Cereal leaf beetle-associated bacteria enhance the survival of their host upon insecticide treatments and respond differently to insecticides with different modes of action.

The cereal leaf beetle (CLB, Oulema melanopus) is one of the major cereal pests. The effect of insecticides belonging to different chemical classes, with different mechanisms of action and the active substances' concentrations on the CLB bacterial microbiome, was investigated. Targeted metagenomic analysis of the V3-V4 regions of the 16S ribosomal gene was used to determine the composition of the CLB bacterial microbiome. Each of the insecticides caused a decrease in the abundance of bacteria of the genus Pantoea, and an increase in the abundance of bacteria of the genus Stenotrophomonas, Acinetobacter, compared to untreated insects. After cypermethrin application, a decrease in the relative abundance of bacteria of the genus Pseudomonas was noted. The dominant bacterial genera in cypermethrin-treated larvae were Lactococcus, Pantoea, while in insects exposed to chlorpyrifos or flonicamid it was Pseudomonas. Insecticide-treated larvae were characterized, on average, by higher biodiversity and richness of bacterial genera, compared to untreated insects. The depletion of CLB-associated bacteria resulted in a decrease in larval survival, especially after cypermethrin and chlorpyrifos treatments. The use of a metagenome-based functional prediction approach revealed a higher predicted function of bacterial acetyl-CoA C-acetyltransferase in flonicamid and chlorpyrifos-treated larvae and tRNA dimethyltransferase in cypermethrin-treated insects than in untreated insects.

The Impact of Oulema melanopus-Associated Bacteria on the Wheat Defense Response to the Feeding of Their Insect Hosts.

Wheat production is threatened by the destructive effects of numerous pests, including Oulema melanopus (cereal leaf beetle, CLB). Both adults and larvae of CLB damage grain crops, but the target of insecticide treatments are the larvae. Insect-associated bacteria are important for many of the insects' life processes and may also modulate plant defense responses to feeding of their insect host. The aim of our study was to elucidate the early wheat plants' reaction to this herbivore feeding and to disclose the CLB-associated bacteria modulation of the wheat-insect interactions. Transcriptome analyses were performed for the leaves wounded mechanically and by feeding of the CLB larvae as well as for the distal leaves to study both, the plant's local and systemic response. Comparative transcriptome analysis indicated that 24 h after the plant treatment, a much larger number of up-regulated DEGs in damaged leaves was noted, especially those on which larvae were fed. It may suggest that at the analysed time point, the local response was stronger than the systemic one. In the leaves on which larvae with natural bacterial flora were fed (local response), the number of up- and down-regulated differentially expressed genes (DEGs) was 7136 and 7411, respectively, in comparison to the dataset obtained for the leaves wounded by larvae with a reduced number of bacteria. In the distal leaves, 3015 up- and 2372 down-regulated DEGs were noted. CLB-associated bacteria were found to affect many aspects of the physiology of wheat plants, especially in wounded leaves, including the expression of genes related to primary metabolism, phytohormone signaling and photosynthesis. We also observed that CLB-associated bacteria mitigated numerous anti-herbivore processes and pathways associated with the synthesis of metabolites and proteins, potentially harmful to the insects. The bacteria also reversed the expression of some genes involved, inter alia, in the phosphorylation of proteins, oxidative stress, cell wall organization, and biogenesis. Understanding the role of CLB-associated bacteria in the plant's defense response will be important to the fields of pest control and herbivore and its host ecology and evolution.

Beetles as Plant Pathogen Vectors.

Herbivorous insects, likewise, other organisms, are exposed to diverse communities of microbes from the surrounding environment. Insects and microorganisms associated with them share a range of relationships, including symbiotic and pathogenic. Insects damage plants by feeding on them and delivering plant pathogens to wounded places, from where pathogens spread over the plant. Thus insects can be considered as both pests and reservoirs or vectors of plant pathogens. Although beetles are not mentioned in the first place as plant pathogen vectors, their transmission of pathogens also takes place and affects the ecosystem. Here we present an overview of beetles as vectors of plant pathogens, including viruses, bacteria, fungi, nematodes, and Oomycota, which are responsible for developing plant diseases that can have a significant impact on crop yield and quality.

The structure of the cereal leaf beetle (Oulema melanopus) microbiome depends on the insect's developmental stage, host plant, and origin.

Cereal leaf beetle (CLB, Oulema melanopus, Coleoptera, Chrysomelidae) is a serious agricultural pest that causes considerable damages to agricultural production. The aim of this study was to characterize the bacterial communities associated with larvae and imagoes of CLB collected from various cereal host species and locations. The bacterial profile was characterized by 16S rRNA gene sequencing at the V3-V4 hypervariable region. Using taxonomy-based analysis, the bacterial community of CLB containing 16 phyla, 26 classes, 49 orders, 78 families, 94 genera, and 63 species of bacteria was identified. The abundance of Wolbachia, Rickettsia, and Lactococcus genus was significantly higher in CLB imagoes than in larvae. Statistical analysis confirmed that the bacterial community of the larvae is more diverse in comparison to imagoes and that insects collected from spring barley and wheat are characterized by a much higher biodiversity level of bacterial genera and species than insects collected from other cereals. Obtained results indicated that the developmental stage, the host plant, and the insect's sampling location affected the CLB's microbiome. Additionally, the CLB core microbiome was determined. It consists of 2 genera (Wolbachia and Rickettsia) shared by at least 90% tested CLB insects, regardless of the variables analysed.

Pantoea ananatis, A New Bacterial Pathogen Affecting Wheat Plants (Triticum L.) in Poland.

Wheat (Triticum aestivum) is one of the most economically important crops in the world. During the routine monitoring of wheat pest, the cereal leaf beetle (CLB, Oulema melanopus, Coleoptera, Chrysomelidae), in the Greater Poland region, it was observed that some leaves wounded by CLB also displayed brownish lesions with clear margins and yellow halo, disease symptoms resembling a bacterial infection. The aim of this study was therefore to investigate those symptoms to establish a causal agent of the disease. The identification based on the results of the Biolog's Gen III system, 16S rRNA, and gyrB genes sequencing, revealed the presence of eight strains of Pantoea ananatis bacteria. Four strains were derived from wheat leaves (Ta024, Ta027, Ta030, Ta046), and four from the CLB's oral secretion (OUC1, OUD2, OUF2, and OUG1). They shared the nucleotide identity ranging from 99 to 100% to P. ananatis strains deposited in the GenBank database. Additionally, the multi-locus sequence analysis (MLSA) of concatenated sequences of partial atpD, fusA, gyrB, rplB, and rpoB genes was performed. All P. ananatis strains isolated in Poland, grouped into one cluster supported with high bootstrap value. Pathogenicity tests performed on four varieties of wheat plants have identified P. ananatis strains as a causal agent of wheat disease. To our knowledge, this is the first report of P. ananatis affecting wheat plants.

Three-way interaction among plants, bacteria, and coleopteran insects.

MAIN CONCLUSION: Coleoptera, the largest and the most diverse Insecta order, is characterized by multiple adaptations to plant feeding. Insect-associated microorganisms can be important mediators and modulators of interactions between insects and plants. Interactions between plants and insects are highly complex and involve multiple factors. There are various defense mechanisms initiated by plants upon attack by herbivorous insects, including the development of morphological structures and the synthesis of toxic secondary metabolites and volatiles. In turn, herbivores have adapted to feeding on plants and further sophisticated adaptations to overcome plant responses may continue to evolve. Herbivorous insects may detoxify toxic phytocompounds, sequester poisonous plant factors, and alter their own overall gene expression pattern. Moreover, insects are associated with microbes, which not only considerably affect insects, but can also modify plant defense responses to the benefit of their host. Plants are also frequently associated with endophytes, which may act as bioinsecticides. Therefore, it is very important to consider the factors influencing the interaction between plants and insects. Herbivorous insects cause considerable damage to global crop production. Coleoptera is the largest and the most diverse order in the class Insecta. In this review, various aspects of the interactions among insects, microbes, and plants are described with a focus on coleopteran species, their bacterial symbionts, and their plant hosts to demonstrate that many factors contribute to the success of coleopteran herbivory.

Identification and partial characterization of proteases in larval preparations of the cereal leaf beetle (Oulema melanopus, Chrysomelidae, Coleoptera).

We determined some biochemical properties of Oulema melanopus larval gut proteases. We found adult midgut enzyme preparations yielded results similar to whole-larval preparations, permitting studies of the very small whole-larval preparations. Protein preparations were analyzed using FITC-casein as a substrate. Acidic pH is optimal for proteolytic activity (range 3.0-4.0). Cysteine protease activity increased at acidic pH and in the presence of ß-mercaptoethanol. Protease activities at all pH values were maximal at 45°C. Enzyme activity in larval preparations was inhibited by addition of Fe(2+) , Ca(2+) , Mg(2+) , Zn(2+) , and K(+) (10 mM). Fe(2+) and Zn(2+) significantly decreased enzyme activity at all pH values, Ca(2+) and Mg(2+) at pH 6.2 and Mg(2+) at pH 4.0. Inhibitors, including pepstatin A, showed the greatest inhibition at pH 4.0; phenylmethylsulfonyl fluoride, N-p-tosyl-l-phenylalanine chloromethyl ketone at pH 6.2; and phenylmethylsulfonyl fluoride, Nα -tosyl-l-lysine chloromethyl ketone hydrochloride, N-p-tosyl-l-phenylalanine chloromethyl ketone, trans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane at pH of 7.6. Inhibition assays indicated that cysteine, aspartyl (cathepsin D), serine (trypsin, chymotrypsin-like) proteases and metalloproteases act in cereal leaf beetle digestion.