Western honey bees (Apis mellifera) are widely reared in apiaries worldwide and produce a variety of honey bee products, such as honey, royal jelly, and propolis. A. mellifera pollinate a substantial quantity of crops and wild flowers, playing a critical role in ecological balance and food security (Potts et al., 2010). Ascosphaera apis is a widespread fungal pathogen which can not only infect bee larvae to causes chalkbrood disease, but also lead to a sharp reduction in colony population and productivity (Aronstein and Murray, 2010). In the last 2 decades, researchers have conducted an array of studies on the interaction between honey bee larvae and A. apis at the morphological, ethological, or biochemical level (Garrido-Bailón et al., 2013; Chaimanee et al., 2017; Guo et al., 2019b). Our group previously systematically investigated the response of Apis mellifera ligustica larval and the infection of A. apis during chalkbrood (Chen D. et al., 2017). For example, Chen D. et al. (2017) revealed the cellular and humoral immune responses of western honey bee larvae to A. apis invasion based on deep sequencing combined with bioinformatics; Chen D. F. et al. (2017) deciphered the transcriptomic dynamics of A. apis during the infection process in A. m. ligustica larvae.

Long non-coding RNAs (lncRNAs) are a class of ncRNA with a length of more than 200 nt containing two or more exons (Zhang et al., 2019). Similar to mRNA structures, most lncRNAs are transcribed by RNA polymerase II and hence contain the 5′ cap and the 3′ polyA tail (Li J. H. et al., 2012). According to the location with respect to protein-coding genes, lncRNA can be classified into sense lncRNA, antisense lncRNA, intergenic lncRNA, intron lncRNA, and other lncRNA (Jarroux et al., 2017). LncRNA can be capable of exerting functions in versatile manners, namely, cis-acting regulation, trans-acting modulation, miRNA precursor, competing endogenous RNA (ceRNA) network, and translation into protein (Gil and Ulitsky, 2020). Evidences suggest that as pivotal regulator, lncRNA palys an important role in a large number of biological processes, including dose compensation (Carmona et al., 2018), cell cycle (Ma et al., 2021), and host-pathogen interaction (Carnero et al., 2016).

With the rapid development of high-throughput sequencing, abundant of lncRNAs have been identified in animals, plants, and microorganisms (Guo et al., 2018; Zhou R. et al., 2021; Fan et al., 2021). In insects, lncRNA-associated studies have mainly focused on model insects such as fruit flies (Drosophila) and silkworms (Bombyx mori); however, knowledge of lncRNA-associated in other insects, including honey bees, is still limited at present. Zhou H. et al. (2021) discovered that lncRNA-CR11538 in Drosophila restored Toll immunity homeostasis via interaction with the transcription factor Dif/Dorsal, further participating in host innate immunity. Previous works demonstrated that lncRNAs in honey bees were potentially involved in behavior (Feng R. R. et al., 2022), caste development (Humann et al., 2013), and host-pathogen/parasite interaction (Chen et al., 2019a). Our previous studies deciphered the responses of both A. m. ligustica and Apis cerana larvae to A. apis invasion at the mRNA and miRNA levels (Chen D. et al., 2017; Guo et al., 2019a). Accumulating evidences have demonstrated that molecules containing miRNA response elements (MREs) such as mRNA, lncRNA, circRNA, can competitively bind miRNA and further regulating the expression of neighboring genes and biological processes (Ala, 2020). However, until now, there was no reported study on the expression profile and regulatory role of lncRNAs during the A. m. ligustica larval response to A. apis invasion.

In the current work, 4-, 5-, and 6-day-old A. m. ligustica larval guts inoculated with A. apis spores and un-inoculated larval guts were subjected to strand-specific cDNA library construction and deep sequencing, followed by identification of lncRNAs and investigation of the expression profile. The potential regulatory functions of host DElncRNAs were then analyzed in combination with previously obtained small RNA-seq (sRNA-seq) data. To our knowledge, this is the first report of the lncRNA-regulated response of honey bee larvae to A. apis invasion. Our findings will shed light on the mechanism underlying the lncRNA-mediated larval response to A. apis infection and offer novel insights into the interaction between A. m. ligustica larvae and A. apis.

Previously, our group identified 6,353 lncRNAs in A. m. ligustica workers’ midguts based on RNA-seq, including 4,749 known and 1,604 novel lncRNAs (Chen et al., 2019a). Here, 3,146 known and 600 novel lncRNAs were identified (Figure 1), further enriching the reservoir of lncRNAs in A. mellifera. In addition, we found that 39 (1.2%) known lncRNAs were shared by the A. m. ligustica larval guts and workers’ midguts, indicative of their key roles in the growth and development of both larval gut and worker’s midgut. It is speculated that those specific lncRNAs exert different functions at different developmental stages of the gut tissue. In animals and plants, the lncRNA expression was suggested to be tissue- and stage-specific (Statello et al., 2021). Hence, the total number of A. m. ligustica lncRNAs should be much greater than the documented ones. It is believed that with the increasing quantity of related studies, more honey bee lncRNAs will be discovered in the future.

LncRNAs were verified to be vital regulators in responses of insects to pathogen/parasite infection (Valadkhan and Valencia-Hipólito, 2016; Meng et al., 2021). For instance, Zhang L. et al. (2020) identified 4,450 DElncRNAs, 66 DEmiRNAs, and 7,448 DEmRNAs in the B. mori BmN cells responding to the B. mori nucleopolyhedrosis virus (BmNPV) infection, and found that DElncRNAs were likely to participate in host response via ceRNA network. Jayakodi et al. (2015) identified 2,470 lincRNAs and 1,514 lincRNAs from Apis cerana and A. mellifera, respectively, in sacbrood virus (SBV) infected A. cerana, 11 lincRNAs are specifically regulated upon viral infection. In this work, 156, 98, and 361 up-regulated lncRNAs were detected in the 4-, 5-, and 6-day-old comparison groups (Figure 2A), which showed that a portion of lncRNAs were induced to activation. Comparatively, 201, 138, and 144 lncRNAs were found to down-regulate in the above-mentioned three comparison groups, indicative of the suppression of these lncRNAs by A. apis. In summary, these DElncRNAs were speculated to be engaged in the host response to A. apis infection and play certain regulatory parts. In addition, three up-regulated lncRNAs (XR_001702513.2 (log₂FC = 8.748 2, p = 0.000 2), XR_003304325.1 (log₂FC = 1.854 1, p = 0.000 4), and XR_411902.3 (log₂FC = 2.667 4, p < 0.000 1)) and two down-regulated lncRNAs (XR_001703713.2 (log₂FC = −7.965 8, p < 0.000 1) and XR_003305757.1 (log₂FC = −7.409 4, p < 0.000 1)) were shared by the aforementioned three comparison groups (Figure 2C), suggestive of their pivotal roles in host response, therefore deserving additional investigation such as RNAi-based functional dissection.

Accumulating evidences have shown that lncRNAs are capable of regulating the transcription of neighboring genes in a cis-acting manner (Gil and Ulitsky, 2020). Chen et al. (2019a) constructed DElncRNA-miRNA-mRNA networks of the A. m. ligustica response to Nosema ceranae infection and found that a portion of DElncRNAs were likely to participate in regulating host material and energy metabolism as well as cellular and humoral immunity during host responses to N. ceranae invasion. Ropri et al. (2021) reported that SE-lncRNAs (RP11-379F4.4 and RP11-465B22.8) played a potential role in the progression of ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) by regulating the expression of neighboring genes. Insect only consumes a small amount of energy to maintain basic activities without activation of the immune system; however, once the immune system is activated under pathogen invasion, the energy consumption rate will greatly increase (Kingsolver et al., 2013). Here, we observed that the largest number of neighboring genes in the AmCK3 vs. AmT3 comparison group were involved in energy metabolism-associated pathways such as oxidative phosphorylation and nitrogen metabolism, indicating the continuous proliferation of A. apis within the host larval gut and the A. apis-caused pressure at the later stage of infection, which resulted in the enhancement of host immune defense and thus the elevation of the energy metabolism rate. To cope with the infection of pathogenic microorganisms, insects have evolved an efficient innate immune system including cellular and humoral immune, and the latter is mediated by multiple signaling pathways (Valanne et al., 2011; Hillyer, 2016). In the present study, neighboring genes regulated by DElncRNAs in the 4-, 5- and 6-day-old larval guts infected by A. apis were observed to be involved in several cellular immune pathways namely cell apoptosis, lysosome, phagosome, ubiquitin-mediated proteolysis, and some humoral immune pathways such as Jak-STAT, MAPK as well as Toll and Imd signaling pathways (Figure 3). These results demonstrated that corresponding DElncRNAs were likely to control the transcription of neighboring genes and further participate in regulating host cellular and humoral immune responses to A. apis infection.

Those lncRNAs with MREs could interact with miRNAs and influence downstream gene expression via ceRNA networks (Tang et al., 2017; Wang et al., 2020). An increasing number of lncRNAs have been verified to be pivotal regulators in the occurrence and development of an array of human diseases through ceRNA mechanisms, such as cancer, Alzheimer’s disease, and cardiovascular disease (Wu et al., 2020; Ma et al., 2020). Additionally, lncRNAs were suggested to mediate insect-pathogen interactions via ceRNA regulatory networks (Chen et al., 2019a; Zhang S. et al., 2020; Mao et al., 2022). For example, Mao et al. (2022) reported that aae-lnc-0165 suppressed by Wolbachia induced the expression of REL1 gene in Aedes aegypti through the sequence-specific binding of aae-miR-980-5p, which contributed to the activation of Toll signaling pathway. Previous findings showed that lncRNA-mediated ceRNA regulatory networks were putatively engaged in midgut development and the N. ceranae response of western honey bee workers (Guo et al., 2018; Chen et al., 2019a). In this current work, 197, 95, and 356 DElncRNAs in 4-, 5-, and 6-day-old larval guts could respectively target 10, eight, and 21 DEmiRNAs and further target 147, 79, and 315 DEmRNAs, forming complex ceRNA regulatory networks (Figure 4). This indicated that these DElncRNAs may participated in the regulation larval response to A. apis invasion via ceRNA mechanism. After being ingested by bee larvae, the A. apis spores enter into the midgut and germinate at low level, the diaphragm between midgut and hindgut disappears at prepupal stage (7- and 8-day-old), and the spores rapidly germinate and at the meantime the mycelia grow in abundance in the hindgut when contacting O₂, thereafter the mycelia penetrate the gut wall and then the body wall, resulting in chalkbrood mummy (Li T. et al., 2012; Jensen et al., 2013). Intriguingly, it is observed that the number of immune-associated targets within ceRNA networks in the 6-day-old comparison group were more than other two comparison groups. This reflected that with the increased time of A. apis infection the host-pathogen interactions were at a higher level at 6-day-old (3 dpi), a timepoint adjacent to the outbreak of chalkbrood disease.

MiR-1 plays an important role in the pathogenesis of heart disease, Liu et al. (2021) indicated that inhibiting miR-1 may relieve right ventricle hypertrophy and fibrosis in model rats used in their research, which also works significantly in plants. Wang et al. (2018) discovered that Connexin43, an extract of Astragalus root, worked by targeting miR-1 to cure viral myocarditis, and overexpression of miR-1 inhibited endogenous Connexin43 expression significantly. Previous studies have found that the expression of miR-1 in A. m. ligustica workers was significantly downregulated 6 days after N. ceranae inoculation, suggesting its potential involvement in the host immune response (Huang et al., 2015). In the Asian honey bee A. ceranae, Chen et al. (2019b) found that the expression of miR-1-x in the worker’s midgut was significantly downregulated at 7 days post inoculation with N. ceranae spores. Here, 35 DElncRNAs in the 6-day-old larval gut could jointly target miR-1-z (highly homologous to ame-miR-1), which can further target 32 DEmRNAs (Figure 4C). It is speculated that these DElncRNAs potentially regulate the expression of downstream target genes by targeting miR-1-z, further modulating the larval immune response to A. apis invasion. Effective knockdown of lncRNAs in insects such as Drosophila, Helicoverpa armigera, and Plutella xylostella was achieved utilizing the RNAi method (Guan et al., 2020; Zhang L. et al., 2020). Recently, our team conducted dsRNA-based knockdown of lncRNA13164 in the A. ceranae larval guts and found that lncRNA13164 regulated the expression of three immune genes (stk, e3µl and or1) via ace-miR-4968 and further mediated the host immune response to A. apis infection (Fu et al., 2022). In the near future, we will perform a functional study on miR-1-z as well as associated DElncRNAs and explore the mechanism underlying host response mediated by the DElncRNA-miR-1-z-DEmRNA axis.

In a nutshell, 3,146 known lncRNAs and 600 novel lncRNAs were identified in the A. mellifera larval guts; additionally, A. apis infection caused overall change of expression profile of lncRNAs in host guts; DElncRNAs potentially participated in larval immune response to A. apis invasion by regulating the expression of neighboring genes or interacting with DEmiRNAs (Figure 7); corresponding DElncRNAs were potentially engaged in host immune response through ceRNA regulatory networks via absorption of miR-1-z.