Functional Analysis of NPC2 in Alarm Pheromone Recognition by the Red Imported Fire Ant, Solenopsis invicta (Formicidae: Solenopsis)

Abstract

The red imported fire ant (Solenopsis invicta) is a dangerous invasive insect. These ants rely on releasing an alarm pheromone, mainly composed of 2-ethyl-3,6-dimethylptrazine (EDMP), to warn nestmates of danger and trigger group defense or escape behaviors. This study found two NPC2 proteins in the ant antennae: SinvNPC2a and SinvNPC2b. SinvNPC2a was highly expressed in the antennae; phylogenetic analysis also suggests that SinvNPC2 likely possesses conserved olfactory recognition functions. By knocking down the SinvNPC2a gene, we found that the electrophysiological response of ant antennae to EDMP became weaker. More importantly, ants lacking SinvNPC2a showed significantly reduced movement range and speed when exposed to EDMP, compared to normal ants not treated with RNAi. These ants did not spread out quickly. Furthermore, tests showed that the purified SinvNPC2a protein could directly bind to EDMP molecules. Computer modeling also showed that they fit together tightly. These findings provide direct evidence that the SinvNPC2a protein plays a key role in helping fire ants detect the EDMP alarm pheromone. It enables the ants to sense this chemical signal, allowing ant colonies to respond quickly. Understanding this mechanism improves our knowledge of how insects smell things. It also suggests a potential molecular target for developing new methods to control fire ants, such as using RNAi to block its function.

Keywords: NPC2; RNA interference; alarm pheromone; electroantennography; fluorescence competitive binding; red imported fire ant.

Figure 1

Sequence structure and sequence comparison analysis of SinvNPC2. (A) A schematic comparison of the domains of SinvNPC2a and SinvNPC2b. SP represents the signal peptide transported by the Sec transposon. (B) Sequence alignment analysis of SinvNPC2a, SinvNPC2b, and the NPC2s from Camponotus japonicus, Polistes dominula, Megachile rotundata, Bombus impatiens, Apis mellifera, Microplitis mediator, Helicoverpa armigera, and Phymastichus coffea. The red regions indicate conserved areas and the protein structure database analysis shows that SinvNPC2a has a β-fold structure compared to other species.

Figure 2

Phylogenetic relationship between SinvNPC and other species NPC2s. The phylogenetic analysis of SinvNPC2a, SinvNPC2b, and other insect NPC2s (red pentagrams represent target genes, blue triangles indicate NPC2s previously shown to bind chemicals in previous studies, green squares indicate NPC2s with Blastp homologous sequences). The phylogenetic tree was constructed using the maximum likelihood method, with values representing percentages based on 1000 repetitions. The NPC2s used for phylogenetic analysis are listed in Table S6.

Figure 3

The tissue-specific expression profile of SinvNPC2a. The mRNA levels of SinvNPC2a in different tissues were analyzed using qPCR. In the qPCR analysis, mRNA levels were normalized to ef1-β levels, with data from three biological replicates, each including four technical replicates. The bars and error bars represent the mean ± standard deviation (n = 4). Different letters on the bars indicate significant differences between tissues, as determined by Tukey’s HSD test (one-way ANOVA, p < 0.05).

Figure 4

The interference efficiency of SinvNPC2a after feeding with gene-specific dsRNA. Expression of SinvNPC2a after feeding with dsSinvNPC2a. The expression levels of SinvNPC2s transcripts were analyzed using qPCR. The data shown are the mean ± SD from three biological replicates, with asterisks indicating significant differences detected by two-tailed t-tests (** p < 0.01, **** p < 0.001).

Figure 5

Worker ants’ response to different concentrations of EDMP (0.01, 0.1, 1.0, 10.0, and 100.0 µg/µL) after 36 h of feeding with dsSinvNPC2a. The bars and error bars represent the mean ± standard deviation (n = 3), and Tukey’s HSD test was used for one-way ANOVA, and different letters indicated that the difference between different treatments was significant, with p < 0.05.

Figure 6

The behavioral responses of worker ants to different concentrations of EDMP after 36 h of feeding with dsEGFP and dsSinvNPC2a. (A) Thermal images showing the trajectories of worker ants responding to 0.1, 1.0, and 10.0 µg/µL EDMP. (B) The movement speed responses of worker ants to 0.1, 1.0, and 10.0 µg/µL EDMP. (C) The number of worker ants that spread to non-region one under 0.1, 1.0, and 10.0 µg/µL EDMP treatments (recorded every 20 s for five minutes). (D) The percentage of ants spreading to each region under 0.1, 1.0, and 10.0 µg/µL EDMP treatments. The bars and error bars represent the mean ± standard deviation (n = 3), with stars (*), indicating significant differences (ns, p > 0.05, * p < 0.05, ** p < 0.01, Student’s t-test).

Figure 7

SinvNPC2a protein docking with EDMP.

Figure 8

Ligand binding assays of SinvNPC2a with n-phenyl-1-naphthylamine (1-NPN) and EDMP at pH 7.4. Bmax represents the maximum fluorescence value. (A) Fluorescence competitive binding curve of SinvNPC2a with 1-NPN. (B) Binding curve of SinvNPC2a with EDMP.