As proposed by the MIR hypothesis, the insect resistance of plants colonized with AMF is increased by activating the defense signaling pathways. For example, Wang et al (2020) found that AMF colonization significantly increased the phenolic content in wheat and reduced the number of wheat aphids. It is generally believed that the "AMF-plant" symbiotic system has ecological specificity (Razak and Gange 2021; Simon et al. 2017). In some cases, this ecological specificity inhibits plant resistance and promotes the growth of harmful insects. In line with our previous studies, the present study revealed that GM and GI treatments respectively induced and decreased the resistance of P. alba × P. berolinensis seedlings to gypsy moth (Jiang et al. 2021a; Jiang et al. 2022). These results, together with the previous studies, demonstrated that specific AMF colonization could induce alterations (either increase or decrease) in the plant resistance to insects. Therefore, for the utilization of AMF in agriculture and forestry production, AMF-plant combinations concerning insect resistance should be selected carefully to avoid the positive effects of AMF colonization on the growth of herbivorous insects.
During the long-term evolution process, insects and their gut microbiota have established a mutually beneficial symbiotic relationship (Engel and Moran 2013; Feng et al. 2011; Jiang et al. 2021b). As one of the main factors to maintain homeostasis in host insects, the gut microbiota is regarded as an important index to measure the growth of host insects. In the present study, different gut microbiota at the genus level were identified between the untreated and treated groups by LefSe analysis, and subsequently, their functions were elucidated. The results showed that the relative abundance of 8 genera in gypsy moth larvae gut in the GM group was decreased as compared to the untreated group. Of these, Staphylococcus, Klebsiella, Lachnospirnceae_ND3007, Ruminococcaceae, Akkermansia, and Lactobacillus are major probiotics that participate in the energy metabolism, detoxification metabolism, or improvement of the gut barrier (Chouaia et al. 2019; Dabbou et al. 2020; Dai et al. 2014; Feng et al. 2011; Liang et al. 2020; Muhammad et al. 2017; Osawa et al. 2006). A decrease of these genera in the GM group indicated that the digestion of food materials and the degradation ability of plant secondary substances were reduced in the gypsy moth larvae. Surprisingly, the abundance of some probiotics (Acinetobacter and Ralstonia) that can promote digestion and absorption in the host significantly increased in the GM group (Briones-Roblero et al. 2017; Paulson et al. 2014), indicating that gypsy moth larvae try to compensate for the nutrition obtained from low-quality food with the help of gut microbiota. The abundance of 20 genera in the GI group decreased significantly as compared to those in the untreated group, including 7 pathogenic bacteria (Chryseobacterium, Fusobacterium, Neisseria, Klebsiella, Streptococcus and Pseudomonas) (Bog et al. 2020; Dijokaite et al. 2021; He et al. 2019; Moman et al. 2021; Tzec-Interian et al. 2020; Teoh et al. 2021; Yip et al. 2021). These results indicate that GI treatment seems to improve the gut environment in gypsy moth larvae by reducing the abundance of pathogenic bacteria in the larval gut. In addition, some probiotics associated with the nutritional metabolism and detoxification were also found to decrease in the GI group, including Bifidobacterium, Bacteroides, Lachnospirnceae and Ruminococcaceae, Brevundimonas, Ralstonia and Lactobacillus (Chen et al. 2021; Chouaia et al. 2019; Dijokaite et al. 2021; Jiang et al. 2021c; Li et al. 2022; Liang et al. 2020; Muhammad et al. 2017; Osawa et al. 2006; Paulson et al. 2014). In our previous studies, GI colonization was shown to improve the leaf quality significantly, as evident by a marked increase in the nutrient content and a decrease in the secondary metabolites content. Therefore, the improvement in leaf quality post GI colonization is responsible for lowering the need for a lot of gut probiotics in gypsy moth larvae to assist nutrition metabolism and the detoxification process.
To further explore the functional diversity of gut microbiota in gypsy moth larvae between AMF-treated and untreated groups, PICRUSt2 function prediction and significance analysis was performed. We observed that glycan biosynthesis and metabolism and digestible system pathways in the GM group were down-regulated, indicating the inhibition of digestion, absorption, and energy metabolism in gut microbiota. In addition, the pathways related to cardiovascular diseases, neurodevelopmental diseases, and cancer were also up-regulated in the GM group, which suggests that the P. alba × P. berolinensis seedlings with GM colonization provide a better environment for the reproduction of pathogenic bacteria in the larval gut. However, the energy metabolism and detoxification pathways in the GI group were significantly up-regulated, including the metabolism of other amino acids, metabolism of terpenoids and polyketides, metabolism of cofactors and vitamins, and energy metabolism. It can be deduced that the ability of larvae to utilize nutrients and adapt to host plants increased in the GI treatment group. In addition, immune-related pathways such as immune system and enzyme families were significantly up-regulated in the GI group, which might be the reason behind the decrease of pathogenic bacteria in the GI group. Altogether, the decrease in gut probiotics and microbial function disorder is one of the main reasons for the growth retardation of gypsy moth larvae in the GM group, while the improved gut environment and the enhanced metabolic functions of gut flora are responsible for the increased larval growth in GI group.
Metabolites, as important markers associated with various physiological and biochemical activities, are closely related to the growth and development of insects. The metabolic levels of gypsy moth larvae in the treated and untreated groups were analyzed by untargeted metabonomics. Our results revealed that 25 DAMs were identified in both GM and GI groups. In the comparative analysis of GM and CK groups, it was observed that several DAMs that are critical for growth, reproduction, and detoxification metabolism were significantly reduced in the GM group, including pantothenic acid, uridine, 5, 6-dihydroxyindole-2-carboxylic acid, isoleucine, penicillamine and hydroxykynurenine (Abolaii et al. 2020; Barek et al. 2018; Li et al. 1999; Li et al. 2020; Ren et al. 2021; Zhang et al. 2019b). These results indicated that the metabolic disorder of gypsy moth larvae in GM treatment group occurred, consistent with the growth retardation of gypsy moth larvae mentioned above. In addition, Corticosterone (a metabolite), which can lead to developmental disorder and a repressed immune system (Cabor et al. 2019), was increased significantly in the fat body of GM larvae, highlighting the possible reason underlying decreased adaptability of larvae to the leaves of P. alba × P. berolinensis colonized with GM. However, GI colonization significantly enhanced the metabolism in gypsy moth larvae. Some DAMs related to growth and development (e.g. (-)-Riboflavin, 3, 4-Dihydroxy-L-phenylalanine, phenylalanine, arginine, and L-tryptophan) showed a marked increase in the GI group, while other DAMs with toxic effects decreased significantly, such as Dodecanoic acid and Pyridine (Du et al. 2021; Ninomiya et al. 2008; Reis et al. 2020; Vatanparast et al. 2020). Among these, amino acids were the majorly up-regulated metabolites in the GI group. Previous studies have demonstrated that amino acids are positively correlated with the digestive level or growth of insects (Pan et al. 2014). Therefore, the increase of amino acid content in the GI treatment group may account for the improvement of larval growth. Functional analysis of DAMs showed that the metabolites affected by GM were mainly involved in the energy metabolism and detoxification pathways, such as citrate cycle (TCA cycle), pyruvate metabolism, and glycerophospholipid metabolism. Combined with the analysis of DAMs, it was found that the energy metabolism and detoxification ability disorder was another reason for the growth inhibition of gypsy moth larvae in the GM treatment group. DAMs increased by GI treatment were mainly involved in the amino acid synthesis and metabolism (e.g., Phenylalanine metabolism, Arginine biosynthesis), and exogenous toxin metabolism (e.g., Nicotinate and nicotinamide metabolism, and Pantothenate and CoA biosynthesis), elucidating that GI colonization further increased the ability of gypsy moth larvae to digest food and adapt to the chemical defense of plants.