Abstract
Bumblebee queens have remarkable spermathecae that store sperm for year-round reproduction. The spermathecal gland is regarded as a secretory organ that could benefit sperm storage. Queen mating provokes substantial physiological, behavioral, and gene expression changes. Here, the transcriptomes of spermathecae were compared between virgins and mated queens of the bumblebee, Bombus terrestris L., at 24 h post mating. Differentially expressed genes were further validated by real time quantitative PCR and immunofluorescence assay. In total, the expression of 11, 069 and 10, 862 genes were identified in virgins and mated queens, respectively. We identified that 176 differentially expressed genes between virgin and mated queen spermathecae: 110 (62.5%) genes were upregulated, and 66 (37.5%) genes were downregulated in mated queens. Most of the differentially expressed genes validated by RT-qPCR were concentrated on immune response [i.e., leucine-rich repeat-containing protein 70 (35.8-fold), phenoloxidase 2 (41.9-fold), and defensin (4.9-fold)] and sperm storage [i.e., chymotrypsin inhibitor (6.2-fold), trehalose transporter Tret1 (1.7-, 1.9-, 2.4-, and 2.4-fold), and heterogeneous nuclear ribonucleoprotein A3 (1.2-, and 2.6-fold)] functions in the spermathecae of mated queens. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) was hypothesized to promote the mating behavior according to RT-qPCR and immunofluorescence assay. The expression levels of most upregulated immune genes were decreased significantly at 3 days post mating. In conclusion, the external sperm transfer into spermathecae led to the significantly upregulated immune response genes in bumblebees. These gene expression differences in queen spermathecae contribute to understanding the bumblebee post mating regulatory network.
Highlights
Long-term sperm storage by reproductive females is common in eusocial insects, such as bumblebees, honeybees, and ants, whereby queens typically mate early in life, store sperm in their spermatheca, and subsequently use the stored sperm throughout their lifetimes (Schoeters and Billen 2000; Chérasse and Aron 2018; Rangel et al, 2021)
The purpose was to start to address the molecular mechanism whereby bumblebee queens store viable sperm for several years after mating. This is the first report of a complete transcriptome of the spermathecae of mated and virgin bumblebee queens, in which over 10,000 genes were identified
The real time quantitative PCR (RT-qPCR) data confirmed some of the genes expression differences at the mRNA level that were first identified in the RNAsequencing data
Summary
Long-term sperm storage by reproductive females is common in eusocial insects, such as bumblebees, honeybees, and ants, whereby queens typically mate early in life, store sperm in their spermatheca, and subsequently use the stored sperm throughout their lifetimes (Schoeters and Billen 2000; Chérasse and Aron 2018; Rangel et al, 2021). There is a more complete protein network that is, conducive to long-term sperm storage (Baer et al, 2009a; Baer et al, 2009b). Hundreds of proteins representing the main components of spermathecal fluid have been identified. They belong to a series of different functional groups, the most obvious of which are energy metabolism enzymes and antioxidant defense enzymes. Mating leads to the biochemical and physiological changes the spermathecae of insects (Baer et al, 2009a; Huo et al, 2020)
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