Contents lists available at ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys Arginine kinase interacts with 2MIT and is involved in Drosophila melanogaster short-term memory Andrea Bozzato a , Ottavia Romoli a,1 , Denis Polo a , Francesca Baggio a,2 , Gabriella M. Mazzotta a , Gianluca Triolo b , Michael P. Myers b , Federica Sandrelli a, ⁎ a Dipartimento di Biologia, Università di Padova, via Ugo Bassi 58/B, 35121 Padova, Italy b Protein Networks Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), AREA Science Park, Padriciano 99, 34012 Trieste, Italy ARTICLEINFO Keywords: Short-term memory Arginine kinase 2MIT Drosophila melanogaster ABSTRACT Mushroom bodies are a higher order center for sensory integration, learning and memory of the insect brain. Memoryisgenerallysubdividedintodiferentphases.Inthemodelorganism Drosophila melanogaster,mushroom bodieshavebeenshowntoplayacentralroleinbothshort-andlong-termmemory.In D. melanogaster, thegene 2mit codes a transmembrane protein carrying an extracellular Leucin-rich-repeat domain, which is highly transcribed in the mushroom and ellipsoid bodies of the adult fy brain and has a role in the early phase of memory. Utilizing coimmunoprecipitation experiments and mass spectrometry analyses, we have shown that 2MIT interacts with Arginine kinase in adult fy heads. Arginine kinase belongs to the family of Phosphagen kinases and plays a fundamental role in energy homeostasis. Using the GAL4/UAS binary system, we demon- strated that a downregulation of Arginine kinase mainly driven in the mushroom bodies afects short-term memory of Drosophila adult fies, in a courtship conditioning paradigm. As 2mit c03963 hypomorphic mutants showed comparable results when analyzed with the same assay, these data suggest that 2MIT and Arginine kinase are both involved in the same memory phenotype, likely interacting at the level of mushroom bodies. 2MIT and Arginine kinase are conserved among insects, the implications of which, along with their potential roles in other insect taxa are also discussed. 1. Introduction Adaptive behaviors have been observed in almost all living organ- isms, from unicellular species to vertebrates. Environmental stimuli activatestereotypedbehaviors,sothatevenaunicellularorganismcan show an adaptive response (Banchetti and Erra 2002). In addition, or- ganisms characterized by a structured nervous system are able to ac- quire and maintain new non-stereotyped behaviors correlated with experience (Gasque et al., 2006). These abilities rely on neuronal plasticity, which determines the capability to perceive and process in- formation about environment (learning) and to store and recover this information over time (memory) (Skoulakis and Davis, 1996; Kandel, 2001).Memoryhasbeengenerallysubdividedinshort-andlong-term phases. While short-term memory (STM) involves the modifcation of existing proteins, long-term memory (LTM) requires protein synthesis (Lynch, 2004; Blum et al., 2009; Bufngton et al., 2014). Drosophila melanogaster isconsideredavaluablemodelorganismfor learningandmemorystudies(Heisenberg,2003;Marguliesetal.,2005; Cognigni et al., 2018). Diferent types of assays have shown the fies capability to associate a conditioned stimulus (CS) with an uncondi- tioned stimulus (US). In classical olfactory conditioning assays the fy learns to associate odors (CS) with a harmful stimulus (US) and modi- fesitsbehaviorwhentheCSisgivenalone(Quinn et al., 1974; Tully and Quinn, 1985). In courtship conditioning assays male fies are in- itially subjected to a training period by courting unreceptive fertilized femalesandarethenassessedforareductionintheircourtshipactivity towardssubsequentfemales,whichcanbebothimmobilizedvirgins(in theretentiontest)and/ormobilematedfemales(intheretrainingtest) (Siegel and Hall, 1979; Joiner and Grifth, 1999; Mehren et al., 2004; Kamyshevetal.,1999).Intheseassays,theattractiveUSistypicalfor bothvirginandfertilizedfemalesandappearstobecomeanaversiveCS after being paired with the aversive US that is produced by the un- receptivefertilizedfemale(Kamyshevetal.,1999).Usingthecourtship conditioning paradigm, it has been possible to detect the diferent https://doi.org/10.1016/j.jinsphys.2020.104118 Received 15 June 2020; Received in revised form 10 September 2020; Accepted 29 September 2020 ⁎ Corresponding author. E-mail address: federica.sandrelli@unipd.it (F. Sandrelli). 1 Current address: Institut Pasteur de la Guyane, 23 Avenue Pasteur, 97306 Cayenne, French Guiana, France. 2 Current address: Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 268 CH-8057 Zurich, Switzerland. Journal of Insect Physiology 127 (2020) 104118 Available online 02 October 2020 0022-1910/ © 2020 Elsevier Ltd. All rights reserved. T