Bacillus Sphaericus Exposure Reduced Vector Competence of Anopheles
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Yu et al. Parasites Vectors (2020) 13:446 https://doi.org/10.1186/s13071-020-04321-w Parasites & Vectors RESEARCH Open Access Bacillus sphaericus exposure reduced vector competence of Anopheles dirus to Plasmodium yoelii by upregulating the Imd signaling pathway Shasha Yu1, Pan Wang1, Jie Qin1, Hong Zheng2, Jing Wang1, Tingting Liu1, Xuesen Yang1 and Ying Wang1* Abstract Background: Vector control with Bacillus sphaericus (Bs) is an efective way to block the transmission of malaria. How- ever, in practical application of Bs agents, a sublethal dose efect was often caused by insufcient dosing, and it is little known whether the Bs exposure would afect the surviving mosquitoes’ vector capacity to malaria. Methods: A sublethal dose of the Bs 2362 strain was administrated to the early fourth-instar larvae of Anopheles dirus to simulate shortage use of Bs in feld circumstance. To determine vector competence, mosquitoes were dissected and the oocysts in the midguts were examined on day 9–11 post-infection with Plasmodium yoelii. Meanwhile, a SYBR quantitative PCR assay was conducted to examine the transcriptional level of the key immune molecules of mosqui- toes, and RNA interference was utilized to validate the role of key immune efector molecule TEP1. Results: The sublethal dose of Bs treatment signifcantly reduced susceptibility of An. dirus to P. yoelii, with the decrease of P. yoelii infection intensity and rate. Although there existed a melanization response of adult An. dirus fol- lowing challenge with P. yoelii, it was not involved in the decrease of vector competence as no signifcant diference of melanization rates and densities between the control and Bs groups was found. Further studies showed that Bs treatment signifcantly increased TEP1 expression in the fourth-instar larvae (L4), pupae (Pu), 48 h post-infection (hpi) and 72 hpi (P < 0.001). Further, gene-silencing of TEP1 resulted in disappearance of the Bs impact on vector compe- tence of An. dirus to P. yoelii. Moreover, the transcriptional level of PGRP-LC and Rel2 were signifcantly elevated by Bs treatment with decreased expression of the negative regulator Caspar at 48 hpi, which implied that the Imd signaling pathway was upregulated by Bs exposure. Conclusions: Bs exposure can reduce the vector competence of An. dirus to malaria parasites through upregulat- ing Imd signaling pathway and enhancing the expression of TEP1. The data could not only help us to understand the impact and mechanism of Bs exposure on Anopheles’ vector competence to malaria but also provide us with novel clues for wiping out malaria using vector control. Keywords: Bacillus sphaericus, Anopheles dirus, Plasmodium yoelii, Vector competence, Malaria, TEP1, Imd signaling pathway *Correspondence: [email protected] 1 Department of Tropical Medicine, College of Military Preventive Medicine, Army Medical University, Chongqing 400038, China Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yu et al. Parasites Vectors (2020) 13:446 Page 2 of 10 Background Plasmodium to Anopheles mosquitoes by eliciting the Malaria which is transmitted by Anopheles mosqui- immune priming remains unknown. Anopheles dirus is toes is still one of the most devastating disease world- one of the most important vectors transmitting malaria wide, causing 228 million infections and approximately in China and Southeast Asia [17, 18]. In this study, we 405,000 deaths in 2018 [1]. Vector control is recognized focused on exploring whether the sublethal dose of Bs as an efective way to control this disease [2]. Chemical (SdBs) will afect vector competence of An. dirus to insecticides have been used for several decades to control malaria, and discuss the interaction mechanism between mosquitoes. However, the extensive and intensive use of SdBs and Anopheles mosquitoes. It will be helpful to chemical insecticides has been greatly impeded due to develop new and better ways in the fght against malaria. development of physiological resistance in the vectors, environmental pollution resulting in bio-amplifcation Methods of food chain contamination and undesirable efects on Bacillus sphaericus, mice and Plasmodium parasite benefcial organisms [3, 4]. Terefore, the need for alter- Bacillus sphaericus (Bs) 2362 strain was provided by native, more efective and environment-friendly control Hubei Kangxin agricultural pharmaceutical company, agents becomes urgent. and the titer of Bs was 650 IU/mg. Kunming outbred Bacillus sphaericus (Bs) has been used as an ideal mos- Swiss Webster mice were bought from the Experimental quito biolarvicide and put into mass production because Animal Center of the Army Medical University, Chong- of its good biological efects [5]. Bs is an aerobic, rod- qing, China. Plasmodium yoelii red fuorescence protein shaped, endospore-forming, gram-positive soil bacte- transgenic BY265 strain (P. yoelii BY265 RFP) was kindly rium, which was frstly reported toxic to mosquito larvae donated by Professor Wenyue Xu from the Army Medi- by Kellen et al. in 1965 [6]. Te mosquito larvicidal activ- cal University, and maintained by passage between Kun- ity of Bs is attributed mainly to binary toxins in most of ming mice and mosquitos. highly toxic strains and Mtx toxins in low toxic strains. In target insect species, the crystal proteins can be activated Mosquito larva rearing and sublethal dose of Bs treatment by the mosquito midgut protease and become poisonous Anopheles dirus (Hainan strain) larvae were fed on a pow- polypeptides, then bind to receptors of midgut epithelial der mix of dry degreased pork liver and yeast at a ratio cells and result in epithelial cell perforations and death of of 1:4 at 28 °C with a 12 h light/dark cycle. A sublethal larvae [7, 8]. Compared with other biological insecticides, dose of the Bs 2362 strain was administrated to the early Bs has several advantages, including low environmental fourth-instar larvae for 48–72 h to simulate feld condi- toxicity due to the high specifcity of Bs toxins, high levels tions with shortage use of Bs, according to the bioassay of efcacy, and environmental persistence. Meanwhile, results of the previous research in our laboratory [19]. the use of Bs recombination agents can also extend the When the surviving larvae developed into pupae, they mosquito-killing spectrum, prolong the action time, and were collected and further emerged to adults in cages. improve the insecticidal efcacy [9–11]. Furthermore, it Te adult mosquitoes were then routinely maintained was reported that Bs successfully decreased the spread under the same environmental and rearing conditions as of mosquito-borne diseases in epidemic areas [12–14]. the group without Bs treatment. Hence, B. sphaericus is attracting more and more atten- tion, and has been considered as one of the most eco- Mosquito adult rearing and infection nomical and efcient biolarvicide. Te Bs-treated and normal control adult mosquitoes However, the fundamental research of Bs application were maintained at 28 °C, 70–80% relative humidity and is limited up to now, especially on the subsequent efect fed on 10% sugar solution with a 12 h light/dark photo- of Bs use. In practical terms, a sublethal dose efect often cycle, according to standard rearing procedures in the existed because of the insufcient dosing and led to some laboratory. For infection with P. yoelii BY265 RFP, 3- to mosquitoes’ survival. Obviously, the sublethal dose efect 5-day-old female adults were removed and kept at 24 °C exerts a selection pressure on the surviving mosquito and fed on yoelii BY265 RFP-infected Kunming mice populations and might change their physiological func- with a gametocytemia above 0.5%. On day 9–11 post- tions. It has been reported that bacterial exposure at infection, mosquitoes were dissected, and the oocysts in the larval stage would induce immune priming in adult the midguts were examined under a fuorescence micro- Aedes aegypti mosquitoes [15, 16], which would fnally scope. Te infection rate and the intensity of Plasmo- afect the vector competence of mosquito-borne dis- dium were then calculated as well as the intensity and the eases, such as dengue virus. Whether the sublethal dose rate of melanization. of Bs treatment would change the vector competence of Yu et al. Parasites Vectors (2020) 13:446 Page 3 of 10 RNA extraction and cDNA synthesis (Drummond Scientifc Co., Bromall, PA, USA). Te Total RNA was extracted from ~10 female mosquitoes mortality caused by injection was about 20% with no of each group using TRIzol (Invitrogen, Carlsbad, CA, signifcant diference among groups. USA) as per the manufacturer’s protocol, after pulver- Tree days after dsRNA injection, the gene silenc- izing the tissues in a homogeniser on ice. Total RNA ing efciency was confrmed by semi-quantitative PCR was fnally dissolved in 40 µl RNase-free water and 1 µg and western blot.