fungi as effective biocontrol agents against insect pests and provide insights into the complex interactions between entomopathogenic fungi, their hosts, and associated microbes. © 2024 Society of Chemical Industry.

Cellular dynamics of host - parasitoid interactions: Insights from the encapsulation process in a partially resistant host.

Cotesia typhae is an eastern African endoparasitoid braconid wasp that targets the larval stage of the lepidopteran stem borer, Sesamia nonagrioides, a maize crop pest in Europe. The French host population is partially resistant to the Makindu strain of the wasp, allowing its development in only 40% of the cases. Resistant larvae can encapsulate the parasitoid and survive the infection. This interaction provides a very interesting frame for investigating the impact of parasitism on host cellular resistance. We characterized the parasitoid ovolarval development in a permissive host and studied the encapsulation process in a resistant host by dissection and histological sectioning compared to that of inert chromatography beads. We measured the total hemocyte count in parasitized and bead-injected larvae over time to monitor the magnitude of the immune reaction. Our results show that parasitism of resistant hosts delayed encapsulation but did not affect immune abilities towards inert beads. Moreover, while bead injection increased total hemocyte count, it remained constant in resistant and permissive larvae. We conclude that while Cotesia spp virulence factors are known to impair the host immune system, our results suggest that passive evasion could also occur.

Lipopolysaccharide with long O-antigen is crucial for Salmonella Enteritidis to evade complement activity and to facilitate bacterial survival in vivo in the Galleria mellonella infection model.

Bacterial resistance to serum is a key virulence factor for the development of systemic infections. The amount of lipopolysaccharide (LPS) and the O-antigen chain length distribution on the outer membrane, predispose Salmonella to escape complement-mediated killing. In Salmonella enterica serovar Enteritidis (S. Enteritidis) a modal distribution of the LPS O-antigen length can be observed. It is characterized by the presence of distinct fractions: low molecular weight LPS, long LPS and very long LPS. In the present work, we investigated the effect of the O-antigen modal length composition of LPS molecules on the surface of S. Enteritidis cells on its ability to evade host complement responses. Therefore, we examined systematically, by using specific deletion mutants, roles of different O-antigen fractions in complement evasion. We developed a method to analyze the average LPS lengths and investigated the interaction of the bacteria and isolated LPS molecules with complement components. Additionally, we assessed the aspect of LPS O-antigen chain length distribution in S. Enteritidis virulence in vivo in the Galleria mellonella infection model. The obtained results of the measurements of the average LPS length confirmed that the method is suitable for measuring the average LPS length in bacterial cells as well as isolated LPS molecules and allows the comparison between strains. In contrast to earlier studies we have used much more precise methodology to assess the LPS molecules average length and modal distribution, also conducted more subtle analysis of complement system activation by lipopolysaccharides of various molecular mass. Data obtained in the complement activation assays clearly demonstrated that S. Enteritidis bacteria require LPS with long O-antigen to resist the complement system and to survive in the G. mellonella infection model.

A fungal pathogen secretes a cell wall-associated ß-N-acetylhexosaminidase that is co-expressed with chitinases to contribute to infection of insects.

BACKGROUND: ß-N-acetylhexosaminidases (HEXs) are widely distributed in fungi and involved in cell wall chitin metabolism and utilization of chitin-containing substrates. However, details of the fungal pathogens-derived HEXs in the interaction with their hosts remain limited. RESULTS: An insect nutrients-induced ß-N-acetylhexosaminidase, BbHex1, was identified from the entomopathogenic fungus Beauveria bassiana, which was involved in cell wall modification and degradation of insect cuticle. BbHex1 was localized to cell wall and secreted, and displayed enzyme activity to degrade the chitinase-hydrolyzed product (GlcNAc)2. Disruption of BbHex1 resulted in a significant decrease in the level of cell wall chitin in the presence of insect nutrients and during infection of insects, with impaired ability to penetrate insect cuticle, accompanying downregulated cell wall metabolism-involved and cuticle-degrading chitinase genes. However, the opposite phenotypes were examined in the gene overexpression strain. Distinctly altered cell wall structures caused by BbHex1 mutation and overexpression led to the easy activation and evasion (respectively) of insect immune response during fungal infection. As a result, BbHex1 contributed to fungal virulence. Bioinformatics analysis revealed that promoters of some co-expressed chitinase genes with the BbHex1 promoter shared conserved transcription factors Skn7, Msn2 and Ste12, and CreA-binding motifs, implying co-regulation of those genes with BbHex1. CONCLUSION: These data support a mechanism that the fungal pathogen specifically expresses BbHex1, which is co-expressed with chitinases to modify cell wall for evasion of insect immune recognition and to degrade insect cuticle, and contributes to the fungal virulence against insects. © 2024 Society of Chemical Industry.

Hemolin increases the immune response of a caterpillar to NPV infection.

Hemolin, a member of the immunoglobulin superfamily, plays a crucial role in the immune responses of insects against pathogens. However, the innate immune response of Hemolin to baculovirus infection varies among different insects, and the antiviral effects of Hemolin in Hyphantria cunea (HcHemolin) remain poorly understood. Our results showed that HcHemolin was expressed throughout all developmental stages, with higher expressions observed during pupal and adult stages of H. cunea. Additionally, HcHemolin was expressed in reproductive and digestive organs. The expression levels of the HcHemolin were induced significantly following H. cunea nucleopolyhedrovirus (HcNPV) infection. The susceptibility of H. cunea larvae to HcNPV decreased upon silencing of HcHemolin, resulting in a 40% reduction in median lifespan compared to the control group. The relative growth rate (RGR), the relative efficiency of consumption rate (RCR), the efficiency of the conversion of ingested food (ECI), and efficiency of the conversion of digested food (ECD) of silenced H. cunea larvae were significantly lower than those of the control group. Immune challenge assays showed that the median lifespan of treated H. cunea larvae was two-fold longer than the control group after HcNPV and HcHemolin protein co-injection. Therefore, we propose that HcHemolin plays a crucial role in regulating the growth, development, and food utilization of H. cunea, as well as in the antiviral immune response against HcNPV. These findings provide implications for the development of targeted nucleic acid pesticides and novel strategies for pollution-free biological control synergists for HcNPV.

The effect of infection with the entomopathogenic fungus Conidiobolus coronatus (Entomopthorales) on eighteen cytokine-like proteins in Galleria mellonella (Lepidoptera) larvae.

Background: In response to the replace mammal research models with insects in preliminary immunological studies, interest has grown in invertebrate defense systems. The immunological response is regulated by cytokines; however, while their role in mammals is well understood, little is known of their function in insects. A suitable target for studies into insect immunology is Galleria mellonella (Lepidoptera), the wax moth: a common host for human fungal and bacterial pathogens. G. mellonella is also a perfect subject for studies into the presence of cytokine-like proteins. Specific objectives: The main goal of present research was detection in insect immunocompetent cells the 18 mammalian cytokines (IL-1α, IL-1ß, IL-2, IL-3, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-17, IL-19, IFN-γ, TNF-α, TNF-ß, GM-CSF, M-CSF, G-CSF), which play important role in immunological response and indication how their level change after fungal infection. Methodology: The changes of cytokine-like proteins level were detected in hemocytes taken from G. mellonella larvae infected with entomopathogenic fungus, C. coronatus. The presence of cytokine-proteins was confirmed with using fluorescence microscopy (in cultured hemocytes) and flow cytometry (in freshly collected hemolymph). The ELISA test was used to detect changes in concentration of examined cytokine-like proteins. Results: Our findings indicated the presence of eighteen cytokine-like molecules in G. mellonella hemocytes during infection with C. coronatus. The hemocytes taken from infected larvae demonstrated higher fluorescence intensity for six cytokine-like proteins (GM-CSF, M-CSF, IL-3, IL-15, IL-1ß and IL-19) compared to untreated controls. ELISA test indicated significantly higher IL-3 and IL-15. M-CSF, IL-1α and IL-19 concentration in the hemolymph after fungal infection, and significantly lower TNF-ß and G-CSF. Conclusions: Our findings confirm that the selected cytokine-like molecules are present in insect hemocytes and that their concentrations change after fungal infection, which might suggest that they play a role in the anti-fungal immunological response.

Molecular characterization, immune functions and DNA protective effects of peroxiredoxin-1 gene in Antheraea pernyi.

Peroxiredoxins are antioxidant proteins that detoxify peroxynitrite, hydrogen peroxide, and organic hydroperoxides, impacting various physiological processes such as immune responses, apoptosis, cellular homeostasis, and so on. In the present study, we identified and characterized peroxiredoxin 1 from Antheraea pernyi (thereafter designated as ApPrx-1) that encodes a predicted 195 amino acid residue protein with a 21.8 kDa molecular weight. Quantitative real-time PCR analysis revealed that the mRNA level of ApPrx-1 was highest in the hemocyte, fat body, and midgut. Immune-challenged larval fat bodies and hemocytes showed increased ApPrx-1 transcript. Moreover, ApPrx-1 expression was induced in hemocytes and the whole body of A. pernyi following exogenous H2O2 administration. A DNA cleavage assay performed using recombinant ApPrx-1 protein showed that rApPrx-1 protein manifests the ability to protect supercoiled DNA damage from oxidative stress. To test the rApPrx-1 protein antioxidant activity, the ability of the rApPrx-1 protein to remove H2O2 was assessed in vitro using rApPrx-1 protein and DTT, while BSA + DDT served as a control group. The results revealed that ApPrx-1 can efficiently remove H2O2 in vitro. In the loss of function analysis, we found that ApPrx-1 significantly increased the levels of H2O2 in ApPrx-1-depleted larvae compared to the control group. We also found a significantly lower survival rate in the larvae in which ApPrx-1 was knocked down. Interestingly, the antibacterial activity was significantly higher in the ApPrx-1 depleted larvae, compared to the control. Collectively, evidence strongly suggests that ApPrx-1 may regulate physiological activities and provides a reference for further studies to validate the utility of the key genes involved in reliving oxidative stress conditions and regulating the immune responses of insects.

Antimicrobial peptide cecropin B functions in pathogen resistance of Mythimna separata.

Mythimna separata (Lepidoptera: Noctuidae) is an omnivorous pest that poses a great threat to food security. Insect antimicrobial peptides (AMPs) are small peptides that are important effector molecules of innate immunity. Here, we investigated the role of the AMP cecropin B in the growth, development, and immunity of M. separata. The gene encoding M. separata cecropin B (MscecropinB) was cloned. The expression of MscecropinB was determined in different developmental stages and tissues of M. separata. It was highest in the prepupal stage, followed by the pupal stage. Among larval stages, the highest expression was observed in the fourth instar. Tissue expression analysis of fourth instar larvae showed that MscecropinB was highly expressed in the fat body and haemolymph. An increase in population density led to upregulation of MscecropinB expression. MscecropinB expression was also upregulated by the infection of third and fourth instar M. separata with Beauveria bassiana or Bacillus thuringiensis (Bt). RNA interference (RNAi) targeting MscecropinB inhibited the emergence rate and fecundity of M. separata, and resulted in an increased sensitivity to B. bassiana and Bt. The mortality of M. separata larvae was significantly higher in pathogen plus RNAi-treated M. separata than in controls treated with pathogens only. Our findings indicate that MscecropinB functions in the eclosion and fecundity of M. separata and plays an important role in resistance to infection by B. bassiana and Bt.

Enhanced baculoviral virulence by suppressing the degradation of an insect immune resolvin, epoxyoctadecamonoenoic acid, in three lepidopteran insects.

Epoxyoctadecamonoenoic acids (EpOMEs) are produced from linoleic acid by a cytochrome P450 monooxygenase (CYP) and play a crucial role in terminating excessive and unnecessary immune responses during the late infection stage in insects. This suggests that an increase in the EpOME level may enhance the virulence of insect pathogens against pests. This study tested this hypothesis using a specific inhibitor against soluble epoxide hydrolase (sEH) to degrade EpOMEs, which leads to elevated endogenous EpOME levels. A baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), was used to infect three different lepidopteran insects (Spodoptera exigua, Maruca vitrata, and Plutella xylostella) by oral feeding or hemocoelic injection treatments. Within one hour, the viral infection induced the expression of three different phospholipase A2 (PLA2) genes and, after 12 h, up-regulated the expressions of CYP and sEH genes in Spodopera exigua. As expected, AcMNPV virulence was suppressed by the addition of arachidonic acid (a catalytic product of PLA2) but was enhanced by the addition of either of the EpOME regioisomers. In addition, treatment with a specific sEH inhibitor (AUDA) increased AcMNPV virulence against three different lepidopteran insects, presumably by increasing endogenous EpOME levels. This enhanced effect of EpOMEs on virulence was further supported by specific RNA interference (RNAi), in which RNAi specific to CYP expression decreased AcMNPV virulence while a specific RNAi against sEH expression significantly enhanced virulence. In response to AcMNPV infection, TUNEL assay results showed that S. exigua larvae exhibited apoptosis in the midgut, fat body, and epidermis. Inhibition of apoptosis by a pan-caspase inhibitor, Z-VAD-FMK, significantly increased virulence. Similarly, the addition of AUDA to the viral treatment suppressed the gene expression of five inducible caspases and cytochrome C to suppress apoptosis, which led to a significant increase in the tissue viral titers. These results indicate that EpOMEs play a role in terminating excessive and unnecessary immune responses against viral infection during the late stage by down-regulating antiviral apoptosis in lepidopteran insects.

Pleiotropic immunoregulation by growth-blocking peptide in Ostrinia furnacalis.

Insects rely on their innate immune system to eliminate pathogenic microbes. As a system component, cytokines transmit intercellular signals to control immune responses. Growth-blocking peptide (GBP) is a member of the stress-responsive peptide family of cytokines found in several orders of insects, including Drosophila. However, the physiological role of GBP in defence against pathogens is not thoroughly understood. In this study, we explored the functions of GBP in a lepidopteran pest, Ostrinia furnacalis. Injection of recombinant O. furnacalis GBP (OfGBP) precursor (proGBP) and chemically synthesised GBP significantly induced the transcription of antimicrobial peptides (AMPs) and other immunity-related genes including immune deficiency (IMD) and Dorsal. The level of OfGBP mRNA was upregulated after bacterial infection. Knockdown of OfGBP expression led to a decrease in IMD, Relish, MyD88 and Dorsal mRNA levels. OfGBP induced phenoloxidase activity and affected hemocyte behaviours in O. furnacalis larvae. In summary, GBP is a potent cytokine, effectively regulating AMP synthesis, melanization response and cellular immunity to eliminate invading pathogens.

Molecular mechanism of Serratia marcescens Bizio infection in Reticulitermes chinensis Snyder based on full-length SMRT transcriptome sequencing.

Reticulitermes chinensis Snyder is an important pest in forestry and construction and is widely distributed in China. We found that Serratia marcescens Bizio strain SM1 has insecticidal activity to R. chinensis, but the pathogenic mechanism of SM1 to R. chinensis is not clear. Therefore, full-length transcriptome sequencing was performed on R. chinensis infected with SM1 and the control group. A total of 230 differentially expressed genes were identified by comparing SM1 infection group and the control group, among which 103 were downregulated and 127 were upregulated. We found downregulated genes in nine metabolic pathway categories, among which carbohydrate metabolism had the most downregulated genes, followed by energy metabolism and amino acid metabolism. We also found that some downregulated genes were related to pattern recognition receptors, cellular immunity, and humoral immunity, indicating that R. chinensis immunity was negatively affected by SM1 infection. In addition, some genes in signal transduction and genetic information processing pathways were downregulated. In this study, high-throughput full-length transcriptome analysis was used to analyse the pathogenic mechanism of SM1 to R. chinensis. The results of this study provide useful information for exploring the relationship between SM1 and R. chinensis, and provide theoretical support for the future application of SM1 and the prevention and treatment of R. chinensis.

Calcium and integrin-binding protein 1-like interacting with an integrin α-cytoplasmic domain facilitates cellular immunity in Helicoverpa armigera.

Integrins are transmembrane receptor heterodimers composed of α and ß subunits. They are known to mediate extracellular signals to promote cell adhesion and spreading, and are therefore essential for cellular immunity. However, proteins that bind to integrin cytoplasmic domains and mediate intracellular signaling to promote cell adhesion require identification. Calcium and integrin-binding protein 1 (CIB1) that binds to the integrin α-cytoplasmic domain has rarely been examined in insects. In this study, we found that 20-hydroxyecdysone promoted cell phagocytosis and spreading in Helicoverpa armigera. Transcriptomic analyses of hemocytes identified an integrin α gene (HaINTα-PS1) whose expression could be induced by either 20-hydroxyecdysone injection or bead challenge. Isothermal titration calorimetry assays showed that H. armigera CIB1-like (HaCIB1-like) weakly bound to the cytoplasmic domain of HaINTα-PS1 in the presence of calcium. HaINTα-PS1 or HaCIB1-like knockdown inhibited hemocytic encapsulation and phagocytosis, and plasmatocyte spreading. Moreover, HaCIB1-like overexpression in a H. armigera epidermal cell line overexpanded cells and impaired cell phagocytosis. Thus, insect CIB1-like potentially interacted with integrin α-cytoplasmic domain and facilitated cell adhesion. This study enriches our understanding of the molecular mechanism underlying integrin-mediated cellular immunity in insects.

Physiological and transcriptional immune responses of a non-model arthropod to infection with different entomopathogenic groups.

Insect immune responses to multiple pathogen groups including viruses, bacteria, fungi, and entomopathogenic nematodes have traditionally been documented in model insects such as Drosophila melanogaster, or medically important insects such as Aedes aegypti. Despite their potential importance in understanding the efficacy of pathogens as biological control agents, these responses are infrequently studied in agriculturally important pests. Additionally, studies that investigate responses of a host species to different pathogen groups are uncommon, and typically focus on only a single time point during infection. As such, a robust understanding of immune system responses over the time of infection is often lacking in many pest species. This study was conducted to understand how 3rd instar larvae of the major insect pest Helicoverpa zea responded through the course of an infection by four different pathogenic groups: viruses, bacteria, fungi, and entomopathogenic nematodes; by sampling at three different times post-inoculation. Physiological immune responses were assessed at 4-, 24-, and 48-hours post-infection by measuring hemolymph phenoloxidase concentrations, hemolymph prophenoloxidase concentrations, hemocyte counts, and encapsulation ability. Transcriptional immune responses were measured at 24-, 48-, and 72-hours post-infection by quantifying the expression of PPO2, Argonaute-2, JNK, Dorsal, and Relish. This gene set covers the major known immune pathways: phenoloxidase cascade, siRNA, JNK pathway, Toll pathway, and IMD pathway. Our results indicate H. zea has an extreme immune response to Bacillus thuringiensis bacteria, a mild response to Helicoverpa armigera nucleopolyhedrovirus, and little-to-no detectable response to either the fungus Beauveria bassiana or Steinernema carpocapsae nematodes.