Transcriptional profiling and physiological roles of Aedes aegypti spermathecal-related genes

Abstract

Background: Successful mating of female mosquitoes typically occurs once, with the male sperm being stored in the female spermatheca for every subsequent oviposition event. The female spermatheca is responsible for the maintenance, nourishment, and protection of the male sperm against damage during storage. Aedes aegypti is a major vector of arboviruses, including Yellow Fever, Dengue, Chikungunya, and Zika. Vector control is difficult due to this mosquito high reproductive capacity.

Results: Following comparative RNA-seq analyses of spermathecae obtained from virgin and inseminated females, eight transcripts were selected based on their putative roles in sperm maintenance and survival, including energy metabolism, chitin components, transcriptional regulation, hormonal signaling, enzymatic activity, antimicrobial activity, and ionic homeostasis. In situ RNA hybridization confirmed tissue-specific expression of the eight transcripts. Following RNA interference (RNAi), observed outcomes varied between targeted transcripts, affecting mosquito survival, egg morphology, fecundity, and sperm motility within the spermathecae.

Conclusions: This study identified spermatheca-specific transcripts associated with sperm storage in Ae. aegypti. Using RNAi we characterized the role of eight spermathecal transcripts on various aspects of female fecundity and offspring survival. RNAi-induced knockdown of transcript AeSigP-66,427, coding for a Na⁺/Ca²⁺ protein exchanger, specifically interfered with egg production and reduced sperm motility. Our results bring new insights into the molecular basis of sperm storage and identify potential targets for Ae. aegypti control.

Keywords: Ae. Aegypti; Insect reproduction; Sperm; Spermatheca; Transcriptome.

Fig. 1

Schematic representation of a section of the Ae. aegypti spermatheca. (c) reservoir cuticle, (D) spermathecal duct, (dc) duct cuticle, (dep) duct epithelium with columnar cells, (dG) individual duct gland cell, (DL) duct lumen, (ep) spermathecal reservoir epithelium with flattened cells, (G) spermathecal gland with prominent cells, (L) reservoir lumen, (m) muscle, (n) nuclei, (spz) spermatozoa in circles, (*) opening of a glandular cell ductule through the cuticle of reservoir. Not to scale

Fig. 2

Upregulation of spermathecal genes in Ae. aegypti. The pattern of differentially expressed genes in female spermathecae from both virgin (Vir) and inseminated (Ins) females, and from male and female whole bodies. Z-score indicates transformed data from transcripts per million for each library. The lateral clusters represent the differentially expressed transcript groups, as shown in Additional file 1: Tables S1, S2, and S3

Fig. 3

RT-PCR of genes expressed in Ae. aegypti spermathecae. Relative expression was determined in the spermathecae from virgin (Vir) or inseminated (Ins) females, and from the material collected in the spermathecal reservoir lumen (Cont) of inseminated females. Bar graphs show the fold-change of each sample normalized to S7 ribosomal gene. Reactions were done in triplicate using two biological replicates. Statistical analyses were performed using one-way ANOVA and Tukey’s multiple comparison test (α = 0.05). a: S7 (F = 1; R²: 0.25; P = 0.4219), b: Gld (F = 477.2; R²: 0.9907; P < 0.001; *P < 0.001; **P < 0.01), c: ChtB4 (F = 54.4; R²: 0.9236; P < 0.001; *P < 0.001; **P < 0.01), d: Atro-1 (F = 17.24; R²: 0.793; P = 0.0008; *P = 0.0031; **P = 0.0011), e: DHR4 (F = 29.27; R²: 0.8667; P = 0.0001. *P = 0.0003; **P = 0.0003), f: GALNT6 (F = 21.91; R²: 0.8296; P = 0.0003. *P = 0.0021; **P = 0.0004), g: ChtBD2 (F = 5.724; R²: 0.5599; P = 0.0249; *P = 0.0303), h: KSPI (F = 75.8; R²: 0.944; P < 0.0001. *P < 0.0001; **P < 0.0001), i: Na⁺/Ca²⁺ (F = 74.28; R²: 0.9429; P < 0.0001. *P < 0.0001; **P = 0.0009; ***P = 0.0003)

Fig. 4

Detection of gene transcripts Gld, ChtB4, Atro-1, DHR4, and GALNT6 in whole mounts of spermathecae of Ae. aegypti (virgin females) by in situ hybridization with red RNA probes and DAPI (blue). EGFP probe was used as control. (D) spermathecal duct, (G) spermathecal gland, (dc) spermathecal duct cells, (ep): epithelial cells, dotted line: spermathecal reservoir. Bar: 50 μm

Fig. 5

Detection of gene transcripts ChtBD2, KSPI, and Na⁺/Ca²⁺ in whole mounts of spermathecae of Ae. aegypti (inseminated females) by in situ hybridization with red RNA probes and DAPI (blue). EGFP probe was used as control. (D): spermathecal duct, (G): spermathecal gland, (dc): spermathecal duct cells, (ep): epithelial cells, dotted area: spermathecal reservoir. The spermathecal cuticle did not allowed the visualization of stained sperm. Bar: 50 μm