The red imported fire ant (Solenopsis invicta Buren, 1972) is a globally significant invasive species, causing extensive agricultural, human health, and biodiversity damage amounting to billions of dollars worldwide. The pathogenic fungus Metarhizium anisopliae (Metchnikoff) Sorokin (1883), widely distributed in natural environments, has been used to control S. invicta populations. However, the interaction between M. anisopliae and the immune system of the social insect S. invicta remains poorly understood. In this study, we employed RNA-seq to investigate the effects of M. anisopliae on the immune systems of S. invicta at different time points (0, 6, 24, and 48 h). A total of 1313 differentially expressed genes (DEGs) were identified and classified into 12 expression profiles using short time-series expression miner (STEM) for analysis. Weighted gene co-expression network analysis (WGCNA) was employed to partition all genes into 21 gene modules. Upon analyzing the statistically significant WGCNA model and conducting Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the modules, we identified key immune pathways, including the Toll and Imd signaling pathways, lysosomes, autophagy, and phagosomes, which may collectively contribute to S. invicta defense against M. anisopliae infection. Subsequently, we conducted a comprehensive scan of all differentially expressed genes and identified 33 immune-related genes, encompassing various aspects such as recognition, signal transduction, and effector gene expression. Furthermore, by integrating the significant gene modules derived from the WGCNA analysis, we constructed illustrative pathway diagrams depicting the Toll and Imd signaling pathways. Overall, our research findings demonstrated that M. anisopliae suppressed the immune response of S. invicta during the early stages while stimulating its immune response at later stages, making it a potential biopesticide for controlling S. invicta populations. These discoveries lay the foundation for further understanding the immune mechanisms of S. invicta and the molecular mechanisms underlying its response to M. anisopliae.

Peptidoglycan recognition proteins (PGRPs) are a class of pattern recognition receptors (PRRs) that activate the innate immune system in response to microbial infection by detection of peptidoglycan, a distinct component of bacterial cell walls. Bioinformatic studies have revealed four PGRPs in the red imported fire ant Solenopsis invicta; nonetheless, the mechanism of the immune response of S. invicta induced by pathogens is still poorly understood. The peptidoglycan recognition protein full-length cDNA (designated as SiPGRP-S1/S2/S3/L) from S. invicta was used in this investigation. According to the sequencing analysis, there was a significant degree of homology between the anticipated amino acid sequence of SiPGRPs and other members of the PGRPs superfamily. Molecular docking studies demonstrated that SiPGRPs show strong binding affinity for a variety of PGN substrates. Additionally, tissue distribution analysis indicated that SiPGRPs are primarily expressed in several tissues of naïve larvae, including fat body, hemocytes, head, and thorax, as detected by quantitative real-time PCR (RT-qPCR). Microbial challenges resulted in variable changes in mRNA levels across different tissues. Furthermore, the antibacterial effects of antimicrobial peptides (AMPs) produced by major ants infected with Metarhizium anisopliae were assessed. These AMPs demonstrated inhibitory effects against M. anisopliae, Staphylococcus aureus, and Escherichia coli, with the most pronounced effect observed against E. coli. In conclusion, SiPGRPs act as pattern recognition receptors (PRRs) that identify pathogens and initiate the expression of AMPs in S. invicta, this mechanism contributes to the development of biopesticides designed for the targeted control of invasive agricultural pests.

Long non-coding RNAs (lncRNAs) represent a class of RNA molecules that do not encode proteins. Generally studied for their regulatory potential in model insects, relatively little is known about their immunoregulatory functions in different castes of eusocial insects, including Solenopsis invicta, a notoriously invasive insect pest. In the current study, we used Metarhizium anisopliae, an entomopathogenic fungus, to infect the polymorphic worker castes (Major and Minor Workers) and subjected them to RNA sequencing at different intervals (6, 24, and 48 h post-infection (hpi)). Comprehensive bioinformatic analysis identified 5719 (1869 known and 3850 novel) lncRNAs in all libraries. Genomic characteristics analysis showed that S. invicta lncRNAs exhibited structural similarities with lncRNAs from other eusocial insects, including lower exon numbers, shorter intron and exon lengths, and a lower expression profile. A comparison of lncRNAs in major and minor worker ants revealed that several lncRNAs were exclusively expressed in one worker caste and remained absent in the other. LncRNAs such as MSTRG.12029.1, XR_005575440.1 (6 h), MSTRG.16728.1, XR_005575440.1 (24 h), MSTRG.20263.41, and MSTRG.11994.5 (48 h) were only present in major worker ants, while lncRNAs such as MSTRG.8896.1, XR_005574239.1 (6 h), MSTRG.20289.8, XR_005575051.1 (24 h), MSTRG.20289.8, and MSTRG.6682.1 (48 h) were only detected in minor workers. Additionally, we performed real-time quantitative PCR and experimentally validated these findings. Functional annotation of cis-acting lncRNAs in major worker ants showed that lncRNAs targeted genes such as serine protease, trypsin, melanization protease-1, spaetzle-3, etc. In contrast, apoptosis and autophagy-related genes were identified as targets of lncRNAs in minor ants. Lastly, we identified several lncRNAs as precursors of microRNAs (miRNAs), such as miR-8, miR-14, miR-210, miR-6038, etc., indicating a regulatory relationship between lncRNAs, miRNAs, and mRNAs in antifungal immunity. These findings will serve as a genetic resource for lncRNAs in polymorphic eusocial ants and provide a theoretical basis for exploring the function of lncRNAs from a unique and novel perspective.