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Temporal production of coloured faeces in wild roof rats (Rattus rattus) following consumption of fluorescent non-toxic bait and a comparison with wild R. norvegicus and Mus musculus
Journal of Stored Products Research 81 (2019) 7e10
Contents lists available at ScienceDirect
Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr
Temporal production of coloured faeces in wild roof rats (Rattus rattus) following consumption of fluorescent non-toxic bait and a comparison with wild R. norvegicus and Mus musculus Marcela Frankova a, *, Barbora Kaftanova b, Radek Aulicky a, Pavel Rodl c, Daniel Frynta b, Vaclav Stejskal a a b c
Crop Research Institute, Drnovska 507, Prague 6, Czech Republic Department of Zoology, Faculty of Science, Charles University, Vinicna 7, Prague 2, Czech Republic National Institute of Public Health, Srobarova 48, Prague 10, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 June 2018 Received in revised form 6 December 2018 Accepted 17 December 2018
In the laboratory, we evaluated the efficacy of the use of fluorescent non-toxic bait for the monitoring of wild roof rats (Rattus rattus). We described the temporal dynamics of the production of fluorescent faeces after the consumption of fluorescent bait and compared it with those of R. norvegicus and Mus musculus. Roof rats produced, on average, 52 faecal pellets per 24 h, ranging from 31 to 81. The dry weight of the produced faeces per 24 h ranged from 1.1 to 3.4 g. The production peak of highly detectable fluorescent faeces was 8e18 h after consuming the bait, and the last detectable faeces were recorded 28e30 h after consuming the bait. Our data showed that roof rats produced the highest proportion of highly fluorescent faeces, indicating that best physiological potential for the use of this type of fluorescent bait exists in R. rattus since R. norvegicus and M. musculus produced 80% and 65% of the fluorescent faeces produced by R. rattus, respectively. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Rodent control Rodent pests IPM Monitoring Faeces Environmental contaminants
1. Introduction Rodents cause enormous amounts of damage to grain stores globally (Hamel, 2010; Stejskal et al. 2014, 2015). For example, roof rats were recently documented to be serious faecal contaminators of stored commodities (Stejskal and Aulicky, 2014); grain kernels were found to be of equal size to rat droppings, so their admixture with grain mass is difficult to separate. Since natural rodenticides and repellents are mostly still in the research phase (e.g., Jokic et al., 2017), synthetic anticoagulant rodenticides are currently most commonly employed for rodent control (Guidobono et al., 2010; Buckle and Eason, 2015; Frankova et al., 2017). However, the widespread and non-targeted use of anticoagulants poses a serious toxicological and environmental threat. Mitigation of these
* Corresponding author. Crop Research Institute, Drnovska 507, CZ-16106, Czech Republic. E-mail addresses: [email protected] (M. Frankova), [email protected] (B. Kaftanova), [email protected] (R. Aulicky), [email protected] (P. Rodl), frynta@ centrum.cz (D. Frynta), [email protected] (V. Stejskal). https://doi.org/10.1016/j.jspr.2018.12.002 0022-474X/© 2018 Elsevier Ltd. All rights reserved.
environmental risks requires implementation of programmes of integrated pest management (IPM) based on efficient monitoring followed by targeted control (Witmer, 2007). Currently, rodent monitoring is mainly based on trapping or observation of bait consumption and/or the distribution of faeces. Although the faeces of rodents are at least 10 larger than that of insects (e.g., Stejskal, 1997), it still may be easily overlooked by inspectors due to its dark colour (Frynta et al., 2012), especially under conditions of dimly lit storage and food facilities. Recently, a new ultraviolet-based technique that enables easy tracking of the highly visible fluorescent faeces was developed for professional urban pest control (Corrigan, 2010). It is based on the use of technology that encapsulates fluorescent particles, protecting the ultraviolet (UV) dye during its passage through the digestive tract. The prerequisite for the use of this method in practice is a thorough knowledge of the physiology of the digestion and defecation of the fluorescent bait that can address practical questions such as “How many fluorescent faeces are produced per day?” or “How quickly does the bait pass through the digestive tract to appear as fluorescent faeces in the environment?” The goal of this study, similar to our preceding studies on fluorescent rodent-monitoring baits, was to determine the answers
8
M. Frankova et al. / Journal of Stored Products Research 81 (2019) 7e10
to these questions. Our previous studies described the physiological reactions and defecation parameters in Mus musculus Linnaeus and Rattus norvegicus Berkenhout (Frynta et al., 2012; Frankova et al., 2015). Therefore, this study, as part of a multispecies project, assessed the temporal dynamics of the production of fluorescent faeces in Rattus rattus Linnaeus and compared the fluorescent faeces production among the three main rodent pests (i.e., M. musculus e Frynta et al. (2012), R. norvegicus e Frankova et al. (2015), and R. rattus e this study). 2. Material and methods 2.1. Laboratory experiment Twenty-three wild roof rats (Rattus rattus; 20 males, 3 females) were trapped in a piggery in the Czech Republic (Palecek, the Central Bohemian region). During a 6-week adaptation period, rats were subjected to the physiological (temperature and light conditions) and behavioural (experimental cages, the presence of human, no handling) acclimatization. Rats were kept separately in wire mesh cages (20 30 24 cm). Standard food (Ssniff Spe€ten GmbH, Soest, Germany) and water were provided ad zialdia libitum. After the adaptation to captivity, the experimental procedures were performed. Standard food was removed from the cage for 20 h prior to the experiment to increase food motivation in the rats. Then, 20 g of fluorescent pellets (ICB Pharma, Poland; for details see Frynta et al., 2012) was placed into each cage, and the floor under the cage was covered with freely accessible clean filter paper. The fluorescent pellets were weighed at 2-h intervals, and their consumption was monitored until the experimental animals had consumed at least 3 g, after which the fluorescent food was removed and replaced with standard food. After the beginning of the consumption of the fluorescent pellets, the paper covering the floor under the cage was replaced with a new sheet, and all faeces were collected. This procedure was performed every two hours until 40 h after the beginning of the consumption of the fluorescent pellets, at which point the experiment was terminated. The collected faeces were then observed under UV illumination (a 21 LED flashlight; ICB Pharma), counted and sorted into those that were (1) highly fluorescent (i.e., clearly detectable at the distance of approximately one metre), (2) poorly fluorescent and (3) exhibiting no sign of fluorescence; Fig. 1. Next, all faeces were dried and weighed. The sorting and weighing were performed in a blinded manner.
Fig. 1. The different types of faeces produced by roof rat (R. rattus): highly fluorescent faeces (first row), poorly fluorescent faeces (second row) and faeces exhibiting no sign of fluorescence (third row).
variable (fixed factor). The rat identity and AR-1 correlation structure were included to avoid pseudoreplication. To evaluate faeces production after the consumption of fluorescent non-toxic bait in three rodent pests, we compared the data (sums of different types of faeces per 24 h) from this experiment with the data from previously published studies on house mice (Frynta et al., 2012) and Norway rats (Frankova et al., 2015). A nonnyi parametric Kruskal-Wallis rank sum test with the Kruskal-Neme post hoc test (the PMCRM package) was used for between-species comparisons of the ratios of different types of faeces. It was employed to avoid an assumption of normal distribution for variables expressed as proportions. All statistical analyses were carried out using the R software (R Core Team, 2015), and graphs were generated in Statistica 12.0 (StatSoft Inc. 2010). 3. Results
2.2. Statistical analysis
3.1. Faeces production in R. rattus
There was a high individual variance in the acceptance of the fluorescent pellets in the laboratory experiment. The experimental animals differed in the amount of food consumed, the latency of consumption and the time interval needed to consume a required amount of food (i.e., minimally 3 g). The recording of fluorescent faeces production in every individual started from the time point when the consumption of 3 g of food had been achieved (i.e., time ¼ 0), allowing comparison among individuals and the graphic presentation of the temporal dynamics. To assess the temporal changes in the proportion of highly fluorescent faeces, we employed marginal models, as implemented in the geeglm package. The course of the experiment was arbitrarily divided into 7 sections, each lasting 6 h and consisting of three elementary time intervals (2 h). The amount of highly fluorescent faeces and the amount of faeces in the remaining categories (poorly and non-fluorescent) for each elementary 2-h time interval were the dependent variables, and the 6-h sections were the explanatory
Rats produced on average 52 faecal pellets per 24 h, ranging from 31 to 81 (SE ¼ 13.8, CI ¼ 46.3e58.2). The dry weight of the produced faeces per 24 h ranged from 1.1. to 3.4 g (mean ¼ 2.3 g, SE ¼ 0.8, CI ¼ 2.0e2.7). The experimental subjects consumed 1.1e13.2 g of the fluorescent pellets (mean ¼ 6.5 g). The first fluorescent faeces were recorded 2e4 h after the consumption of the required number of fluorescent pellets. The production peak of highly detectable fluorescent faeces was 8e18 h, and the last detectable faeces were recorded 28e30 h after the consumption of the bait in most of the experimental animals (Fig. 2). However, one individual produced fluorescent faecal pellets up to 38 h after consuming the bait. The proportion of highly fluorescent pellets to the total produced pellets per 24 h was 48%, and during the peak period, it increased to over 76%. The geeglm analysis confirmed the statistical significance of the observed temporal variation in the proportion of highly fluorescent
M. Frankova et al. / Journal of Stored Products Research 81 (2019) 7e10
Fig. 2. Temporal dynamics of produced faeces after the consumption of fluorescent non-toxic bait in roof rats (R. rattus). Data are given as the means and 95% confidence intervals.
faeces (factor section: df ¼ 6, c2 ¼ 77.4, P < 0.01). Compared to the first section (0e6 h, intercept ¼ 1.893, SE ¼ 0.406, P < 0.01), this proportion was significantly elevated in the 6e12 h (intercept ¼ 2.820, SE ¼ 0.516, P < 0.01), 12e18 h (intercept ¼ 3.097, SE ¼ 0.545, P < 0.001) and 18e24 h (intercept ¼ 1.243, SE ¼ 0.529, P ¼ 0.02) sections. 3.2. Comparison of fluorescent faeces production in three rodent pests The three studied rodent pest species produce different total amounts (H2,76 ¼ 7.35, P ¼ 0.03) and weight (H2,76 ¼ 60.67, P < 0.01; for details see Table 1) of faeces. Roof rats produce significantly fewer faecal pellets than house mice, while they produce a similar number of faecal pellets as Norway rats (Fig. 3). The Kruskal-Wallis rank sum test revealed significant differences in the proportions of highly fluorescent (H ¼ 10.77, P < 0.01) and non-fluorescent (H ¼ 16.11, P < 0.01) faeces, while the proportion of poorly fluorescent faeces did not differ among rodent pests (H ¼ 3.63; P ¼ 0.16). Post hoc tests showed that roof rats produced a significantly higher proportion of highly fluorescent faeces than house mice (P < 0.05) and a significantly lower proportion of non-fluorescent faeces than Norway rats and house mice (both P < 0.01). The best physiological potential (i.e., the number of highly fluorescent faeces vs. total amount of produced faeces) for the use of fluorescent bait was assessed in R. rattus (25 of 52), followed R. norvegicus (24 of 66) and M. musculus (21 of 72). 4. Discussion Currently, there are several alternative methods available for monitoring rodents, including the use of uncoloured faeces as
9
Fig. 3. Numbers of different types of faeces (highly fluorescent, poorly fluorescent, non-fluorescent) produced (during a time period of 24 hours) after the consumption of fluorescent non-toxic bait in three rodent pests: R. rattus (n ¼ 23 individuals; this study); data for M. musculus (n ¼ 20 individuals) and R. norvegicus (n ¼ 33 individuals) are from our previously published studies (Frynta et al., 2012; Frankova et al., 2015). The means, standard errors and 95% confidence intervals are given for each species.
indirect indices of the presence of rodents. It was previously suggested that using brightly fluorescent faecal pellets that are produced by rodents after the ingestion of fluorescent monitoring baits, may allow easy and quick detection of rodent activity under dark conditions in urban environments, e.g., sewers, technical ducts, cellars (Corrigan, 2010). It was experimentally documented that e unlike naturally grey-coloured faeces e the fluorescent faecal pellets were visible even from the distance of several metres (Frynta et al., 2012). However, the prerequisite for fluorescent bait working effectively in practice is a good acceptance of the bait by a target pest rodent and effective passage of the fluorescent bait into the produced faeces. Therefore, in this work we quantified the amount of fluorescent faeces, described the temporal dynamics of the faeces produced after the consumption of fluorescent bait by roof rats and compared those dynamics with the dynamics previously obtained for R. norvegicus and M. musculus (Frynta et al., 2012; Frankova et al., 2015). We found that the peak production of highly fluorescent faeces in R. rattus was detected 8e18 h after consuming the bait. Similar temporal dynamics were detected in comparably sized Norway rats (i.e., 9e15 h; Frankova et al., 2015). On the other hand, smaller sized house mice with faster gut transit time produced fluorescent faeces earlier (peak 5e8 h after the consumption of fluorescent bait; Frynta et al., 2012). The probability of the successful detection of fluorescent faeces is affected by the total amount of produced faeces, faeces deposition (visible vs. hidden faeces; Frankova et al., 2015) and the spatiotemporal defecation dynamics (Aulicky et al., 2015). Our results showed that R. rattus produced, on average, 52 faecal pellets per day. This was 25% less than the number produced by
Table 1 Weight of different types of faeces produced (during a time period of 24 hours) after the consumption of fluorescent non-toxic bait in three rodent pests: R. rattus (n ¼ 23 individuals; this study); data for M. musculus (n ¼ 20 individuals) and R. norvegicus (n ¼ 33 individuals) were extracted from our previously published data (Frynta et al., 2012; Frankova et al., 2015).
Rattus rattus Rattus norvegicus Mus musculus
Highly fluorescent faeces (mg) (Mean ± SE)
Poorly fluorescent faeces (mg) (Mean ± SE)
Non-fluorescent faeces (mg) (Mean ± SE)
0.993 ± 0.108 1.976 ± 0.129 0.132 ± 0.015
0.853 ± 0.132 1.039 ± 0.085 0.192 ± 0.036
0.486 ± 0.063 1.974 ± 0.191 0.285 ± 0.051
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M. Frankova et al. / Journal of Stored Products Research 81 (2019) 7e10
R. norvegicus (Frankova et al., 2015) and nearly 30% less than the number produced by M. musculus (Frynta et al., 2012). Based on a simple quantification of the faecal production, the baiting of R. rattus would be potentially less effective in practice than the baiting of R. norvegicus and M. musculus due to the likelihood of the presence of fewer fluorescent faecal pellets. On the other hand, the proportion of highly fluorescent pellets was higher in roof rats (49%) than in house mice (32%; significant difference revealed by post hoc test) and Norway rats (39%; non-significant difference). The presented work may have multiple outcomes for rodent control practice. The primary goal of practical usage of fluorescent baits is to detect and regularly monitor rodent activity in stores and food industry facilities based on their faeces distribution. The second usage is to increase food safety by early detection of contaminated human food resources by rodent faeces. Although both of previous processes can be achieved by a direct search for presence of non-fluorescent “normal” faeces, we believe that fluorescent dye in faeces may help to increase human searching efficacy profoundly. It has been previously demonstrated by psychologists that rare items are often missed when searched visually by human (Wolfe et al., 2005). Rodent faeces may be relatively rare (e.g., during initial infestation at low rodent population density) and hard to see because of their dark colour. However, fluorescent dye may increase probability of detection of even small No. of faeces via better visibility of rodent faeces through changing its contrast against the dark background. We are convinced, based on previous experiments (e.g., Frynta et al., 2012; Varadinova et al., 2015), that fluorescent dye may decrease probability of over-looking of the present rodent faeces notably under dark conditions of stores and complex food industry buildings. In summary, we completed a unique dataset on the specific aspects of defecation physiology related to rodent monitoring in wild populations of the three most important rodent pests under laboratory conditions. The laboratory tests demonstrated promising potential (i.e., rodents accepted the fluorescent bait and then defecated high numbers of fluorescent faecal pellets) for the use of fluorescent baits for rodent monitoring. The data indicates that the best physiological monitoring potential exists for R. rattus since R. norvegicus and M. musculus produced only 80% and 65% of the fluorescent faeces produced by R. rattus, respectively. In the next stage, this potential should be further validated under diverse field conditions (e.g., Corrigan, 2010), such as various types of food stores, farms, and food industry premises. The authors believe that in many situations, fluorescent non-toxic bait may help to increase the effectiveness of rodent monitoring, allowing better targeting of chemical control measures. The availability of new monitoring tools for precision-targeting of toxic rodent baits seems to be even more important now, as the usage of anticoagulant baits is currently starting to be legislatively restricted in the EU (i.e., Commission Regulation (EU) 2016/1179). Ethical note Harm to experimental animals was avoided, and only noninvasive methods were used for sample collection. The experiments were performed in accordance with Czech law (Act No 246/
1992 Coll., on the protection of animals against cruelty), adhered to all corresponding European Union regulations (Directive 2010/63/ EU on the protection of animals used for scientific purposes).
Acknowledgements The project was supported by the Ministry of Agriculture of the Czech Republic (project MZe - No. RO 0419).
References Aulicky, R., Stejskal, V., Pekar, S., 2015. Risk evaluation of spatial distribution of faecal mice contaminants in simulated agricultural and food store. Pakistan J. Zool. 47, 1037e1043. Buckle, A., Eason, C., 2015. Control methods: chemical. In: Buckle, A., Smith, R. (Eds.), Rodent Pests and Their Control, second ed. CAB International, Wallingford, pp. 123e154. Commission Regulation (EU) 2016/1179 of 19 July 2016 Amending, for the Purposes of its Adaptation to Technical and Scientific Progress, Regulation (EC) No 1272/ 2008 of the European Parliament and of the Council on Classification, Labelling and Packaging of Substances and Mixtures. Corrigan, B., 2010. Biomonitoring for rodents. Annual Rodent Control Issue 27e29. Frankova, M., Kaftanova Eliasova, B., Rodl, P., Aulicky, R., Frynta, D., Stejskal, V., 2015. Monitoring of Rattus norvegicus based on non-toxic bait containing encapsulated fluorescent dye: laboratory and semi-field validation study. J. Stored Prod. Res. 64, 103e108. Frankova, M., Stejskal, V., Aulicky, R., 2017. Suppression of food intake by house mouse (Mus musculus) following ingestion of brodifacoum-based rodenticide bait. Crop Protect. 100, 134e137. Frynta, D., Eliasova, B., Frankova, M., Aulicky, R., Rodl, P., Stejskal, V., 2012. Production of UV-light-detectable faeces from house mice (Mus musculus domesticus) after consumption of encapsulated fluorescent pigment in monitoring bait. Pest Manag. Sci. 68, 355e361. n, V., Go mez Villafane, I.E., Busch, M., 2010. Bromadiolone Guidobono, J.S., Leo susceptibility in wild and laboratory Mus musculus L. (house mice) in Buenos Aires, Argentina. Pest Manag. Sci. 66, 162e167. Hamel, D., 2010. Rodents e health risk and control measures. In: Proc. International European Symposium on Stored Product Protection "Stress on chemical products" Julius-Kühn-Archiv, 429, pp. 18e20. Jokic, G., Blazic, T., Durovic-Pejcev, R., Dordevic, T., Dedovic, S., Vuksa, M., 2017. A method for reducing environmental pollution by using essential oils in rodent pest management program. Environ. Sci. Pollut. Control Ser. 24, 18257e18262. R Core Team, 2015. R: a Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project. org/. Statistica 12.0 (2010) StatSoft, Inc., Tulsa, OK, USA. Stejskal, V., Aulicky, R., Kucerova, Z., 2014. Pest control strategies and damage potential of seed-infesting pests in the Czech stores e a review. Plant Protect. Sci. 50, 165e173. Stejskal, V., Hubert, J., Aulicky, R., Kucerova, Z., 2015. Overview of present and past and pest-associated risks in stored food and feed products: European perspective. J. Stored Prod. Res. 64, 122e132. Stejskal, V., 1997. Distribution of faeces of the German cockroach, Blattella germanica, in a new refuge. Entomol. Exp. Appl. 84, 201e205. Stejskal, V., Aulicky, R., 2014. Field evidence of roof rat (Rattus rattus) faecal contamination of barley grain stored in silos in the Czech Republic. J. Pest. Sci. 87, 117e124. Varadinova, Z., Frynta, D., Aulicky, R., Stejskal, V., 2015. Detection of cockroach faeces: consumption of fluorescent bait and production of UV-light-detectable faeces from German cockroach, Blattella germanica. Entomol. Exp. Appl. 155, 167e175. Witmer, G., 2007. The ecology of vertebrate pests and integrated pest management (IPM). In: Kogan, M., Jepson, P. (Eds.), Perspectives in Ecological Theory and Integrated Pest Management. Cambridge University Press, Cambridge, pp. 393e410. Wolfe, J.M., Horowitz, T.S., Kenner, N.M., 2005. Rare items often missed in visual searches. Nature 435, 439e440.
Contents lists available at ScienceDirect
Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr
Temporal production of coloured faeces in wild roof rats (Rattus rattus) following consumption of fluorescent non-toxic bait and a comparison with wild R. norvegicus and Mus musculus Marcela Frankova a, *, Barbora Kaftanova b, Radek Aulicky a, Pavel Rodl c, Daniel Frynta b, Vaclav Stejskal a a b c
Crop Research Institute, Drnovska 507, Prague 6, Czech Republic Department of Zoology, Faculty of Science, Charles University, Vinicna 7, Prague 2, Czech Republic National Institute of Public Health, Srobarova 48, Prague 10, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 June 2018 Received in revised form 6 December 2018 Accepted 17 December 2018
In the laboratory, we evaluated the efficacy of the use of fluorescent non-toxic bait for the monitoring of wild roof rats (Rattus rattus). We described the temporal dynamics of the production of fluorescent faeces after the consumption of fluorescent bait and compared it with those of R. norvegicus and Mus musculus. Roof rats produced, on average, 52 faecal pellets per 24 h, ranging from 31 to 81. The dry weight of the produced faeces per 24 h ranged from 1.1 to 3.4 g. The production peak of highly detectable fluorescent faeces was 8e18 h after consuming the bait, and the last detectable faeces were recorded 28e30 h after consuming the bait. Our data showed that roof rats produced the highest proportion of highly fluorescent faeces, indicating that best physiological potential for the use of this type of fluorescent bait exists in R. rattus since R. norvegicus and M. musculus produced 80% and 65% of the fluorescent faeces produced by R. rattus, respectively. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Rodent control Rodent pests IPM Monitoring Faeces Environmental contaminants
1. Introduction Rodents cause enormous amounts of damage to grain stores globally (Hamel, 2010; Stejskal et al. 2014, 2015). For example, roof rats were recently documented to be serious faecal contaminators of stored commodities (Stejskal and Aulicky, 2014); grain kernels were found to be of equal size to rat droppings, so their admixture with grain mass is difficult to separate. Since natural rodenticides and repellents are mostly still in the research phase (e.g., Jokic et al., 2017), synthetic anticoagulant rodenticides are currently most commonly employed for rodent control (Guidobono et al., 2010; Buckle and Eason, 2015; Frankova et al., 2017). However, the widespread and non-targeted use of anticoagulants poses a serious toxicological and environmental threat. Mitigation of these
* Corresponding author. Crop Research Institute, Drnovska 507, CZ-16106, Czech Republic. E-mail addresses: [email protected] (M. Frankova), [email protected] (B. Kaftanova), [email protected] (R. Aulicky), [email protected] (P. Rodl), frynta@ centrum.cz (D. Frynta), [email protected] (V. Stejskal). https://doi.org/10.1016/j.jspr.2018.12.002 0022-474X/© 2018 Elsevier Ltd. All rights reserved.
environmental risks requires implementation of programmes of integrated pest management (IPM) based on efficient monitoring followed by targeted control (Witmer, 2007). Currently, rodent monitoring is mainly based on trapping or observation of bait consumption and/or the distribution of faeces. Although the faeces of rodents are at least 10 larger than that of insects (e.g., Stejskal, 1997), it still may be easily overlooked by inspectors due to its dark colour (Frynta et al., 2012), especially under conditions of dimly lit storage and food facilities. Recently, a new ultraviolet-based technique that enables easy tracking of the highly visible fluorescent faeces was developed for professional urban pest control (Corrigan, 2010). It is based on the use of technology that encapsulates fluorescent particles, protecting the ultraviolet (UV) dye during its passage through the digestive tract. The prerequisite for the use of this method in practice is a thorough knowledge of the physiology of the digestion and defecation of the fluorescent bait that can address practical questions such as “How many fluorescent faeces are produced per day?” or “How quickly does the bait pass through the digestive tract to appear as fluorescent faeces in the environment?” The goal of this study, similar to our preceding studies on fluorescent rodent-monitoring baits, was to determine the answers
8
M. Frankova et al. / Journal of Stored Products Research 81 (2019) 7e10
to these questions. Our previous studies described the physiological reactions and defecation parameters in Mus musculus Linnaeus and Rattus norvegicus Berkenhout (Frynta et al., 2012; Frankova et al., 2015). Therefore, this study, as part of a multispecies project, assessed the temporal dynamics of the production of fluorescent faeces in Rattus rattus Linnaeus and compared the fluorescent faeces production among the three main rodent pests (i.e., M. musculus e Frynta et al. (2012), R. norvegicus e Frankova et al. (2015), and R. rattus e this study). 2. Material and methods 2.1. Laboratory experiment Twenty-three wild roof rats (Rattus rattus; 20 males, 3 females) were trapped in a piggery in the Czech Republic (Palecek, the Central Bohemian region). During a 6-week adaptation period, rats were subjected to the physiological (temperature and light conditions) and behavioural (experimental cages, the presence of human, no handling) acclimatization. Rats were kept separately in wire mesh cages (20 30 24 cm). Standard food (Ssniff Spe€ten GmbH, Soest, Germany) and water were provided ad zialdia libitum. After the adaptation to captivity, the experimental procedures were performed. Standard food was removed from the cage for 20 h prior to the experiment to increase food motivation in the rats. Then, 20 g of fluorescent pellets (ICB Pharma, Poland; for details see Frynta et al., 2012) was placed into each cage, and the floor under the cage was covered with freely accessible clean filter paper. The fluorescent pellets were weighed at 2-h intervals, and their consumption was monitored until the experimental animals had consumed at least 3 g, after which the fluorescent food was removed and replaced with standard food. After the beginning of the consumption of the fluorescent pellets, the paper covering the floor under the cage was replaced with a new sheet, and all faeces were collected. This procedure was performed every two hours until 40 h after the beginning of the consumption of the fluorescent pellets, at which point the experiment was terminated. The collected faeces were then observed under UV illumination (a 21 LED flashlight; ICB Pharma), counted and sorted into those that were (1) highly fluorescent (i.e., clearly detectable at the distance of approximately one metre), (2) poorly fluorescent and (3) exhibiting no sign of fluorescence; Fig. 1. Next, all faeces were dried and weighed. The sorting and weighing were performed in a blinded manner.
Fig. 1. The different types of faeces produced by roof rat (R. rattus): highly fluorescent faeces (first row), poorly fluorescent faeces (second row) and faeces exhibiting no sign of fluorescence (third row).
variable (fixed factor). The rat identity and AR-1 correlation structure were included to avoid pseudoreplication. To evaluate faeces production after the consumption of fluorescent non-toxic bait in three rodent pests, we compared the data (sums of different types of faeces per 24 h) from this experiment with the data from previously published studies on house mice (Frynta et al., 2012) and Norway rats (Frankova et al., 2015). A nonnyi parametric Kruskal-Wallis rank sum test with the Kruskal-Neme post hoc test (the PMCRM package) was used for between-species comparisons of the ratios of different types of faeces. It was employed to avoid an assumption of normal distribution for variables expressed as proportions. All statistical analyses were carried out using the R software (R Core Team, 2015), and graphs were generated in Statistica 12.0 (StatSoft Inc. 2010). 3. Results
2.2. Statistical analysis
3.1. Faeces production in R. rattus
There was a high individual variance in the acceptance of the fluorescent pellets in the laboratory experiment. The experimental animals differed in the amount of food consumed, the latency of consumption and the time interval needed to consume a required amount of food (i.e., minimally 3 g). The recording of fluorescent faeces production in every individual started from the time point when the consumption of 3 g of food had been achieved (i.e., time ¼ 0), allowing comparison among individuals and the graphic presentation of the temporal dynamics. To assess the temporal changes in the proportion of highly fluorescent faeces, we employed marginal models, as implemented in the geeglm package. The course of the experiment was arbitrarily divided into 7 sections, each lasting 6 h and consisting of three elementary time intervals (2 h). The amount of highly fluorescent faeces and the amount of faeces in the remaining categories (poorly and non-fluorescent) for each elementary 2-h time interval were the dependent variables, and the 6-h sections were the explanatory
Rats produced on average 52 faecal pellets per 24 h, ranging from 31 to 81 (SE ¼ 13.8, CI ¼ 46.3e58.2). The dry weight of the produced faeces per 24 h ranged from 1.1. to 3.4 g (mean ¼ 2.3 g, SE ¼ 0.8, CI ¼ 2.0e2.7). The experimental subjects consumed 1.1e13.2 g of the fluorescent pellets (mean ¼ 6.5 g). The first fluorescent faeces were recorded 2e4 h after the consumption of the required number of fluorescent pellets. The production peak of highly detectable fluorescent faeces was 8e18 h, and the last detectable faeces were recorded 28e30 h after the consumption of the bait in most of the experimental animals (Fig. 2). However, one individual produced fluorescent faecal pellets up to 38 h after consuming the bait. The proportion of highly fluorescent pellets to the total produced pellets per 24 h was 48%, and during the peak period, it increased to over 76%. The geeglm analysis confirmed the statistical significance of the observed temporal variation in the proportion of highly fluorescent
M. Frankova et al. / Journal of Stored Products Research 81 (2019) 7e10
Fig. 2. Temporal dynamics of produced faeces after the consumption of fluorescent non-toxic bait in roof rats (R. rattus). Data are given as the means and 95% confidence intervals.
faeces (factor section: df ¼ 6, c2 ¼ 77.4, P < 0.01). Compared to the first section (0e6 h, intercept ¼ 1.893, SE ¼ 0.406, P < 0.01), this proportion was significantly elevated in the 6e12 h (intercept ¼ 2.820, SE ¼ 0.516, P < 0.01), 12e18 h (intercept ¼ 3.097, SE ¼ 0.545, P < 0.001) and 18e24 h (intercept ¼ 1.243, SE ¼ 0.529, P ¼ 0.02) sections. 3.2. Comparison of fluorescent faeces production in three rodent pests The three studied rodent pest species produce different total amounts (H2,76 ¼ 7.35, P ¼ 0.03) and weight (H2,76 ¼ 60.67, P < 0.01; for details see Table 1) of faeces. Roof rats produce significantly fewer faecal pellets than house mice, while they produce a similar number of faecal pellets as Norway rats (Fig. 3). The Kruskal-Wallis rank sum test revealed significant differences in the proportions of highly fluorescent (H ¼ 10.77, P < 0.01) and non-fluorescent (H ¼ 16.11, P < 0.01) faeces, while the proportion of poorly fluorescent faeces did not differ among rodent pests (H ¼ 3.63; P ¼ 0.16). Post hoc tests showed that roof rats produced a significantly higher proportion of highly fluorescent faeces than house mice (P < 0.05) and a significantly lower proportion of non-fluorescent faeces than Norway rats and house mice (both P < 0.01). The best physiological potential (i.e., the number of highly fluorescent faeces vs. total amount of produced faeces) for the use of fluorescent bait was assessed in R. rattus (25 of 52), followed R. norvegicus (24 of 66) and M. musculus (21 of 72). 4. Discussion Currently, there are several alternative methods available for monitoring rodents, including the use of uncoloured faeces as
9
Fig. 3. Numbers of different types of faeces (highly fluorescent, poorly fluorescent, non-fluorescent) produced (during a time period of 24 hours) after the consumption of fluorescent non-toxic bait in three rodent pests: R. rattus (n ¼ 23 individuals; this study); data for M. musculus (n ¼ 20 individuals) and R. norvegicus (n ¼ 33 individuals) are from our previously published studies (Frynta et al., 2012; Frankova et al., 2015). The means, standard errors and 95% confidence intervals are given for each species.
indirect indices of the presence of rodents. It was previously suggested that using brightly fluorescent faecal pellets that are produced by rodents after the ingestion of fluorescent monitoring baits, may allow easy and quick detection of rodent activity under dark conditions in urban environments, e.g., sewers, technical ducts, cellars (Corrigan, 2010). It was experimentally documented that e unlike naturally grey-coloured faeces e the fluorescent faecal pellets were visible even from the distance of several metres (Frynta et al., 2012). However, the prerequisite for fluorescent bait working effectively in practice is a good acceptance of the bait by a target pest rodent and effective passage of the fluorescent bait into the produced faeces. Therefore, in this work we quantified the amount of fluorescent faeces, described the temporal dynamics of the faeces produced after the consumption of fluorescent bait by roof rats and compared those dynamics with the dynamics previously obtained for R. norvegicus and M. musculus (Frynta et al., 2012; Frankova et al., 2015). We found that the peak production of highly fluorescent faeces in R. rattus was detected 8e18 h after consuming the bait. Similar temporal dynamics were detected in comparably sized Norway rats (i.e., 9e15 h; Frankova et al., 2015). On the other hand, smaller sized house mice with faster gut transit time produced fluorescent faeces earlier (peak 5e8 h after the consumption of fluorescent bait; Frynta et al., 2012). The probability of the successful detection of fluorescent faeces is affected by the total amount of produced faeces, faeces deposition (visible vs. hidden faeces; Frankova et al., 2015) and the spatiotemporal defecation dynamics (Aulicky et al., 2015). Our results showed that R. rattus produced, on average, 52 faecal pellets per day. This was 25% less than the number produced by
Table 1 Weight of different types of faeces produced (during a time period of 24 hours) after the consumption of fluorescent non-toxic bait in three rodent pests: R. rattus (n ¼ 23 individuals; this study); data for M. musculus (n ¼ 20 individuals) and R. norvegicus (n ¼ 33 individuals) were extracted from our previously published data (Frynta et al., 2012; Frankova et al., 2015).
Rattus rattus Rattus norvegicus Mus musculus
Highly fluorescent faeces (mg) (Mean ± SE)
Poorly fluorescent faeces (mg) (Mean ± SE)
Non-fluorescent faeces (mg) (Mean ± SE)
0.993 ± 0.108 1.976 ± 0.129 0.132 ± 0.015
0.853 ± 0.132 1.039 ± 0.085 0.192 ± 0.036
0.486 ± 0.063 1.974 ± 0.191 0.285 ± 0.051
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R. norvegicus (Frankova et al., 2015) and nearly 30% less than the number produced by M. musculus (Frynta et al., 2012). Based on a simple quantification of the faecal production, the baiting of R. rattus would be potentially less effective in practice than the baiting of R. norvegicus and M. musculus due to the likelihood of the presence of fewer fluorescent faecal pellets. On the other hand, the proportion of highly fluorescent pellets was higher in roof rats (49%) than in house mice (32%; significant difference revealed by post hoc test) and Norway rats (39%; non-significant difference). The presented work may have multiple outcomes for rodent control practice. The primary goal of practical usage of fluorescent baits is to detect and regularly monitor rodent activity in stores and food industry facilities based on their faeces distribution. The second usage is to increase food safety by early detection of contaminated human food resources by rodent faeces. Although both of previous processes can be achieved by a direct search for presence of non-fluorescent “normal” faeces, we believe that fluorescent dye in faeces may help to increase human searching efficacy profoundly. It has been previously demonstrated by psychologists that rare items are often missed when searched visually by human (Wolfe et al., 2005). Rodent faeces may be relatively rare (e.g., during initial infestation at low rodent population density) and hard to see because of their dark colour. However, fluorescent dye may increase probability of detection of even small No. of faeces via better visibility of rodent faeces through changing its contrast against the dark background. We are convinced, based on previous experiments (e.g., Frynta et al., 2012; Varadinova et al., 2015), that fluorescent dye may decrease probability of over-looking of the present rodent faeces notably under dark conditions of stores and complex food industry buildings. In summary, we completed a unique dataset on the specific aspects of defecation physiology related to rodent monitoring in wild populations of the three most important rodent pests under laboratory conditions. The laboratory tests demonstrated promising potential (i.e., rodents accepted the fluorescent bait and then defecated high numbers of fluorescent faecal pellets) for the use of fluorescent baits for rodent monitoring. The data indicates that the best physiological monitoring potential exists for R. rattus since R. norvegicus and M. musculus produced only 80% and 65% of the fluorescent faeces produced by R. rattus, respectively. In the next stage, this potential should be further validated under diverse field conditions (e.g., Corrigan, 2010), such as various types of food stores, farms, and food industry premises. The authors believe that in many situations, fluorescent non-toxic bait may help to increase the effectiveness of rodent monitoring, allowing better targeting of chemical control measures. The availability of new monitoring tools for precision-targeting of toxic rodent baits seems to be even more important now, as the usage of anticoagulant baits is currently starting to be legislatively restricted in the EU (i.e., Commission Regulation (EU) 2016/1179). Ethical note Harm to experimental animals was avoided, and only noninvasive methods were used for sample collection. The experiments were performed in accordance with Czech law (Act No 246/
1992 Coll., on the protection of animals against cruelty), adhered to all corresponding European Union regulations (Directive 2010/63/ EU on the protection of animals used for scientific purposes).
Acknowledgements The project was supported by the Ministry of Agriculture of the Czech Republic (project MZe - No. RO 0419).
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