Using the Culex pipiens sperm proteome to identify elements essential for mosquito reproduction

PLOS ONE RESEARCH ARTICLE Using the Culex pipiens sperm proteome to identify elements essential for mosquito reproduction Catherine D. Thaler1, Kaira Carstens2, Gabrielle Martinez3, Kimberly Stephens3, Richard A. Cardullo1,2,3* 1 Department of Evolution, Ecology and Organismal Biology, University of California, Riverside, Riverside, CA, United States of America, 2 Department of Biochemistry, University of California, Riverside, Riverside, CA, United States of America, 3 Department of Entomology, University of California, Riverside, Riverside, CA, United States of America * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Thaler CD, Carstens K, Martinez G, Stephens K, Cardullo RA (2023) Using the Culex pipiens sperm proteome to identify elements essential for mosquito reproduction. PLoS ONE 18(2): e0280013. https://doi.org/10.1371/journal. pone.0280013 Abstract Mature sperm from Culex pipiens were isolated and analyzed by mass spectrometry to generate a mature sperm proteome dataset. In this study, we highlight subsets of proteins related to flagellar structure and sperm motility and compare the identified protein components to previous studies examining essential functions of sperm. The proteome includes 1700 unique protein IDs, including a number of uncharacterized proteins. Here we discuss those proteins that may contribute to the unusual structure of the Culex sperm flagellum, as well as potential regulators of calcium mobilization and phosphorylation pathways that regulate motility. This database will prove useful for understanding the mechanisms that activate and maintain sperm motility as well as identify potential molecular targets for mosquito population control. Editor: Alexander J. Travis, Cornell University College of Veterinary Medicine, UNITED STATES Received: August 11, 2022 Accepted: December 19, 2022 Published: February 16, 2023 Copyright: © 2023 Thaler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Funding for this project was provided by the University of California, Riverside to RAC. Competing interests: The authors have declared that no competing interests exist. Introduction Sperm are terminally differentiated cells that lack protein synthetic machinery and are thereby constrained to function with a defined, and limited, array of proteins. This imposes specific constraints upon these cells. In particular, with limited energy stores, sperm are usually quiescent until signals to activate motility are received. Thus, while many sperm proteins are components of the eukaryotic 9+2 axoneme that powers the sperm flagellum and constitute a known set of proteins, the proteins that regulate flagellar motility in this non-regenerative system may differ from other cells that use cilia or flagella for motility. Moreover, motility regulators may vary across species depending upon the particular motility behaviors exhibited by the sperm. Some insect sperm, reportedly those with accessory microtubules surrounding the axoneme (the 9+9+2 axoneme), exhibit unusual waveforms when motile [1]. Sperm from species of Culicidae that have been examined to date, including Culex pipiens and Culex quinquefasciatus, possess a 9+9+1 axoneme in which the central pair of microtubules is replaced by a single central “rod” [2–4]. Culex spp. sperm exhibit the unusual waveforms described in other insect sperm: a double waveform flagellar beating pattern (a low PLOS ONE | https://doi.org/10.1371/journal.pone.0280013 February 16, 2023 1 / 24 PLOS ONE Proteomic analysis of mosquito sperm amplitude, short wavelength wave superimposed upon a high amplitude, long wavelength), a single helical waveform with forward progressive motility, and, interestingly, the ability to swim backwards (progressive motility with head trailing) [5]. Given that the major structural features of the axoneme are highly conserved across eukaryotes, it is likely that particular regulatory components govern the ability of these insect sperm to produce these unusual beating patterns, possibly, in part, by interactions with the accessory microtubules and associated proteins surrounding the axoneme. We conducted a proteomic analysis of mature Culex pipiens sperm as a first step in identifying and characterizing the major regulatory components of the flagellum and for comparison to proteomes of sperm from other species. This analysis identified a number of kinases, phosphatases, and Ca2+ regulators that may be important in controlling flagellar waveform, including an Erk1/2 MAPK, which controls switching between flagellar waveforms, as shown by pharmacological treatments in our earlier studies [5]. In addition, we detected a protein, previously annotated only as a conserved hypothetical protein, that bears homology to an axonemal dynein heavy chain (DHC10) and we hypothesize that the protein may reside on the accessory microtubules of the axoneme and contribute to waveform patterns. Furthermore, the Culex sperm proteome contains an unusual β-tubulin isoform with a predicted mass of 70 kDa due to a C-terminal extension of nearly 200 amino acids. No other tubulins in the non-redundant protein databases with such a feature have been found, and the localization of this 70 kDa tubulin will be important for ultimately determining its function. Here we summarize these and other important features of the Culex sperm proteome. Materials and methods Animals Mosquito colony. Culex pipiens males were obtained 5–7 days post eclosion from a colony maintained by our colleague Dr. Edward Platzer (Departments of Nematology and Evolution, Ecology and Organismal Biology, University of California, Riverside). The colony is an autogenous, stenogamous population isolated in Dixon, CA and maintained in the lab since 1973 [6]. Animals were anaesthetized using CO2 and 3–5 males placed in small Solo1 cups. Dilute sugar water was added to each cup to humidify the chamber and provide nutrients until animals were dissected to collect tissues. Dissections. Animals were euthanized by placing a small piece of chloroform soaked cotton in the Solo1 cup. The male reproductive tracts were dissected from the animals. For mass spectroscopy, the testes and external genitalia were removed, leaving the paired seminal vesicles and accessory glands. The accessory glands were dissected away from the seminal vesicles. The seminal vesicles were transferred to a droplet of PBS and forceps were used to squeeze sperm out of each seminal vesicle into the buffer. The seminal vesicle tissue was then removed. After isolating sperm from several animals (~30), the sample was transferred to a 0.65 mL microfuge tube and sto (...truncated)