Comparative Transcriptomics Analysis Reveals Rusty Grain Beetle’s Aggregation Pheromone Biosynthesis Mechanism in Response to Starvation

Insect pheromones, including moth sex pheromones, beetle aggregation pheromones, ant tracking pheromones, and aphid alarm pheromones, play important roles in insect courtship, foraging, egg laying, alarm, and defense processes [1]. Sex pheromones and aggregation pheromones, produced by female and male insects, respectively, have been widely used as pest control strategies, including monitoring, mass trapping, and disrupting the mating process of pests. This is owing to their species specificity, activity at very low concentrations, and environmental safety. Most previous studies on insect pheromones have focused on identifying pheromone components and understanding the factors affecting pheromone synthesis [2,3,4]. Aggregation pheromones have been identified in more than 300 non-social arthropods [5]. Although the compositions of these pheromones vary widely between families, they are structurally conserved within the same family. Sex pheromones can be classified into two structurally different classes: type I pheromones, which are long, straight-chain aliphatic alcohols, aldehydes, and esters, and type II pheromones, which are epoxides without terminal functional groups. Comparatively, the structures of aggregation pheromones are more complex and diverse than those of sex pheromones, for example, the bis-epoxidized heterocyclic structure of the aggregation pheromones in the family Coryphaenidae or the macrolide structure featured in the family Platypodidae [6,7]. Several structurally similar macrolide pheromones have been identified in flat grain beetles. The synthesis and release of these pheromones are influenced by exogenous factors, such as insect maturity, the presence or absence of heterosexuality and food, population density, rhythm, light, temperature, humidity, and gut microbes. Additionally, endogenous substances, such as hormones, the activating neuropeptide PBAN, and androgen appendages play a role [4,8,9,10]. Food intake is a crucial aspect of insect reproduction, requiring coordination between finding food and a mate. Furthermore, insects undergo behavioral and physiological changes as an effective survival strategy during starvation. Behaviors, such as feeding and egg laying, are adjusted based on an insect’s nutritional status [11,12,13,14]. Additionally, sensory responses to sexual and food signals are consistently modulated by food [15]. For instance, compared with satiated Drosophila melanogaster fasted D. melanogaster exhibited a higher attraction to food odor sources to promote food seeking [16]. They were also observed to change their food preferences in response to starvation [15]. Moreover, in D. melanogaster females, insulin signaling partially controls pheromone perception in the antennal lobe (AL) and modulates cis-vaccenyl acetate (cVA) attractiveness according to the nutritional status. After 72 h without food, D. melanogaster males showed a significant decrease in the number, duration, and occurrence of mating attempts [17]. Male-produced aggregation pheromones attract and group males and females of the same species according to the food source for feeding and mating [18]. However, Rhodnius prolixus in a starved state shows repulsion to aggregated pheromones [19]. Similarly, the feeding status significantly affects the quantity of pheromone produced by the tobacco budworm (Heliothis virescens (Fabricius)) [20], German cockroaches (Blattella germanica (Linnaeus)) [21], and boll weevil (Anthonomus grandis grandis Boheman) [22]. In aphids, the weight and quantity of the alarm pheromone EβF are regulated by insulin receptor genes (InsR1/2) and downstream genes (PI3K and Akt), which encode kinases and key enzymes in the glycolysis (HK, A6PFK, and PK) and the isoprenoid (ACSS, HMGR, FPPS1, FPPS2, GGPPS, and DPPS) pathways, and EβF is significantly reduced by nutritional stress [23]. In addition, satiated male mountain pine beetles (Dendroctonus ponderosae), produced frontalin ((1R,5R)-1,5-dimethyl-6,8dioxabicyclo [3.2.1]octane) by feeding males via the MVA pathway, acting as an anti-aggregation pheromone signal to halt attacks and prevent the overcrowding of trees [24]. Thus, starvation is often utilized to analyze pheromone biosynthesis pathways in insects [22,24].

Pheromones can be synthesized de novo via the FAS pathway or the MVA pathway and/or by further processing their precursors in food [20]. It is generally accepted that pheromone synthesis is mainly regulated by the activated neuropeptide PBAN in Lepidoptera [8,25], by ecdysteroids in Diptera (houseflies) [7], and by juvenile hormone (JH) III in Coleoptera [26,27]. The regulation of pheromone biosynthesis by endogenous factors can be achieved by affecting the activities of the key enzymes or the expressions of genes at the transcriptional level. For instance, ecdysteroids regulate hydrocarbon properties by affecting the activity of one or more fatty acyl-CoA elongases [7]. On the other hand, JH III juvenile hormone modulates the expression levels of 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGS) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) or the enzymatic activity of geranylgeranyl diphosphate synthase (GPPS), thereby controlling the synthesis of pheromones in Ips paraconfusus [28]. Consequently, the identification and analysis of genes in the pheromone biosynthesis pathway are a crucial prerequisite for unraveling the molecular mechanisms of pheromone synthesis and regulation.

The rusty grain beetle, Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae), is among the most prevalent grain pests, infesting stored and marketed cereal grains. C. ferrugineus has been reported in more than 110 countries [29]. Previous studies have documented the two main aggregation pheromone components of C. ferrugineus: cucujoid I (4,8-dimethyl-(E,E)-4,8-decadienolide) and cucujoid II ((S)-(Z)-3-dodecen-11-olide), which have been isolated from frass [30]. Isotope-labeling experiments have demonstrated that these two compounds are derived from the de novo synthesis of fatty acids and terpenoids, respectively, and are regulated by the feeding state [31]. However, the molecular biosynthesis and regulation mechanisms of cucujoid I and cucujoid II have not been determined yet. Therefore, in this study, we conducted a comparative transcriptome analysis on rusty grain beetles under normal conditions and during starvation stress, identifying significantly differentially expressed pathways and potential key genes involved in the biosynthesis and regulation of pheromones.