Insect Immunity Research Articles

MicroRNA expression profiling of Plutella xylostella after challenge with B. thuringiensis

The diamondback moth, Plutella xylostella, the main pest of brassica crops, has developed resistance to almost all major classes of insecticides as the farmers rely on insecticides to control this pest. An extensive use of broad-spectrum insecticides against P. xylostella promotes the selection of insecticide resistance, destroy natural enemies, and pollute the environment. In this scenario, it is imperative to use genetic methods such as gene silencing technology as an alternate approach against this pest. Evidence shows that microRNAs play pivotal roles in the regulation of target genes at the post-transcription level and show differential expression under various biological processes. However, the knowledge of their role in insect immunity is still in its infancy. In the present study, we aimed at exploring the response of P. xylostella miRNAs against B. thuringiensis at different time courses (6, 12, 18, 24, and 36 h) by using small RNA sequencing. After data filtration, a combined set of 149 miRNAs was identified from all the libraries. Interestingly, a couple of conserved miRNAs such as miR-1, Let-7, miR-275, miR-184, and miR-10 were listed as abundantly expressed miRNAs after exposure to B. thuringiensis. It is worth mentioning that the differential expression analysis revealed that miR-2, a conserved miRNA, was up-regulated following infection. Furthermore, we experimentally validated the involvement of miR-2b-3p in the regulation of corresponding target trypsin. Our luciferase assay results revealed that miR-2b-3p mimic significantly down-regulated the target gene trypsin indicating that it might play a crucial role in the defense mechanism of P. xylostella against B. thuringiensis infection. On the whole, our findings provide insights into the possible regulatory role of miRNAs in insect immunity in response to microorganisms.

Upregulation of Helicoverpa armigera core RNA interference genes by bacterial infections and its effect on the insect–bacteria interaction

RNA interference (RNAi) is an extremely conserved defence mechanism. The antiviral role of the RNAi pathway in insects is well documented; however, the relevance of this pathway in other aspects of insect immunity is largely unknown. In this study, we questioned whether RNAi has any function during insect-bacteria interactions. For this, we assessed induction of the RNAi pathway in response to bacterial infections by monitoring the expression of dicer1/argonaute1 and dicer2/argonaute2, which are important genes in the microRNA and short interfering RNA sub-pathways respectively. Bacterial cells of Bacillus thuringiensis and Serratia marcescens were injected into the haemocoel of fifth-instar larvae of Helicoverpa armigera, whereas double-distilled water was injected into control insects. Expression levels of the RNAi-related genes increased in the bacteria-injected larvae compared with controls. Transcript knockdown of dicer1 reduced the replication of B.thuringiensis; as a consequence, larval mortality decreased compared with the control. However, replication of S.marcescens increased following dicer1 silencing, which led to higher rates of larval mortality when compared with the control. RNAi of dicer2 promoted replication of both bacteria in the larvae and also enhanced larval mortality. Therefore, dicer1 and dicer2 affected larval survival and the replication rates of the pathogenic bacteria, suggesting their roles in the interactions.

Role of a serine protease gene (AccSp1) from Apis cerana cerana in abiotic stress responses and innate immunity.

Clip-domain serine proteases (Clip-SPs) mediate innate immunity and embryonic development in insects. However, the function of Clip-SPs in Apis cerana cerana is little known. Here, a Clip-SP gene, AccSp1, was identified. AccSp1 was mainly detected in third and sixth day instar larvae, dark-eyed pupae, and adults (1and 30days post-emergence). In addition, AccSp1 was expressed at its highest level in the venom gland and epidermis than tentacle, abdomen, muscle, honey sac, head, leg, chest, hemolymph, rectum, and midgut. AccSp1 was induced by 4, 24, and 44°C; H2O2; CdCl2; HgCl2; and pesticides (paraquat, pyridaben, and methomyl) and was inhibited by UV light and cyhalothrin treatments. When adults that had been pretreated with dsRNA 6h prior (knocking AccSp1 down) were challenged with Bacillus bombysepticus for 18h, the survival rate of bees greatly decreased, the activity of PO (phenoloxidase) was reduced, revealing that AccSp1 may play a critical role in assisting bees to survive the microbial infection and participate in regulating PO activity. The antioxidant enzymatic activities of catalase, peroxidase, and superoxide dismutase; the contents of hydrogen peroxide and malondialdehyde; and the ratio of NADP+/NADPH were all lower in samples containing dsRNA-AccSp1 interference than in control groups, but the content of carbonyl was not significantly different. These findings suggest the knockdown of AccSp1 may influence melanization so that the antioxidant enzyme activities and the harmful metabolites decreased. These results collectively suggest that AccSp1 plays critical roles in abiotic stresses responses and resistance to pathogens.

Immune priming and clearance of orally acquired RNA viruses in Drosophila.

Immune responses in insects are differentially triggered depending on the infection route used by the pathogen. In most studies involving Drosophila melanogaster and viruses, infection is done by injection, while oral infection, which is probably the most common route of viral entry in nature, remains unexplored. Here, we orally infected adults and larvae from wild-type and RNA interference (RNAi) mutant flies with different RNA viruses. We found that, in contrast with what is observed following virus injection, oral infections initiated at larval or adult stages are cleared in adult flies. Virus elimination occurred despite a larger infectious dose than for injected flies and evidence of viral replication. RNAi mutant flies suffered greater mortality relative to wild-type flies following oral infection, but they also eliminated the virus, implying that RNAi is not essential for viral clearance and that other immune mechanisms act during oral infections. We further showed that information of infection by RNA viruses acquired orally leaves a trace under a DNA form, which confers protection against future reinfection by the same virus. Together, this work presents evidence of clearance and immune priming for RNA viruses in insects and challenges the current view of antiviral immunity in insects.

An improved method for testing invertebrate encapsulation response as shown in the honey bee

In 1882, Metchnikoff documented the encapsulation response (ER) of the invertebrate immune system. Since then, researchers have used Metchnikoff’s method to quantify immune function—and examine its relationship with ecological and behavioral factors—across various insect taxa. While scientists continue to uncover information regarding invertebrate immunity, behavioral ecology, and ecological immunology, the basics of Metchnikoff’s method have remained unchanged. All but two previous studies investigating insect immunity have used sterile or PBS-coated inducers, although we know that the immune system recognizes specific pathogens. To account for the specificity of the immune system, we modified Metchnikoff’s method and coated nylon monofilaments with pathogen-associated molecular patterns (PAMPs). Using honey bees (Apis mellifera), we examined ER using implants coated with PAMPs (“PAMPlants”) found on known honey bee parasites and pathogens. Lipopolysaccharide (LPS), peptidoglycan (PGN), and β-1, 3-glucan (B13G) PAMPlants mimicked an infection with Gram-negative bacteria, Gram-positive bacteria, and fungi, respectively. Our PAMPlants induced stronger responses than the control implants in both singly- (one PBS-coated or PAMP-coated implant) and doubly- (internal control; one PBS-coated and one PAMP-coated implant) implanted animals. In doubly-implanted individuals, there was a significant increase in response to B13G and LPS when compared with internal controls. The PGN and BSA did not differ from the internal controls in the doubly implanted individuals. These methods provide an improvement when exploring responses to specific pathogens and exploring topics within the field of invertebrate ecological immunity. When applied to social systems, these methods can be used to examine the evolution of disease resistance in societies.

Molecular characterisation of Apolipophorin-III gene in Samia cynthia ricini and its roles in response to bacterial infection.

Apolipophorin-III (ApoLp-III) is an abundant hemolymph protein mainly involved in lipid transport and innate immunity in insects. In the present study, the gene Samia cynthia ricini ApoLp-III (ScApoLp-III) was identified from a transcriptome database, and contained 790 nucleotides with a putative open reading frame (ORF) of 561 bp encoding 186 amino acid residues. Phylogenetic analysis revealed that ScApoLp-III had significant homology with ApoLp-III protein from Antheraea pernyi. Higher ScApoLp-III expression levels were found in the fat body and silk gland by reverse transcription quantitative PCR (RT-qPCR). Injection of Staphylococcus aureus induced up-regulation of ScApoLp-III in the midgut, fat body and hemocytes. However, ScApoLp-III was down-regulated in the midgut and fat body after Pseudomonas aeruginosa injection, indicating that ScApoLp-III may contribute to the host's defense against invading pathogens. Additionally, recombinant ScApoLp-III was found to bind different bacteria, including E. coli, P. aeruginosa, S. aureus and B. subtilis. Bactericidal tests showed that recombinant ScApoLp-III strongly inhibited Gram-negative bacteria, including Escherichia coli and P. aeruginosa. However, it had no obvious influence on Gram-positive bacteria. Taken together, our results suggest that the ScApoLp-III might play an important role in the innate immunity of S. c. ricini.

Silkworm serpin32 functions as a negative-regulator in prophenoloxidase activation

The extracellular serine protease cascade is an essential component of insect humoral immunity. Serine protease inhibitors (serpins) play an important regulatory role in the process of insect immunity by regulating the serine protease cascade pathway. We aimed to clarify the function of Bmserpin32 in this study. First, we performed homologous sequence alignment and phylogenetic analysis of Bmserpin32. Bmserpin32 was found to share 64% amino acid sequence identity with Manduca sexta serpin7, an immunomodulatory protein. Bmserpin32 cDNA was cloned, and the recombinant Bmserpin32 protein was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid affinity and gel filtration chromatography. The activity assay showed that Bmserpin32 had significant inhibitory activity against trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and site-directed mutagenesis combined with activity assays indicated that the cleavage site of Bmserpin32 is between Arg359 and Ile360. After infection with E. coli or Micrococcus luteus, the expression level of Bmserpin32 in immune-related tissues was significantly upregulated. In addition, Bmserpin32 could delay or inhibit the melanization of hemolymph by inhibiting the activation of prophenoloxidase in larval hemolymph. Furthermore, a physiological target of Bmserpin32 was identified as the clip protease, BmPAP3, an apparent ortholog of M. sexta propenoloxidase-activating protease-3. Our observations enable a better understanding of the physiological role of Bmserpin32 in regulating melanization in silkworm.

Insects Go on a STING Operation to Tackle Intracellular Invaders

Insect immunity to extracellular microbes relies largely on the TOLL and IMD pathways. In this issue of Immunity, Goto etal. (2018) report that the IKKβ-Relish module of the IMD pathway hitches up the intracellular sensor STING to activate antiviral responses in Drosophila.

Electrophysiological evidence of RML12 mosquito cell line towards neuronal differentiation by 20-hydroxyecdysdone

Continuous cell lines from insect larval tissues are widely used in different research domains, such as virology, insect immunity, gene expression, and bio pharmacology. Previous study showed that introduction of 20-hydroxyecdysone to Spodoptera cell line induced a neuron-like morphology with neurite extensions. Despite some results suggesting potential presence of neuro-receptors, no study so far has shown that these neuron-induced cells were functional. Here, using microelectrode arrays, we showed that the mosquito cell line, RML12, differentiated with 20-hydroxyecdysone, displays spontaneous electrophysiological activity. Results showed that these cells can be stimulated by GABAergic antagonist as well as nicotinic agonist. These results provide new evidence of neuron-like functionality of 20-hydroxyecdysone induced differentiated mosquito cell line. Finally, we used this new model to test the effects of two insecticides, temephos and permethrin. Our analysis revealed significant changes in the spiking activity after the introduction of these insecticides with prolonged effect on the neuronal activity. We believe that this differentiated mosquito neuronal cell model can be used for high-throughput screening of new pesticides on insect nervous system instead of primary neurons or in vivo studies.

Stimulator of interferon genes (STING) provides insect antiviral immunity by promoting Dredd caspase–mediated NF-κB activation

The antiviral cGMP-AMP (cGAMP)-stimulator of interferon genes (STING) pathway is well characterized in mammalian cells. However, whether this pathway also plays a role in insect antiviral immunity is unknown. In this study, we found that cGAMP is produced in silkworm (Bombyx mori) cells infected with nucleopolyhedrovirus (NPV). In searches for STING-related sequences, we identified BmSTING, a potential cGAMP sensor in B. mori We observed that BmSTING overexpression effectively inhibits NPV replication in silkworm larvae, whereas dsRNA-mediated BmSTING knockdown resulted in higher viral load. Cleavage and nuclear translocation of BmRelish, a NF-κB-related transcription factor, was also observed when BmSTING was overexpressed and was enhanced by cGAMP stimulation or viral infection of B. mori larvae. Moreover, we identified a caspase-8-like protein (BmCasp8L) as a BmSTING-interacting molecule and as a suppressor of BmSTING-mediated BmRelish activation. Interestingly, cGAMP stimulation decreased BmCasp8L binding to BmSTING and increased BmRelish activity. Of note, an interaction between death-related ced-3/Nedd2-like caspase (BmDredd) and BmSTING promoted BmRelish cleavage for efficient antiviral signaling and protection of insect cells from viral infection. Our findings have uncovered BmSTING as a critical mediator of antiviral immunity in the model insect B. mori and have identified several BmSTING-interacting proteins that control antiviral defenses.

Transcriptome analysis provides insights into the immunity function of venom glands in Pardosa pseudoannulata in responses to cadmium toxicity.

Due to some similarity of innate immunity between insects and mammals, the study of the molecular mechanism of innate immunity in insects has become a focus of research. However, the exact molecular and cellular basis of immune system in insect remains poorly understood. Characterization of the transcriptomic response to Cd of spider is an effective approach to understanding the innate immunity mechanisms. In this study, we carried out transcriptome sequencing and gene expression analyses to develop molecular resources for Pardosa pseudoannulata venom glands with and without Cd treatments. A total of 92,778 assembled unigenes and 237 Cd stress-associated differentially expressed genes between the Cd-treated and control groups were obtained. Expression profile analysis demonstrated that immunity-related genes involved in bacterial invasion of epithelial cells, leukocyte transendothelial migration, platelet activation, apoptosis, phagosome, and Rap1 signaling pathway were upregulated by Cd exposure, except the genes involved in PPAR signaling pathway were downregulated. Our results provide the first comprehensive transcriptome dataset of venom glands in P. pseudoannulata response to Cd, which is valuable for throws light on the immunotoxicity mechanism of Cd, and the innate immunity complexity.

Galleria mellonella Reveals Niche Differences Between Highly Pathogenic and Closely Related Strains of Francisella spp.

Francisella tularensis, a highly virulent bacteria that causes the zoonotic disease tularemia, is considered a potential agent of biological warfare and bioterrorism. Although the host range for several species within the Francisella is known, little is known about the natural reservoirs of various Francisella species. The lack of knowledge regarding the environmental fates of these pathogens greatly reduces the possibilities for microbial risk assessments. The greater wax moth (Galleria mellonella) is an insect of the order Lepidoptera that has been used as an alternative model to study microbial infection during recent years. The aim of this study was to evaluate G. mellonella as a model system for studies of human pathogenic and closely related opportunistic and non-pathogenic strains within the Francisella genus. The employed G. mellonella larvae model demonstrated differences in lethality between human pathogenic and human non-pathogenic or opportunistic Francisella species. The F. novicida, F. hispaniensis and F. philomiragia strains were significantly more virulent in the G. mellonella model than the strains of human pathogens F. t. holarctica and F. t. tularensis. Our data show that G. mellonella is a possible in vivo model of insect immunity for studies of both opportunistic and virulent lineages of Francisella spp., that produces inverse results regarding lethality in G. mellonella and incapacitating disease in humans. The results provide insight into the potential host specificity of F. tularensis and closely related members of the same genus, thus increasing our present understanding of Francisella spp. ecology.

P38b and JAK-STAT signaling protect against Invertebrate iridescent virus 6 infection in Drosophila.

The fruit fly Drosophila melanogaster is a powerful model system for the study of innate immunity in vector insects as well as mammals. For vector insects, it is particularly important to understand all aspects of their antiviral immune defenses, which could eventually be harnessed to control the transmission of human pathogenic viruses. The immune responses controlling RNA viruses in insects have been extensively studied, but the response to DNA virus infections is poorly characterized. Here, we report that infection of Drosophila with the DNA virus Invertebrate iridescent Virus 6 (IIV-6) triggers JAK-STAT signaling and the robust expression of the Turandots, a gene family encoding small secreted proteins. To drive JAK-STAT signaling, IIV-6 infection more immediately induced expression of the unpaireds, a family of IL-6-related cytokine genes, via a pathway that required one of the three Drosophila p38 homologs, p38b. In fact, both Stat92E and p38b were required for the survival of IIV-6 infected flies. In addition, in vitro induction of the unpaireds required an NADPH-oxidase, and in vivo studies demonstrated Nox was required for induction of TotA. These results argue that ROS production, triggered by IIV-6 infection, leads to p38b activation and unpaired expression, and subsequent JAK-STAT signaling, which ultimately protects the fly from IIV-6 infection.

The interplay between dose and immune system activation determines fungal infection outcome in the African malaria mosquito, Anopheles gambiae

The Toll pathway is a central regulator of antifungal immunity in insects. In mosquitoes, the Toll pathway affects infections with the fungal entomopathogen, Beauveria bassiana, which is considered a potential mosquito biopesticide. We report here the use of B. bassiana strain I93-825 in Anopheles gambiae to analyze the impact of Toll pathway modulation on mosquito survival. Exposure to a narrow dose range of conidia by direct contact decreased mosquito longevity and median survival. In addition, fungal exposure dose correlated positively and linearly with hazard ratio. Increased Toll signaling by knockdown of its inhibitor, cactus, decreased survivorship of uninfected females, increased mosquito survival after low dose B. bassiana exposure, but had little effect following exposure to higher doses. This observed trade-off could have implications for development of B. bassiana as a prospective vector control tool. On the one hand, selection for small increases in mosquito immune signaling across a narrow dose range could impair efficacy of B. bassiana. On the other hand, costs of immunity and the capacity for higher doses of fungus to overwhelm immune responses could limit evolution of resistance.

Research progress on the immune mechanism of the silkworm Bombyx mori

AbstractThe silkworm Bombyx mori L., representing an important economic insect and one of the best models for studying insect immunity, possesses an efficient and sophisticated innate immune system against invasive microorganisms. The innate immune system basically includes humoural immunity and cellular immunity. The humoural immunity, which functions via molecules including humoural factors, lysozymes, phenoloxidase, hemolin, lectins and, in particular, antimicrobial peptides, plays a central role in eliminating the invading pathogens. The cellular immunity is primarily carried out and mediated by plasmatocytes and granular cells of haemocytes in the haemolymph, usually followed by melanization. Additionally, apoptosis, a primary viral defence for insects lacking adaptive immunity, comprises an important part of the silkworm immune system. Currently, there is still the lack of a comprehensive and systematic understanding of the molecular mechanisms of silkworm immunity. We review the latest research progress on silkworm immune mechanisms, including phenoloxidase‐dependent melanization and apoptosis, which is conducive to improving our understanding of the silkworm immune mechanism, clarifying the relationship of various immune mechanisms, and also providing a theoretical basis and reference for the future research of insect immunity.

Laccase 1 gene from Plutella xylostella (PxLac1) and its functions in humoral immune response

Laccase (EC 1.10.3.2) is a phenoloxidase found in many insect species. The Laccase 1 gene from Plutella xylostella (PxLac1) was cloned, and its expression patterns and functions were determined using qPCR and RNAi methods. The results showed that the expression levels of PxLac1 were consistently high in all larval stages, and the most abundant was in the midgut during the 4th instar stage. Moreover, the expression of PxLac1 was up-regulated in response to bacterial infection, and decreased 24 h after being parasitized by Cotesia vestalis. Further analyses indicated that the effect of parasitization on PxLac1 was induced by active C. vestalis Bracovirus (CvBV). Haemocyte-free hemolymph phenoloxidase (PO) activity was suppressed when PxLac1 was treated with RNAi. Our results provide evidence for a connection between the Laccase 1 gene and insect immunity, and revealed that parasitoid polydnavirus suppresses host PO activity via PxLac1 regulation.

Toll immune signal activates cellular immune response via eicosanoids

Upon immune challenge, insects recognize nonself. The recognition signal will propagate to nearby immune effectors. It is well-known that Toll signal pathway induces antimicrobial peptide (AMP) gene expression. Eicosanoids play crucial roles in mediating the recognition signal to immune effectors by enhancing humoral immune response through activation of AMP synthesis as well as cellular immune responses, suggesting a functional cross-talk between Toll and eicosanoid signals. This study tested a cross-talk between these two signals. Two signal transducing factors (MyD88 and Pelle) of Toll immune pathway were identified in Spodoptera exigua. RNA interference (RNAi) of either SeMyD88 or SePelle expression interfered with the expression of AMP genes under Toll signal pathway. Bacterial challenge induced PLA2 enzyme activity. However, RNAi of these two immune factors significantly suppressed the induction of PLA2 enzyme activity. Furthermore, RNAi treatment prevented gene expression of cellular PLA2. Inhibition of PLA2 activity reduced phenoloxidase activity and subsequent suppression in cellular immune response measured by hemocyte nodule formation. However, immunosuppression induced by RNAi of Toll signal molecules was significantly reversed by addition of arachidonic acid (AA), a catalytic product of PLA2. The addition also significantly reduced the enhanced fungal susceptibility of S. exigua treated by RNAi against two Toll signal molecules. These results indicate that there is a cross-talk between Toll and eicosanoid signals in insect immunity.

Natural Variation in Resistance to Virus Infection in Dipteran Insects.

The power and ease of Drosophila genetics and the medical relevance of mosquito-transmitted viruses have made dipterans important model organisms in antiviral immunology. Studies of virus–host interactions at the molecular and population levels have illuminated determinants of resistance to virus infection. Here, we review the sources and nature of variation in antiviral immunity and virus susceptibility in model dipteran insects, specifically the fruit fly Drosophila melanogaster and vector mosquitoes of the genera Aedes and Culex. We first discuss antiviral immune mechanisms and describe the virus-specificity of these responses. In the following sections, we review genetic and microbiota-dependent variation in antiviral immunity. In the final sections, we explore less well-studied sources of variation, including abiotic factors, sexual dimorphism, infection history, and endogenous viral elements. We borrow from work on other pathogen types and non-dipteran species when it parallels or complements studies in dipterans. Understanding natural variation in virus–host interactions may lead to the identification of novel restriction factors and immune mechanisms and shed light on the molecular determinants of vector competence.

Effect of entomopathogenic nematode of Heterorhabditis indica infection on immune and antioxidant system in lepidopteran pest Spodoptera litura (Lepidoptera: Noctuidae).

Entomopathogenic nematodes form excellent tools to study insect immunity in response to during infection. Insects activate as several defense mechanisms, namely Phenoloxidase, haemocytes, detoxification and antioxidant enzymes. However little mechanistic information is available about the sublethal effects of entomopathogenic nematodes infection on detoxification and immune mechanisms in lepidopteran insects. In the present study, the effects of infection on antioxidant, detoxification and immune systems of Spodoptera litura larvae were studied. Results show a significant reduction in Total Haemocyte Count observed after 3h of infection. A significant increase Superoxide dismutase, Catalase, Glutathione S-transferase, Glutathione Peroxidase and Acid phosphatase were observed 6h after infection and, progressive decrease in Peroxidase, Alkaline phosphatase and Lipid peroxidation was also observed. This study shows that increased detoxification enzyme levels in response to nematode infection are a protective mechanism in insects. Nematode infection suppresses insect immune response, which is evident from low haemocyte count and Phenoloxidase levels to ultimately cause larval mortality.

Dicer-2-Dependent Generation of Viral DNA from Defective Genomes of RNA Viruses Modulates Antiviral Immunity in Insects

SummaryThe RNAi pathway confers antiviral immunity in insects. Virus-specific siRNA responses are amplified via the reverse transcription of viral RNA to viral DNA (vDNA). The nature, biogenesis, and regulation of vDNA are unclear. We find that vDNA produced during RNA virus infection of Drosophila and mosquitoes is present in both linear and circular forms. Circular vDNA (cvDNA) is sufficient to produce siRNAs that confer partially protective immunity when challenged with a cognate virus. cvDNAs bear homology to defective viral genomes (DVGs), and DVGs serve as templates for vDNA and cvDNA synthesis. Accordingly, DVGs promote the amplification of vDNA-mediated antiviral RNAi responses in infected Drosophila. Furthermore, vDNA synthesis is regulated by the DExD/H helicase domain of Dicer-2 in a mechanism distinct from its role in siRNA generation. We suggest that, analogous to mammalian RIG-I-like receptors, Dicer-2 functions like a pattern recognition receptor for DVGs to modulate antiviral immunity in insects.