Published online May 10, 2023
Journal of Ecology and Environment (2023) 47:04
Nam-Ho Roh1# , Jongsun Kim2# , Jaejin Park2 and Daesik Park2*
1Department of Biological Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea
2Division of Science Education, Kangwon National University, Chuncheon 24341, Republic of Korea
Correspondence to:Daesik Park
#These authors equally contributed to this work.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Several species of amphibians in agricultural areas are often infected with ranaviruses; however, the biological or ecological factors that cause this infection are not well understood. In this study, we investigated whether local tadpole density, Gosner developmental stage, and weather conditions affected ranavirus infection in Dryophytes japonicus tadpoles in rice paddies over three months.
Results: During the study, eight samplings were undertaken between June 6 and August 21, 2022. No die-off of tadpoles occurred, but 20 of 110 tadpoles (18.8%) were found to be infected with ranavirus. The tadpole density at the sampling site and Gosner stage of the sampled tadpoles were not related to the daily ranavirus infection rate. The mean daily highest temperature during the two weeks prior to the sampling date and the mean daily lowest and highest temperatures during the week prior to the sampling date were negatively related to the daily infection rate.
Conclusions: Our results suggest that low and extreme temperatures caused by flooding and draining of paddy fields or climate change in summer could be a significant risk factor for ranavirus infection in summer-breeding frogs in agricultural areas.
Keywords: Asia, climate change, infectious disease, iridoviridae, rice paddy
The mortality of amphibians due to ranavirus infection has been reported in North America and Europe, and recently in South America (Bartlett et al. 2021; Brunner et al. 2021; Duffus et al. 2015). In Asian countries, ranavirus infection and ranavirus-associated mortality have also increased since the first case of ranavirus infection in cultured pig frogs (
Various biological and ecological factors are involved in ranavirus infection and propagation. For example, metamorphosing amphibians, whose Gosner development stage is approximately 44–46, are often more vulnerable to ranavirus infections (Haislip et al. 2011; Kwon et al. 2017). In addition, a high individual density can increase individual contact among tadpoles or frogs, resulting in an increased ranavirus infection rate (Brunner et al. 2015; Millikin et al. 2023; Peace et al. 2019). In addition, ecological factors such as exposure to anthropogenic contaminants and extreme weather conditions could increase the possibility of ranavirus infection in amphibians (Cohen et al. 2020; Daszak et al. 2001; Davis et al. 2020), possibly by suppressing the immune system (Carey et al. 1999; Humphries et al. 2022). Recent climate change has often shifted local weather conditions, such as increasing extreme temperatures and drought periods, resulting in altered seasonal rain patterns (Altizer et al. 2013; Kamruzzaman et al. 2020; Tegegne et al. 2020). These factors may affect the ranavirus infection rate of amphibians in the field.
In our previous study, we found that ranavirus is present in 16.1%–50% of populations of
We determined the tadpole density at the sampling site by dividing the number of tadpoles caught by the number of nettings and the size of each net in square meters (Loman 1997). We conducted, on average, 6.7 nettings (1–10) for the calculation before each sampling. We individually preserved sampled tadpoles in 99% EtOH after euthanasia by submerging them for more than 15 minutes in 0.5% MS222 (Galex et al. 2020) and washing them out using paddy water. In the laboratory, we determined their Gosner developmental stage (Gosner 1960), extracted liver tissues, and preserved the tissues at −80°C.
Additionally, we analyzed the weather data for the average daily lowest, highest (Fig. 2), and mean air temperatures and average daily mean relative humidity during one, two, and four weeks prior to the sampling date. We selected these timeframes to evaluate whether weather conditions during tadpole growth influenced potential ranavirus infection and propagation. We downloaded the weather data from the Cheongju Meteorological Center (www.weather.go.kr), which is 7.6 km away from the sampling site.
We extracted genomic DNA from liver tissue with the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol, quantified it with a Qubit3 Fluorometer (Invitrogen, Waltham, MA, USA) using the Qubit 1X dsDNA HS Assay Kit (Invitrogen, Waltham, MA, USA), and then stored it at −80°C until quantitative PCR (qPCR) experiments. For qPCR amplification of the MCP gene, which is conserved among
All samples were run in triplicates, with sterile molecular-grade water as a negative control and a standard set of gBlocks to measure the viral load of each sample in viral copy number equivalents. We constructed a synthetic double-stranded DNA standard as the gBlock copy by synthesizing a 126 bp fragment of the MCP gene (gBlocks Gene Fragments; Integrated DNA TechnologiesTM, Coralville, IA, USA) and evaluated the standard curve in our system using a 10-fold dilution series from 5 × 109 down to 5 × 101 gene copies of the gBlocks. We used a set of gBlocks (5 × 106, 5 × 104, and 5 × 102 gene copies) as a standard for each sample analysis.
If there was a positive reaction in two or more of the three replicate samples, the melting temperature of the melting curve was the same as the positive control, and the cycle threshold was 35 or less, we considered that the tadpole had been infected by ranavirus. If a positive reaction was confirmed in only one well, the test was rerun and the tadpole was considered to be infected only when the above conditions were satisfied.
For analyses, we log-transformed the tadpole density and viral concentration of the infected samples, which were determined using the quantity of gBlock copies. Considering that most of the data did not pass the normality test (
The mean tadpole density at the sampling site was 219.2 ± 83.8 ind./m2 (range: 78–780 ind./m2; n = 11) (Table 1). The mean Gosner developmental stage of the collected tadpoles was 33.6 ± 1.3 (range: 24–42; n = 110). Among the 110 tadpoles examined, we found a total of 20 tadpoles (18.2% ± 3.3%; range: 0.0%–40.0%; n = 8) (Table 1), which were infected with ranavirus over seven samplings, except a sample on 7 August. No mass mortality of
Neither the daily infection rate nor the viral concentration in the infected tadpoles had a significant relationship with tadpole density at the site or their Gosner developmental stage (
In this study, we examined the effects of tadpole density, Gosner developmental stage, and weather conditions on the rate of ranavirus infection in
The effect of tadpole density on ranavirus infection in
Ranavirus infection rates of
Temperature affects the rate of ranavirus infection in
Even though 40% of
In this study, we found no die-offs in
We thank Hyerim Kwon, Min-Woo Park, and Hojun Jeong for their help during the dissection.
FV3: Frog virus 3
MCP: Major capsid protein
NHR did data curation, investigation, and writing-original draft. JK did data curation, formal analysis, and writing-review and editing. JP did data analysis, funding acquisition, and writing-review and editing. DP did conceptualization, supervision, writing-original draft, and writing-review and editing.
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2020R1A6A3A13060949).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
This study was reviewed and approved by the Institutional Animal Care and Use Committee of Kangwon National University (KW-200618-3).
The authors declare that they have no competing interests.
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