Inumaru et al. Malar J (2021) 20:136 https://doi.org/10.1186/s12936-021-03669-3 Malaria Journal RESEARCH Open Access Vector incrimination and transmission of avian malaria at an aquarium in Japan: mismatch in parasite composition between mosquitoes and penguins Mizue Inumaru1, Atsushi Yamada2, Misa Shimizu1, Ayana Ono1, Makiko Horinouchi1, Tatsuki Shimamoto1, Yoshio Tsuda3, Koichi Murata4 and Yukita Sato1* Abstract Background: Captive populations of penguins outside of their natural distributions are often maintained in outdoor facilities, such as zoos and aquariums. Consequently, such penguins in captivity are constantly exposed to mosquito vectors and risk of avian malarial infection during their active period from spring to autumn, which can be lethal to these naïve birds. Previous studies have investigated parasite prevalence in mosquitoes or penguins, but simultane- ous investigations, which would be crucial to monitor the transmission dynamics and cycle within a facility, have not been done. To identify dominant lineages and trends, multiple-year surveys are recommended. Methods: Avian malaria parasites (Plasmodium spp.) and related haemosporidia were tested in penguins and mos- quitoes at an aquarium in Japan through multiple years from 2011 to 2018. Prevalence and dynamics were confrmed, and molecular analyses targeting the protozoal cytb gene were used to reveal the transmission cycle. Blood meals of mosquitoes were also identifed using molecular methods. Results: Parasite detection in penguins tended to fuctuate within an individual. Two Plasmodium lineages were consistently detected in mosquitoes that had fed on penguins and wild birds observed around the aquarium. Plas- modium lineage CXPIP09 was detected from both mosquitoes and penguins, suggesting active transmission at this facility. However, Plasmodium cathemerium PADOM02 was only detected in mosquitoes, which may be due to host, vector or parasite-related factors, or detection methods and their limits. Additionally, Haemoproteus larae SPMAG12 was detected from penguins, suggesting active transmission via biting midges. Conclusions: The mismatch in parasite composition between penguins and mosquitoes shows that multiple aspects such as captive birds, wild birds and vector insects should be monitored in order to better understand and control avian malarial infection within ex-situ conservation facilities. Furthermore, morphological analyses would be needed to confrm competency and infection dynamics of avian malaria parasites. Keywords: Avian malaria, Blood meal, Japan, Mosquito, Penguin, Plasmodium, Transmission cycle Background *Correspondence: [email protected] Many wild populations of penguins are at risk of extinc- 1 Laboratory of Biomedical Science, Department of Veterinary Medicine, tion as a result of multiple threats, including pollution, College of Bioresource Sciences, Nihon University, 1866 Kameino, habitat loss, climate change, and infectious diseases. Fujisawa, Kanagawa 252-0880, Japan Full list of author information is available at the end of the article Many species have been designated as endangered © The Author(s) 2021. 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. Inumaru et al. Malar J (2021) 20:136 Page 2 of 12 species [1]. As part of conservation eforts, ex-situ pop- recommended to determine transmission within a study ulations have been established in zoos, aquariums and area. other breeding facilities throughout the world. Avian In this study, the prevalence and infection dynamics malaria, caused by Plasmodium parasites, is mainly of avian malaria and related haemosporidian parasites transmitted by Culex mosquitoes [2–5]. Due to their were investigated in both penguins and mosquitoes at evolutionary background in environments of little con- an aquarium in northern Japan. Parasite lineages from tact with vector species, penguins are mostly naïve to penguins and mosquitoes were compared across multi- avian malaria and many lethal cases of avian malarial ple years to determine the transmission dynamics, iden- infections have been reported in captive penguins [6, 7]. tify vector species (vector incrimination), and reveal the While indoor enclosures can substantially decrease risk role of captive penguins within the transmission cycle. of infection by lowering contact with mosquito vectors Blood meal analysis in mosquitoes was also used to com- [6], penguins at most facilities are kept in open-air enclo- pare results between penguins and mosquitoes in order sures throughout the year where mosquito vectors can to evaluate how well such indirect evaluations using mos- freely access these captive birds. Mosquitoes can there- quitoes refect the actual host parasite prevalence. fore transmit avian malaria parasites among and between captive penguins and wild birds that inhabit surrounding Methods areas [2, 8, 9]. Local mosquitoes and wild birds maintain Study site a constant transmission cycle of avian malaria, which Te study took place at Niigata City Aquarium Marine- can consequently be transmitted to captive individuals pia Nihonkai (37°55′ N 139°01′ E), located on the coast via vectors. Previous studies have indicated that Culex of the Sea of Japan. Penguins are the only avian species mosquitoes are the primary vectors transmitting Plasmo- kept at the facility and are kept in an outdoor enclosure dium to captive penguins [7–9]. However, most studies during the whole year, where free access of mosquitoes have investigated the prevalence in mosquitoes following is possible. Te enclosure is connected to a shack, where mortality in penguins, and simultaneous investigations of penguins are able to go in and out freely. Two species, mosquitoes and penguins have not been done. Te role Humboldt penguins (Spheniscus humboldti) and south- of captive penguins in avian malarial parasite transmis- ern rockhopper penguins (Eudyptes chrysocome), are sion in zoos and aquariums has been poorly investigated. kept at the aquarium. Te facility is surrounded by for- In order to better understand the infection dynamics and ested areas. Roughly 20 km southwest of the aquarium transmission within zoo and aquarium facilities, simulta- is the Sakata wetland, where avian malaria parasite DNA neous investigations of haemosporidia in both penguins was previously detected from mosquitoes [20]. A fresh- and mosquitoes would be needed. Blood-feeding pat- water biotope area was created within the facility in 2013 terns of mosquitoes are an important factor in explain- for recreational and educational purposes. ing transmission dynamics of avian malaria parasites and other vector-borne pathogens [10–13]. Blood meal anal- Penguin samples ysis can help to indirectly reveal the transmission cycle Penguins were sampled from 2012 to 2018, mainly during within the survey area. Whether blood meal analysis can summer and spring, for their routine medical check-ups. accurately estimate parasite prevalence in avian hosts has No anti-malarial medications were used during the study, not been assessed. and the penguins were in good health. All penguins were In Japan, over 3000 penguins are kept at zoos and born within Japan. In total, 104 Humboldt penguins aquariums, making one of the largest ex-situ popula- and two southern rockhopper penguins were sampled. tions in the world [14, 15]. Sporadic detections of avian Some individuals were sampled multiple times during malaria parasites have been reported within the coun- the course of the study, although sampling frequency and try [16]. However, infection status had not been moni- interval varied between individuals. Blood samples were tored over multiple years and infection dynamics are taken from the metatarsal vein and placed in microtubes unknown. Likewise, parasite prevalence in mosquitoes containing 70% ethanol. Te samples were sent to Nihon caught at zoological gardens have been investigated in University College of Bioresource Sciences Laboratory of only a single year [8, 12]. Parasite intensity in host birds Biomedical Science, Fujisawa, Japan and kept at − 20 °C is known to fuctuate in relation to circannual changes in until further processes. Blood smears were not prepared both the host and parasite physiology [2, 17–19]. A pre- in this study and microscopic observation of parasites vious study demonstrated changes in Plasmodium line- was not possible. age composition in Culex mosquitoes between years, Deoxyribonucleic acid (DNA) extraction was done by and also noted that dominant lineages were consistently standard phenol–chloroform