Comparative Analyses of Reproductive Caste Types Reveal Vitellogenin Genes Involved in Queen Fertility in Solenopsis invicta

Abstract

The red imported fire ant (Solenopsis invicta Buren) is a social pest species with a robust reproductive ability that causes extensive damage. Identification of the genes involved in queen fertility is critical in order to better understand the reproductive biology and screening for the potential molecular targets in S. invicta. Here, we used the mRNA deep sequencing (RNA-seq) approach to identify differentially expressed genes (DEGs) in the transcriptomes of three reproductive caste types of S. invicta, including queen (QA) and winged female (FA) and male (MA) ants. The genes that were specific to and highly expressed in the queens were then screened, and the Vg2 and Vg3 genes were chosen as targets to explore their functions in oogenesis and fertility. A minimum of 6.08 giga bases (Gb) of clean reads was obtained from all samples, with a mapping rate > 89.78%. There were 7524, 7133, and 977 DEGs identified in the MA vs. QA, MA vs. FA, and FA vs. QA comparisons, respectively. qRT-PCR was used to validate 10 randomly selected DEGs, including vitellogenin 2 (Vg2) and 3 (Vg3), and their expression patterns were mostly consistent with the RNA-seq data. The S. invicta Vgs included conserved domains and motifs that are commonly found in most insect Vgs. SiVg2 and SiVg3 were highly expressed in queens and winged females and were most highly expressed in the thorax, followed by the fat body, head, and epidermis. Evaluation based on a loss-of-function-based knockdown analysis showed that the downregulation of either or both of these genes resulted in smaller ovaries, less oogenesis, and less egg production. The results of transcriptional sequencing provide a foundation for clarifying the regulators of queen fertility in S. invicta. The functions of SiVg2 and SiVg3 as regulators of oogenesis highlight their importance in queen fecundity and their potential as targets of reproductive disruption in S. invicta control.

Keywords: Solenopsis invicta; Vgs; fertility; oogenesis; transcriptome.

Figure 1

Principal component analysis and qPCR-based validation of transcriptomic data. (A) Venn diagram showing the expression of different genes in three samples. (B) Correlation analyses showing the relationship between biological replicate samples. (C) Scatter plots indicating PC1 and PC2 from the principal component analysis. (D) qPCR-based validation. QA: queens, FA: winged female ants, MA: winged male ants; the same below.

Figure 2

Analysis of differentially expressed genes (DEGs). (A) DEGs between MA and QA samples. (B) DEGs between MA and FA samples. (C) DEGs between FA and QA samples. Volcano map showing the DEGs between pairs of groups of samples (p value ≤ 0.05 and |log2 (fold change)|≥ 1).

Figure 3

Functional classification of the differentially expressed genes (DEGs) based on KEGG analysis. The top 20 pathway-based gene numbers are displayed; (A) MA vs. QA, (B) MA vs. FA, (C) FA vs. QA. (A,C) enrichment based on the upregulated genes in QA; (B) enrichment based on the upregulated genes in FA. The horizontal axis is the gene ratio; the sizes and colours of the dots represent the gene number and p value, respectively; p value ≤ 0.05.

Figure 4

Sequence structure and phylogenetic tree of insect VgRs. (A) Diagrammatic comparison of typical domains between SiVg2 and SiVg3. Cs indicates the signal peptides transported by the Sec translocon. (B) Phylogenetic tree of insect Vgs based on the neighbour-joining (NJ) method with a bootstrap value from 1000 replicates. The asterisk highlights the S. invicta Vgs. Sequences were deposited in the GenBank database, which included the Vgs of Rhyparobia maderae (RmVg, BAB19327.1), Periplaneta americana (PaVg, BAA86656.1), Bemisia tabaci (BtVg, ADU04393.1), Lethocerus deyrollei (LdVg, BAG12118.1), Riptortus clavatus (RcVg, AAB72001.1), Nilaparvata lugens (NlVg, BAF75351.1), Laodelphax striatella (LsVg, AGJ26478.1), Tribolium castaneum (TcVg, XP_971398.1), Harmonia axyridis (HaVg, ASO96848.1), Colaphellus bowringi (CbVg, AMK38869.1), Anthonomus grandis (AgVg, AAA27740.1), Rhynchophorus ferrugineus (RfVg, ALN38803.1), Vespula vulgaris (VvVg, AER70365.1), Osmia cornifrons (OcVg, AIU68826.1), Apis mellifera (AmVg, CAD56944.1), Bombus ignitus (BiVg, ACQ91623.1), Bombus hypocrita (BhVg, ACU00433.1), Anopheles culicifacies (AcVg, AEO51020.1), Anopheles gambiae (AgVg, AAF82131.1), Culex quinquefasciatus (CqVg, XP_001857970.1), Aedes aegypti (AaVg, AAA18221.1), Thitarodes pui (TpVg, AWJ95280.1), Cadra cautella (CcVg, ALN38805.1), Danaus plexippus (DpVg, OWR44310.1), Helicoverpa armigera (HaVg, AGL08685.1), Spodoptera litura (SlVg, ABU68426.1), Lymantria dispar (LdVg, AAC02818.1), Bombyx mori (BmVg, NP_001037309.1), Actias selene (AsVg, ADB94560.1), Cnaphalocrocis medinalis (CmVg, AEM75020.1), Plutella xylostella (PxVg, MN539627), and Solenopsis invicta (SiVg, NM_001304584.1, NM_001304585).

Figure 5

Developmental and tissue-specific expression profiles of SiVgs. The expression profiles of SiVg transcripts were analysed using qRT–PCR. (A,B) The expression profiles of SiVg2 and SiVg3 in the different developmental stages, respectively. (C,D) The expression profiles of SiVg2 and SiVg3 in the different tissues, respectively. The mRNA level was normalised relative to the ef1-beta level in qRT–PCR analysis. Data represent three biological replicates and each replication includes four technical replicates. The bars show the mean ± SE. Different letters indicate significant differences detected using one-way ANOVA with Tukey’s multiple range test for multiple comparisons at the p < 0.05 level.

Figure 6

SiVg expression and oogenesis after isolation of the queens. (A) The expression profiles of SiVg transcripts were analysed using qRT–PCR. The mRNA level was normalised relative to the ef1-beta level in qRT–PCR analysis. Data represent three biological replicates and each replication includes four technical replicates. The bars show the mean ± SE. Different letters indicate significant differences detected using one-way ANOVA with Tukey’s multiple range test for multiple comparisons at the p < 0.05 level. (B) Ovary development was observed using a DMi8 stereomicroscope (Leica, Wetzlar, Germany); bar = 200 µm.

Figure 7

Silencing efficiency of SiVg2 and SiVg3 after gene-specific dsRNA injection. (A) Silencing efficiency of SiVg2 after dsVg2 injection. (B) Silencing efficiency of SiVg3 after dsVg3 injection. (C) Silencing efficiency of SiVg2 after dsVg2 and dsVg3 injection. (D) Silencing efficiency of SiVg3 after dsVg2 and dsVg3 injection. The expression levels of SiVgs transcripts were analysed using qRT-PCR. The mRNA level was normalised relative to the ef1-beta level in qRT-PCR analysis. Data represent three biological replicates and each replication includes four technical replicates. The data shown are the means ± SEs. Asterisks indicate significant differences detected using a two-tailed t test; ns p > 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 8

The effects of SiVg2 and SiVg3 knockdown on ovary development and reproduction. (A) Total egg production within 72h after dsRNA injection. The data shown are the mean ± SE. Different letters indicate significant differences detected using one-way ANOVA with Tukey’s multiple range test for multiple comparisons at the p < 0.05 level. (B) Daily egg production within 72h after dsRNA injection. Asterisks indicate significant differences detected using a two-tailed t test; * p < 0.05, ** p < 0.01, and *** p < 0.001. (C) Ovary development was observed using a DMi8 stereomicroscope (Leica, Wetzlar, Germany); bar = 200 µm.