Steinernema feltiae K1 (Filipjev) (Nematode: Steinernematidae), an entomopathogenic nematode, was isolated and identified based on its morphological and molecular diagnostic characteristics. Its infective juveniles (IJs) were highly pathogenic to three lepidopteran (LC50 = 23.7–25.0 IJs/larva) and one coleopteran (LC50 = 39.3 IJs/larva) insect species. Infected larvae of the diamondback moth, Plutella xylostella (L.) (Insecta: Lepidoptera), exhibited significant reduction in phospholipase A2 (PLA2) activity in their plasma. The decrease of PLA2 activity was followed by significant septicemia of the larvae infected with S. feltiae. Insecticidal activity induced by S. feltiae was explained by significant immunosuppression in cellular immune responses measured by hemocyte nodule formation and total hemocyte count (THC). Although S. feltiae infection suppressed nodule formation and THC in the larvae, an addition of arachidonic acid (AA, a catalytic product of PLA2) rescued these larvae from fatal immunosuppression. In contrast, an addition of dexamethasone (a specific PLA2 inhibitor) enhanced the nematode’s pathogenicity in a dose-dependent manner. To discriminate the immunosuppressive activity of a symbiotic bacterium (Xenorhabdus bovienii (Proteobacteria: Enterobacterales)) from the nematode, kanamycin was applied to after nematode infection. It significantly inhibited the bacterial growth in the hemolymph. Compared to nematode treatment alone, the addition of antibiotics to nematode infection partially rescued the immunosuppression measured by phenol oxidase activity. Consequently, treatment with antibiotics significantly rescued the larvae from the insecticidal activity of S. feltiae. These results suggest that immunosuppression induced by infection of S. feltiae depends on its symbiotic bacteria by inhibiting eicosanoid biosynthesis, resulting in significant insect mortality. However, the addition of antibiotics or AA could not completely rescue the virulence of the nematode, suggesting that the nematode itself also plays a role in its insecticidal activity.
Xenorhabdus hominickii ANU1 is known to be an entomopathogenic bacterium symbiotic to nematode Steinernema monticolum. Another bacterial strain X. hominickii DY1 was isolated from a local population of S. monticolum. This bacterial strain X. hominickii DY1 was found to exhibit high insecticidal activities against lepidopteran and coleopteran species after hemocoelic injection. However, these two X. hominickii strains exhibited significant variations in insecticidal activities, with ANU1 strain being more potent than DY1 strain. To clarify their virulence difference, bacterial culture broths of these two strains were compared for secondary metabolite compositions. GC-MS analysis revealed that these two strains had different compositions, including pyrrolopyrazines, piperazines, cyclopeptides, and indoles. Some of these compounds exhibited inhibitory activities against phospholipase A2 to block eicosanoid biosynthesis and induce significant immunosuppression. They also exhibited significant insecticidal activities after oral feeding, with indole derivatives being the most potent. More kinds of indole derivatives were detected in the culture broth of ANU1 strain. To investigate variations in regulation of secondary metabolite production, expression level of leucine-responsive regulatory protein (Lrp), a global transcription factor, was compared. ANU1 strain exhibited significantly lower Lrp expression level than DY1 strain. To assess genetic variations associated with secondary metabolite synthesis, bacterial loci encoding non-ribosomal protein synthase and polyketide synthase (NRPS-PKS) were compared. Three NRPS and four PKS loci were predicted from the genome of X. hominickii. The two bacterial strains exhibited genetic variations (0.12∼0.67%) in amino acid sequences of these NRPS-PKS. Most NRPS-PKS genes exhibited high expression peaks at stationary phase of bacterial growth. However, their expression levels were significantly different between the two strains. These results suggest that differential virulence of the two bacterial strains is caused by the difference in Lrp expression level, leading to difference in the production of indole compounds and other NRPS-PKS-associated secondary metabolites.
Virgin female moths are known to release sex pheromones to attract conspecific males. Accurate sex pheromones are required for their chemical communication. Sex pheromones of Spodoptera exigua, a lepidopteran insect, contain unsaturated fatty acid derivatives having a double bond at the 12th carbon position. A desaturase of S. exigua (SexiDES5) was proposed to have dual functions by forming double bonds at the 11th and 12th carbons to synthesize Z9,E12-tetradecedienoic acid, which could be acetylated to be a main sex pheromone component Z9,E12-tetradecenoic acetate (Z9E12-14:Ac). A deletion of SexiDES5 using CRISPR/Cas9 was generated and inbred to obtain homozygotes. Mutant females could not produce Z9E12-14:Ac along with Z9-14:Ac and Z11-14:Ac. Subsequently, pheromone extract of mutant females did not induce a sensory signal in male antennae. They failed to induce male mating behavior including hair pencil erection and orientation. In the field, these mutant females did not attract any males while control females attracted males. These results indicate that SexiDES5 can catalyze the desaturation at the 11th and 12th positions to produce sex pheromone components in S. exigua. This study also suggests an application of the genome editing technology to insect pest control by generating non-attractive female moths.
Western flower thrips, Frankliella occidentalis, is a serious pest by directly infesting host crops. It can also give indirect damage to host crops by transmitting a plant virus called tomato spotted wilt virus. A fungal pathogen, Beauveria bassiana, can infect thrips. It has been used as a biopesticide. However, little is known on the defense of thrips against this fungal pathogen. This study assessed the defense of thrips against the fungal infection with respect to immunity by analyzing immune-associated genes of F. occidentalis in both larvae and adults. Immunity-associated genes of western flower thrips were selected from three immunity steps: nonself recognition, mediation, and immune responses. For the pathogen recognition step, dorsal switch protein 1 (DSP1) was chosen. For the immune mediation step, phospholipase A2 (PLA2) and prostaglandin E2 synthase were also selected. For the step of immune responses, two phenoloxidases (PO) genes and four proPO-activating peptidase genes involved in melanization against pathogens were chosen. Dual oxidase gene involved in the production of reactive oxygen species and four antimicrobial peptide genes for executing humoral immune responses were selected. All immunity-associated genes were inducible to the fungal infection. Their expression levels were induced higher in adults than in larvae by the fungal infections. However, inhibitor treatments specific to DSP1 or PLA2 significantly suppressed the inducible expression of these immune-associated genes, leading to significant enhancement of fungal pathogenicity. These results suggest that immunity is essential for thrips to defend against B. bassiana, in which DSP1 and eicosanoids play a crucial role in eliciting immune responses.
Innate immune responses are effective for insect survival to defend against entomopathogens including a fungal pathogen, Metarhizium rileyi, that infects a lepidopteran Spodoptera exigua. In particular, the fungal virulence was attenuated by cellular immune responses, in which the conidia were phagocytosed by hemocytes (insect blood cells) and hyphal growth was inhibited by hemocyte encapsulation. However, the chemokine signal to drive hemocytes to the infection foci was little understood. The hemocyte behaviors appeared to be guided by a Ca2+ signal stimulating cell aggregation to the infection foci. The induction of the Ca2+ signal was significantly inhibited by the cyclooxygenase (COX) inhibitor. Under the inhibitory condition, the addition of thromboxane A2 or B2 (TXA2 or TXB2) among COX products was the most effective to recover the Ca2+ signal and hemocyte aggregation. TXB2 alone induced a microaggregation behavior of hemocytes under in vitro conditions. Indeed, TXB2 titer was significantly increased in the plasma of the infected larvae. The elevated TXB2 level was further supported by the induction of phospholipase A2 (PLA2) activity in the hemocytes and subsequent up-regulation of COX-like peroxinectins (SePOX-F and SePOX-H) in response to the fungal infection. Finally, the expression of a thromboxane synthase (Se-TXAS) gene was highly expressed in the hemocytes. RNA interference (RNAi) of Se-TXAS expression inhibited the Ca2+ signal and hemocyte aggregation around fungal hyphae, which were rescued by the addition of TXB2. Without any ortholog to mammalian thromboxane receptors, a prostaglandin receptor was essential to mediate TXB2 signal to elevate the Ca2+ signal and mediate hemocyte aggregation behavior. Specific inhibitor assays suggest that the downstream signal after binding TXB2 to the receptor follows the Ca2+-induced Ca2+ release pathway from the endoplasmic reticulum of the hemocytes. These results suggest that hemocyte aggregation induced by the fungal infection is triggered by TXB2via a Ca2+ signal through a PG receptor.
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