bioRxiv preprint doi: https://doi.org/10.1101/802397; this version posted October 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Larviposition site selection mediated by volatile semiochemicals of larval origin in Glossina 2 palpalis gambiensis 3 4 Gimonneau Geoffrey1,2,3, Romaric Ouedraogo1, Salou Ernest1,4, Rayaisse Jean-Baptiste1, Bruno 5 Buatois5, Philippe Solano2,3, Laurent Dormont5, Olivier Roux6,7 and Jérémy Bouyer2,3,8,9 6 7 1. Centre International de Recherche – Développement sur l’Elevage en zone subhumide, BP 454, Bobo- 8 Dioulasso 01, Burkina Faso. 9 2. CIRAD, UMR INTERTRYP, F-34398, Montpellier, France. 10 3. INTERTRYP, Univ Montpellier, CIRAD, IRD, Montpellier, France 11 4. Département de Sciences biologiques/UFR-ST, Université Polytechnique de Bobo - Dioulasso (UPB), 12 Bobo-Dioulasso, Burkina Faso 13 5. CEFE, Université Paul Valéry Montpellier 3, CNRS, Université de Montpellier, EPHE, IRD, Montpellier, 14 France 15 6. MIVEGEC Unit, IRD-CNRS-Université de Montpellier, Montpellier, France. 16 7. Institut de Recherche des Sciences de la Santé (IRSS), Bobo-Dioulasso, Burkina Faso. 17 8. CIRAD, UMR ASTRE CIRAD-INRA « AnimalS, health, Territories, Risks and Ecosystems », Campus 18 international de Baillarguet, 34398 Montpellier cedex 05, France. 19 9. Insect Pest Control Laboratory, Joint FAO/IAEA Programme of Nuclear Techniques in Food and 20 Agriculture, A-1400, Vienna, Austria 21 22 Short title: Larval aggregation in tsetse flies 23 Corresponding author: Geoffrey Gimonneau, Centre International de Recherche – 24 Développement sur l’Elevage en zone subhumide, BP 454, Bobo-Dioulasso 01, Burkina Faso. 25 Phone: 00226 64 14 75 00; Email address: [email protected] 26 1 bioRxiv preprint doi: https://doi.org/10.1101/802397; this version posted October 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 27 Abstract 28 Tsetse flies (Diptera: Glossinidae) are the cyclical vectors of trypanosomes, the causative 29 agents of African animal trypanosomosis or nagana in animals and human African 30 trypanosomosis or sleeping sickness in humans. Tsetse flies are K-strategist species with the 31 deposition of a single larva at 10 days intervals in specific sites. As larviposition site selection 32 will strongly impact reproductive success, it is obvious that the selection of larviposition sites 33 is not random and is under strong selective processes, probably mediated by specific cues as 34 suggested by the existence of an aggregation factor in the Morsitans and Fusca groups. This 35 study aimed to highlight the existence of an aggregation effect in the Palpalis group and to 36 test for its chemical nature. We studied the larviposition site selection of Glossina palpalis 37 gambiensis according to the presence of conspecific and heterospecific larvae buried in 38 substrates in different settings. Three sets of experiments were performed with either 39 individual or grouped (n = 50) gravid females, and with physical access to substrate or not. 40 In both individual and grouped larviposition experiments, females selected significantly more 41 often trays conditioned by larvae (P<0.005), either conspecific or heterospecific even in the 42 absence of physical contact with the substrate. These results highlight the first evidence for 43 larviposition site selection mediated by volatile semiochemicals of larval origin in Glossina 44 palpalis gambiensis. However, these compounds seem not to be species-specific and 45 therefore offer new avenues for the behavioural manipulation of these vectors and for the 46 development of new vector control tools targeting gravid females. 47 48 Keywords: tsetse, aggregation, Human and African animal trypanosomosis, reproduction, 49 behaviour 50 2 bioRxiv preprint doi: https://doi.org/10.1101/802397; this version posted October 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 51 Author summary 52 Larviposition site selection in tsetse flies is govern by several biotic and abiotic factors that 53 lead to an aggregation effect of larvae. Among those, larvae are suspected to produce 54 chemicals that drive females to breeding site but little information is available. This study 55 aimed to highlight the existence of an aggregation effect of larval origin in the Palpalis group 56 and to test for its chemical nature. Through behavioural larviposition choice experiments, we 57 showed that females of Glossina palpalis gambiensis deposit their larvae significantly more 58 often in trays conditioned either by conspecific or heterospecific larvae, even in the absence 59 of physical contact with the substrate. These results highlight the first evidence for 60 aggregation effect in Glossina palpalis gambiensis mediated by volatile semiochemicals of 61 larval origin. Isolation and identification of these chemicals should offer new avenues for the 62 behavioural manipulation of these vectors and for the development of new vector control 63 tools targeting gravid females. 64 65 66 67 68 69 70 71 72 73 74 3 bioRxiv preprint doi: https://doi.org/10.1101/802397; this version posted October 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 75 Introduction 76 Oviposition in insects is one of the most important aspects of their reproduction as female 77 breeding site selection can have far-reaching consequences for the life history of the offspring, 78 especially in insects with no parental care (1). To make the best choice for its progeny, females 79 use environmental information mainly driven by olfactory and visual cues. Olfactory cues 80 linked to oviposition are generally volatiles semiochemicals produced by eggs, larvae, habitats, 81 microbes and plants that provide information about the location suitability of breeding sites 82 (2). In hematophagous insects, oviposition pheromones, which mediate interactions between 83 members of the same species, are generally produced by ovipositing female or by conspecific 84 larvae (3). Such compounds are detected by several odour receptors located on the antennae 85 as well as by contact chemoreceptors on tarsi, mouthparts and antennae. Gravid females 86 therefore lay eggs or larvae in suitable breeding sites through information provided by such 87 conspecific cues. 88 Tsetse flies belong to a unique family in Diptera called Glossinidae, which is composed of a 89 single genus, Glossina. It has been divided into three “groups”, or subgenera, called the 90 Palpalis, Morsitans, and Fusca groups, based on morphological and eco-geographical 91 characteristics. All the 31 species of tsetse flies are theoretically vectors of trypanosomes but 92 only a small number (5 to 6 species) have an epidemiological significance nowadays. They 93 mainly belong to the Palpalis and Morsitans groups of the Glossina genus (4). Tsetse flies infest 94 most part of sub-Saharan Africa where they are vectors of African trypanosomoses affecting 95 both humans (HAT: Human African Trypanosomiasis) and livestock (AAT: Animal African 96 Trypanosomosis), two major scourges which impede development in Africa (5). Indeed, HAT 97 is a major neglected tropical disease (6), with more than 50 million people at risk of infection 98 (7) and AAT is considered among the greatest obstacles to the development and 4 bioRxiv preprint doi: https://doi.org/10.1101/802397; this version posted October 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 99 intensification of livestock in sub-Saharan Africa (8). There is no vaccine and these diseases 100 lead to the death of patients if left untreated. In the context of WHO's objectives to eliminate 101 HAT by 2020 (7) and to improve productivity in the African livestock sector to achieve food 102 security (9), innovative one health vector control solutions must be developed. 103 Tsetse fly control is currently achieved through methods based on insecticide-treated cattle 104 and insecticide-treated attracting devices (traps and targets) and focuses on a unique 105 behavioural target, the “host seeking behaviour” of adults only (10). Other basic life history, 106 behavioural or ecological traits remain largely unexplored despite their importance to tsetse 107 biology and parasite transmission. Although it plays a central role in tsetse population 108 dynamics, larviposition has been one of the most overlooked behaviour. 109 Compared to other Diptera, which basically lay a large number of eggs, the tsetse fly 110 reproductive rate is very low producing a single larva at once. Tsetse flies are larviparous 111 insects and their mode of reproduction is adenotrophic viviparity (11), i.e. the larva develops 112 feeding from the uterine glands of the mother. The first larva is deposited in about 20 days 113 and the subsequent larvipositions occurs at 9 to 10 days intervals thereafter (12). As a result, 114 searching for and choosing a larviposition site is a crucial task for females because the 115 consequences of that choice directly impact their reproductive success and tsetse population 116 dynamics. From an evolutionary point of view, it is obvious that the selection of larviposition 117 sites is not random and is under strong selective pressures to respond to specific cues.