The Early Sex-Specific Expression of the Fruitless Gene in the Asian Tiger Mosquito Aedes albopictus (Skuse) and Its Functional Conservation in Male Courtship
Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Insect Rearing
2.2. Preparation of Double-Stranded RNA (dsRNA) with In Vitro Transcription
2.3. Embryonic Microinjections of dsRNA
2.4. RNA Extraction and cDNA Preparation
2.5. RT-PCR and RT-qPCR
2.6. Sequence Analysis
2.7. Behavioral Assays
- (a)
- 1 wild-type male × 5 wild-type females (WT);
- (b)
- 1 GFP-knockdown male × 5 wild-type females (GFP);
- (c)
- 1 fru-knockdown male × 5 wild-type females (fru).
- (1)
- Courtship and mating;
- (2)
- feeding;
- (3)
- fecundity.
- (1)
- The fraction of time spent by the male on various aspects of courtship and mating behavior was recorded through direct observation using manual scoring under controlled laboratory conditions. In each observation session, cages were monitored starting from the moment males and females were introduced, with a total observation time of 30 min per session conducted twice per day, in the early morning and late afternoon.
- (2)
- Following a three-day mating period, the cages were examined for feeding behavior. Mosquitoes had constant access to 10% (w/v) sucrose, and warm porcine blood was offered at the top of the cages for 30 min. Feeding was assessed based on visual observation of the abdomen of the male and female mosquitoes.
- (3)
- Three days after the blood meal, plastic cups containing deionized water and lined with germination paper were provided in each cage for 48 h. Eggs were counted and examined under an optical stereomicroscope.
2.8. Statistical Analysis
3. Results and Discussion
3.1. The Molecular Characterization of the Aalfru Gene
3.2. The Developmental Expression Analysis of the Aalfru Gene
3.3. The Conservation of the Aalfru Genomic Organization and Sex-Specific Splicing Regulation
3.4. The In Vivo Functional Analysis of the fru Gene in Ae. albopictus by RNAi Knockdown
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Fuxjager, M.J.; Fusani, L.; Schlinger, B.A. Physiological innovation and the evolutionary elaboration of courtship behaviour. Anim. Behav. 2022, 184, 185–195. [Google Scholar] [CrossRef]
- Salvemini, M.; Polito, C.; Saccone, G. Fruitless alternative splicing and sex behaviour in insects: An ancient and unforgettable love story? J. Genet. 2010, 89, 287–299. [Google Scholar] [CrossRef] [PubMed]
- Brockmann, H.J.; Thornhill, R.; Alcock, J. The Evolution of Insect Mating Systems. Fla Entomol. 1984, 67, 180. [Google Scholar] [CrossRef]
- Shuker, D.; Simmons, L. (Eds.) The Evolution of Insect Mating Systems; Oxford University Press: Oxford, UK, 2014. [Google Scholar]
- McKelvey, E.G.; Fabre, C.C. Recent neurogenetic findings in insect courtship behaviour. Curr. Opin. Insect Sci. 2019, 36, 103–110. [Google Scholar] [CrossRef]
- Shirangi, T.R.; Taylor, B.J.; McKeown, M. A double-switch system regulates male courtship behavior in male and female Drosophila melanogaster. Nat. Genet. 2006, 38, 1435–1439. [Google Scholar] [CrossRef] [PubMed]
- Auer, T.O.; Benton, R. Sexual circuitry in Drosophila. Curr. Opin. Neurobiol. 2016, 38, 18–26. [Google Scholar] [CrossRef]
- Saccone, G.; Salvemini, M.; Polito, L.C. The transformer gene of Ceratitis capitata: A paradigm for a conserved epigenetic master regulator of sex determination in insects. Genetica 2011, 139, 99–111. [Google Scholar] [CrossRef]
- Saccone, G. A history of the genetic and molecular identification of genes and their functions controlling insect sex determination. Insect Biochem. Mol. Biol. 2022, 151, 103873. [Google Scholar] [CrossRef]
- Dornan, A.J.; Gailey, D.A.; Goodwin, S.F. GAL4 enhancer trap targeting of the Drosophila sex determination gene fruitless. Genesis 2005, 42, 236–246. [Google Scholar] [CrossRef]
- Anand, A.; Villella, A.; Ryner, L.C.; Carlo, T.; Goodwin, S.F.; Song, H.J.; A Gailey, D.; Morales, A.; Hall, J.C.; Baker, B.S.; et al. Molecular Genetic Dissection of the Sex-Specific and Vital Functions of the Drosophila melanogaster Sex Determination Gene fruitless. Genetics 2001, 158, 1569–1595. [Google Scholar] [CrossRef]
- Song, H.J.; Billeter, J.C.; Reynaud, E.; Carlo, T.; Spana, E.P.; Perrimon, N.; Goodwin, S.F.; Baker, B.S.; Taylor, B.J. The fruitless Gene Is Required for the Proper Formation of Axonal Tracts in the Embryonic Central Nervous System of Drosophila. Genetics 2002, 162, 1703–1724. [Google Scholar] [CrossRef] [PubMed]
- Chowdhury, Z.S.; Sato, K.; Yamamoto, D. The core-promoter factor TRF2 mediates a Fruitless action to masculinize neurobehavioral traits in Drosophila. Nat. Commun. 2017, 8, 1480. [Google Scholar] [CrossRef]
- Ito, H.; Sato, K.; Koganezawa, M.; Ote, M.; Matsumoto, K.; Hama, C.; Yamamoto, D. Fruitless Recruits Two Antagonistic Chromatin Factors to Establish Single-Neuron Sexual Dimorphism. Cell 2012, 149, 1327–1338. [Google Scholar] [CrossRef] [PubMed]
- Ito, H.; Sato, K.; Kondo, S.; Ueda, R.; Yamamoto, D. Fruitless Represses robo1 Transcription to Shape Male-Specific Neural Morphology and Behavior in Drosophila. Curr. Biol. 2016, 26, 1532–1542. [Google Scholar] [CrossRef]
- Goto, J.; Mikawa, Y.; Koganezawa, M.; Ito, H.; Yamamoto, D. Sexually Dimorphic Shaping of Interneuron Dendrites Involves the Hunchback Transcription Factor. J. Neurosci. 2011, 31, 5454–5459. [Google Scholar] [CrossRef] [PubMed]
- Sato, K.; Goto, J.; Yamamoto, D. Sex Mysteries of the Fly Courtship Master Regulator Fruitless. Front. Behav. Neurosci. 2019, 13, 245. [Google Scholar] [CrossRef]
- Gailey, D.A.; Billeter, J.C.; Liu, J.H.; Bauzon, F.; Allendorfer, J.B.; Goodwin, S.F. Functional Conservation of the fruitless Male Sex-Determination Gene Across 250 Myr of Insect Evolution. Mol. Biol. Evol. 2006, 23, 633–643. [Google Scholar] [CrossRef]
- Meier, N.; Käppeli, S.C.; Hediger Niessen, M.; Billeter, J.C.; Goodwin, S.F.; Bopp, D. Genetic Control of Courtship Behavior in the Housefly: Evidence for a Conserved Bifurcation of the Sex-Determining Pathway. PLoS ONE 2013, 8, e62476. [Google Scholar] [CrossRef]
- Petrella, V.; Aceto, S.; Colonna, V.; Saccone, G.; Sanges, R.; Polanska, N.; Volf, P.; Gradoni, L.; Bongiorno, G.; Salvemini, M. Identification of sex determination genes and their evolution in Phlebotominae sand flies (Diptera, Nematocera). BMC Genom. 2019, 20, 522. [Google Scholar] [CrossRef]
- Salvemini, M.; D’Amato, R.; Petrella, V.; Aceto, S.; Nimmo, D.; Neira, M.; Alphey, L.; Polito, L.C.; Saccone, G. The Orthologue of the Fruitfly Sex Behaviour Gene Fruitless in the Mosquito Aedes aegypti: Evolution of Genomic Organisation and Alternative Splicing. PLoS ONE 2013, 8, e48554. [Google Scholar] [CrossRef]
- Bertossa, R.C.; van de Zande, L.; Beukeboom, L.W. The Fruitless Gene in Nasonia Displays Complex Sex-Specific Splicing and Contains New Zinc Finger Domains. Mol. Biol. Evol. 2009, 26, 1557–1569. [Google Scholar] [CrossRef] [PubMed]
- Ueno, M.; Nakata, M.; Kaneko, Y.; Iwami, M.; Takayanagi-Kiya, S.; Kiya, T. fruitless is sex-differentially spliced and is important for the courtship behavior and development of silkmoth Bombyx mori. Insect Biochem. Mol. Biol. 2023, 159, 103989. [Google Scholar] [CrossRef] [PubMed]
- Nguantad, S.; Chumnanpuen, P.; Thancharoen, A.; Vongsangnak, W.; Sriboonlert, A. Identification of potential candidate genes involved in the sex determination cascade in an aquatic firefly, Sclerotia aquatilis (Coleoptera, Lampyridae). Genomics 2020, 112, 2590–2602. [Google Scholar] [CrossRef]
- Boerjan, B.; Tobback, J.; Vandersmissen, H.P.; Huybrechts, R.; Schoofs, L. Fruitless RNAi knockdown in the desert locust, Schistocerca gregaria, influences male fertility. J. Insect Physiol. 2012, 58, 265–269. [Google Scholar] [CrossRef] [PubMed]
- Wang, B.; Mao, Z.; Chen, Y.; Ying, J.; Wang, H.; Sun, Z.; Li, J.; Zhang, C.; Zhuo, J. Identification and Functional Analysis of the fruitless Gene in a Hemimetabolous Insect, Nilaparvata lugens. Insects 2024, 15, 262. [Google Scholar] [CrossRef]
- Zhang, W.; Wang, J.; Liu, Q.; Gong, Z. A Review of Pathogens Transmitted by the Container-Inhabiting Mosquitoes, Aedes albopictus, A Global Public Health Threat. China CDC Wkly. 2023, 5, 984–990. [Google Scholar] [CrossRef]
- Basrur, N.S.; De Obaldia, M.E.; Morita, T.; Herre, M.; von Heynitz, R.K.; Tsitohay, Y.N.; Vosshall, L.B. Fruitless mutant male mosquitoes gain attraction to human odor. eLife 2020, 9, e63982. [Google Scholar] [CrossRef]
- Puggioli, A.; Carrieri, M.; Dindo, M.L.; Medici, A.; Lees, R.S.; Gilles, J.R.L.; Bellini, R. Development of Aedes albopictus (Diptera: Culicidae) Larvae Under Different Laboratory Conditions. J. Med. Entomol. 2017, 54, 142–149. [Google Scholar] [CrossRef]
- Bellini, R.; Medici, A.; Puggioli, A.; Balestrino, F.; Carrieri, M. Pilot field trials with Aedes albopictus irradiated sterile males in Italian urban areas Urban Areas. J. Med. Entomol. 2013, 50, 317–325. [Google Scholar] [CrossRef]
- Maïga, H.; Yamada, H.; Severin, B.-S.N.; de O. Carvalho, D.; Mamai, W.; Herrero, R.A.; Bourtzis, K.; Bouyer, J. Guidelines for Routine Colony Maintenance of Aedes Mosquito Species, 1st ed.; Insect Pest Control Section of the Joint FAO/IAEA Division IAEA, Ed.; Food and Agriculture Organization of the United Nations International Atomic Energy Agency: Vienna, Austria, 2017. [Google Scholar]
- Xi, Z.; Dean, J.L.; Khoo, C.; Dobson, S.L. Generation of a novel Wolbachia infection in Aedes albopictus (Asian tiger mosquito) via embryonic microinjection. Insect Biochem. Mol. Biol. 2005, 35, 903–910. [Google Scholar] [CrossRef]
- Calle-Tobón, A.; Holguin-Rocha, A.F.; Moore, C.; Rippee-Brooks, M.; Rozo-Lopez, P.; Harrod, J.; Fatehi, S.; Rua-Uribe, G.L.; Park, Y.; Londoño-Rentería, B. Blood Meals With Active and Heat-Inactivated Serum Modifies the Gene Expression and Microbiome of Aedes albopictus. Front. Microbiol. 2021, 12, 724345. [Google Scholar] [CrossRef]
- Livak, K.J.; Schmittgen, T.D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
- Lee, G.; Foss, M.; Goodwin, S.F.; Carlo, T.; Taylor, B.J.; Hall, J.C. Spatial, temporal, and sexually dimorphic expression patterns of the fruitless gene in the Drosophila central nervous system. J. Neurobiol. 2000, 43, 404–426. [Google Scholar] [CrossRef]
- Gomulski, L.M.; Mariconti, M.; Di Cosimo, A.; Scolari, F.; Manni, M.; Savini, G.; Malacrida, A.R.; Gasperi, G. The Nix locus on the male-specific homologue of chromosome 1 in Aedes albopictus is a strong candidate for a male-determining factor. Parasites Vectors 2018, 11, 647. [Google Scholar] [CrossRef] [PubMed]
- Moffett, S.B.; Moffett, D.F. Comparison of immunoreactivity to serotonin, FMRFamide and SCPb in the gut and visceral nervous system of larvae, pupae and adults of the yellow fever mosquito Aedes aegypti. J. Insect Sci. 2005, 5, 1–12. [Google Scholar] [CrossRef]
- Mysore, K.; Flister, S.; Müller, P.; Rodrigues, V.; Reichert, H. Brain development in the yellow fever mosquito Aedes aegypti: A comparative immunocytochemical analysis using cross-reacting antibodies from Drosophila melanogaster. Dev. Genes Evol. 2011, 221, 281–296. [Google Scholar] [CrossRef] [PubMed]
- Ziemer, T.; Wetjen, F.; Herbst, A. The Antenna Base Plays a Crucial Role in Mosquito Courtship Behavior. Front. Trop. Dis. 2022, 3, 803611. [Google Scholar] [CrossRef]
- Yeo, G.; Burge, C.B. Maximum Entropy Modeling of Short Sequence Motifs with Applications to RNA Splicing Signals. J. Comput. Biol. 2004, 11, 377–394. [Google Scholar] [CrossRef]
- Hoshijima, K.; Inoue, K.; Higuchi, I.; Sakamoto, H.; Shimura, Y. Control of doublesex Alternative Splicing by transformer and transformer-2 in Drosophila. Science 1991, 252, 833–836. [Google Scholar] [CrossRef]
- Lam, B.J.; Bakshi, A.; Ekinci, F.Y.; Webb, J.; Graveley, B.R.; Hertel, K.J. Enhancer-dependent 5′-Splice Site Control of fruitless Pre-mRNA Splicing. J. Biol. Chem. 2003, 278, 22740–22747. [Google Scholar] [CrossRef]
- Salvemini, M.; Mauro, U.; Lombardo, F.; Milano, A.; Zazzaro, V.; Arcà, B.; Polito, L.C.; Saccone, G. Genomic organization and splicing evolution of the doublesex gene, a Drosophila regulator of sexual differentiation, in the dengue and yellow fever mosquito Aedes aegypti. BMC Evol. Biol. 2011, 11, 41. [Google Scholar] [CrossRef]
- Petrella, V.; Aceto, S.; Musacchia, F.; Colonna, V.; Robinson, M.; Benes, V.; Cicotti, G.; Bongiorno, G.; Gradoni, L.; Volf, P.; et al. De novo assembly and sex-specific transcriptome profiling in the sand fly Phlebotomus perniciosus (Diptera, Phlebotominae), a major Old World vector of Leishmania infantum. BMC Genom. 2015, 16, 847. [Google Scholar] [CrossRef] [PubMed]
- Kennerdell, J.R.; Carthew, R.W. Use of dsRNA-Mediated Genetic Interference to Demonstrate that frizzled and frizzled 2 Act in the Wingless Pathway. Cell 1998, 95, 1017–1026. [Google Scholar] [CrossRef] [PubMed]
- Koo, J.; Palli, S.R. Recent advances in understanding of the mechanisms of RNA interference in insects. Insect Mol. Biol. 2024, 1–14. [Google Scholar] [CrossRef] [PubMed]
- Jin, B.; Zhao, Y.; Dong, Y.; Liu, P.; Sun, Y.; Li, X.; Zhang, X.; Chen, X.; Gu, J. Alternative splicing patterns of doublesex reveal a missing link between Nix and doublesex in the sex determination cascade of Aedes albopictus. Insect Sci. 2021, 28, 1601–1620. [Google Scholar] [CrossRef]
- Kyrou, K.; Hammond, A.M.; Galizi, R.; Kranjc, N.; Burt, A.; Beaghton, A.K.; Nolan, T.; Crisanti, A. A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes. Nat. Biotechnol. 2018, 36, 1062–1066. [Google Scholar] [CrossRef]
- Larrosa-Godall, M.; Ang, J.X.D.; Leftwich, P.T.; Gonzalez, E.; Shackleford, L.; Nevard, K.; Noad, R.; Anderson, M.A.E.; Alphey, L. Challenges in developing a split drive targeting dsx for the genetic control of the invasive malaria vector Anopheles stephensi. Parasites Vectors 2025, 18, 46. [Google Scholar] [CrossRef]
Name | Sequence (5′-3′) | Purpose |
---|---|---|
Aalrp49+ | GACGAAGAAGTTCATCCGCC | cDNA amplification |
Aalrp49− | GTTCTGCTGCGAGCGCAG | cDNA amplification |
Aalnix+ | GTTGTTCGTTACAGACTGATG | cDNA amplification |
Aalnix− | CAAAGCTAATGTAAACCATGAC | cDNA amplification |
Aalfru_M2+ | GAACGGCTACCCTCAGATGA | cDNA amplification and sequencing |
Aalfru_C3− | ACCTGCGATTCGTATCCACC | cDNA amplification and sequencing |
Aalfru_C5+ | ACATGCCTCCGCTGAACGA | cDNA amplification and sequencing |
Aalfru_ZnfA− | CCGTTGTTTGTTCCGGGC | cDNA amplification and sequencing |
Aalfru_ZnfB− | AGCACTGCTCGACTGGC | cDNA amplification and sequencing |
Aalfru_ZnfC− | CGGTAGGTGTCGCCTTGTT | cDNA amplification and sequencing |
Aalfru_real+ | CGGCAATGCCCATCTACAG | qRT-PCR |
Aalfru_real− | GGTCGACACCGTTCATCTGA | qRT-PCR |
AalRp49_real+ | AGAAGTTCCTGGTCCACAAC | qRT-PCR |
AalRp49_real− | GTTCTGCTGCGAGCGCAG | qRT-PCR |
Aalfru_MB_T7+ | taatacgactcactatagggAGTTCTCAATAGTATGTCATCG | dsRNA synthesis |
Aalfru_MB_T7− | taatacgactcactatagggGCGGTGCGATTGCAGTTTTC | dsRNA synthesis |
eGFP_T7+ | taatacgactcactatagggGGTGAACTTCAAGATCCGCC | dsRNA synthesis |
eGFP_T7− | taatacgactcactatagggGCATGGACGAGCTGTACAAG | dsRNA synthesis |
Name | Injected Embryos | Larvae | Pupae | Adults | Males | Females | Larval Survival Rate (%) |
---|---|---|---|---|---|---|---|
AalfruM dsRNA | 1375 | 22 * | 9 | 9 | 8 | 1 | 1.60 |
GFP dsRNA | 516 | 7 † | 7 | 7 | 4 | 3 | 1.36 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Varone, M.; Di Lillo, P.; Nikolouli, K.; Özel, A.E.; Lucibelli, F.; Volpe, G.; Mazzucchiello, S.M.; Carfora, A.; Aceto, S.; Saccone, G.; et al. The Early Sex-Specific Expression of the Fruitless Gene in the Asian Tiger Mosquito Aedes albopictus (Skuse) and Its Functional Conservation in Male Courtship. Insects 2025, 16, 280. https://doi.org/10.3390/insects16030280
Varone M, Di Lillo P, Nikolouli K, Özel AE, Lucibelli F, Volpe G, Mazzucchiello SM, Carfora A, Aceto S, Saccone G, et al. The Early Sex-Specific Expression of the Fruitless Gene in the Asian Tiger Mosquito Aedes albopictus (Skuse) and Its Functional Conservation in Male Courtship. Insects. 2025; 16(3):280. https://doi.org/10.3390/insects16030280
Chicago/Turabian StyleVarone, Marianna, Paola Di Lillo, Katerina Nikolouli, Ayca Eda Özel, Francesca Lucibelli, Gennaro Volpe, Sarah Maria Mazzucchiello, Angela Carfora, Serena Aceto, Giuseppe Saccone, and et al. 2025. "The Early Sex-Specific Expression of the Fruitless Gene in the Asian Tiger Mosquito Aedes albopictus (Skuse) and Its Functional Conservation in Male Courtship" Insects 16, no. 3: 280. https://doi.org/10.3390/insects16030280
APA StyleVarone, M., Di Lillo, P., Nikolouli, K., Özel, A. E., Lucibelli, F., Volpe, G., Mazzucchiello, S. M., Carfora, A., Aceto, S., Saccone, G., Bourtzis, K., & Salvemini, M. (2025). The Early Sex-Specific Expression of the Fruitless Gene in the Asian Tiger Mosquito Aedes albopictus (Skuse) and Its Functional Conservation in Male Courtship. Insects, 16(3), 280. https://doi.org/10.3390/insects16030280