Research Note: Genetic analysis, pathology, and vectors of echinostomiasis, a zoonotic helminth infection in chickens in Bangladesh.

Echinostomes (Trematoda: Echinostomatidae) are food-borne zoonotic flatworms that affect birds, animals and humans, and has been classified as neglected tropical diseases (NTDs) by the World Health Organization (WHO), which cause severe enteritis in poultry and hamper production. Here, we confirmed the species of echinostomes affecting chickens in Bangladesh along with their genetic analyses, pathology and vectors. We isolated and identified adult worms from chickens, cercariae from fresh water snails and metacerariae (MC) from some wild fishes. We recovered Echinostoma revolutum (10.3%) and Hypoderaeum conoideum (6.0%) from chickens. Zoonotic E. revolutum was confirmed by amplifying nad1 gene and subsequent sequencing. Several mutations were detected in nad1 gene and our isolates belonged to the Euro-Asian clade. We observed thickening of mucosal layer, hyperplasia of goblet cells, infiltration of eosinophils, lymphocytes and must cells in the infected intestine. About 5.3% snails were infected and the highest percentage of infection was found in Lymnaea luteola (12.1%). Echinostome infection in snails was the highest in November (9.6%) and lowest in February (3.1%) in Bangladesh. MC of echinostomes were identified from blue panchax (Aplocheilus panchax) and tank goby (Glossogobius giuris). In conclusion, echinostomiasis is a notable big problem in indigenous chickens in Bangladesh and people, especially, villagers are at risk.

Timing and order of exposure to two echinostome species affect patterns of infection in larval amphibians.

The study of priority effects with respect to coinfections is still in its infancy. Moreover, existing coinfection studies typically focus on infection outcomes associated with exposure to distinct sets of parasite species, despite that functionally and morphologically similar parasite species commonly coexist in nature. Therefore, it is important to understand how interactions between similar parasites influence infection outcomes. Surveys at seven ponds in northwest Pennsylvania found that multiple species of echinostomes commonly co-occur. Using a larval anuran host (Rana pipiens) and the two most commonly identified echinostome species from our field surveys (Echinostoma trivolvis and Echinoparyphium lineage 3), we examined how species composition and timing of exposure affect patterns of infection. When tadpoles were exposed to both parasites simultaneously, infection loads were higher than when exposed to Echinoparyphium alone but similar to being exposed to Echinostoma alone. When tadpoles were sequentially exposed to the parasite species, tadpoles first exposed to Echinoparyphium had 23% lower infection loads than tadpoles first exposed to Echinostoma. These findings demonstrate that exposure timing and order, even with similar parasites, can influence coinfection outcomes, and emphasize the importance of using molecular methods to identify parasites for ecological studies.

[Characterization of Echinostoma miyagawai in domestic ducks in Wuhu City].

OBJECTIVE: To investigate the morphological characteristics of Echinostoma miyagawai in domestic ducks in Wuhu area, and to explore the feasibility of the cytochrome oxidase subunit-1 (Cox1) gene as a molecular marker for the identification of E. miyagawai. METHODS: E. miyagawai was isolated from free-ranged domestic ducks in Wuhu area, and the parasites were stained and identified. In addition, the mitochondrial Cox1 gene of E. miyagawai was amplified using a PCR assay, and the amplification product was sequenced and aligned with the GenBank database to yield the homology for the identification of parasite species in combination with morphological findings. Intra-species comparison was done based on the Cox1 gene sequence. RESULTS: The prevalence of E. miyagawai infection was 16.67% in domestic ducks in Wuhu area, and the adult E. miyagawai was 6.6 to 13.2 mm in length. The size of the E. miyagawai Cox1 gene was approximately 660 bp, which had a 99.68% homology to the E. miyagawai accessed in GenBank. The morphological findings were in agreement with molecular identification. CONCLUSIONS: E. miyagawai infection is common in domestic ducks in Wuhu area, and the mitochondrial Cox1 gene is a feasible marker of intra- and inter-species molecular identification of Echinostoma.

Sub-chronic exposure to a neonicotinoid does not affect susceptibility of larval leopard frogs to infection by trematode parasites, via either depressed cercarial performance or host immunity.

Little information is available on the effects of neonicotinoid insecticides on vertebrates. Previous work using amphibians found chronic exposure to some neonicotinoids had no detrimental effects on fitness-relevant traits. However, there is some evidence of more subtle effects of neonicotinoids on immune traits and evidence that other pesticides can suppress tadpole immunity resulting in elevated levels of parasitism in the exposed tadpoles. The objective of our study was to assess whether neonicotinoid exposure affected tadpole immunometrics and susceptibility to parasitic helminths. We assessed northern leopard frog tadpole (Lithobates pipiens) levels of parasitism and leukocyte profiles following exposure to environmentally relevant concentrations of clothianidin and free-living infective cercariae of a helminth parasite, an Echinostoma sp. trematode. When comparing tadpoles from controls to either 1 or 100 µg/L clothianidin treatments, we found similar measures of parasitism (i.e. prevalence, abundance and intensity of echinostome cysts) and similar leukocyte profiles. We also confirmed that clothianidin was not lethal for cercariae; however, slight reductions in swimming activity were detected at the lowest exposure concentration of 0.23 µg/L. Our results show that exposure to clothianidin during the larval amphibian stage does not affect leukocyte profiles or susceptibility to parasitism by larval trematodes in northern leopard frogs although other aspects such as length of host exposure require further study.

Suburbanization Increases Echinostome Infection in Green Frogs and Snails.

An important contribution to infectious disease emergence in wildlife is environmental degradation driven by pollution, habitat fragmentation, and eutrophication. Amphibians are a wildlife group that is particularly sensitive to land use change, infectious diseases, and their interactions. Residential suburban land use is now a dominant, and increasing, form of land cover in the USA and globally, contributing to increased pollutant and nutrient loading in freshwater systems. We examined how suburbanization affects the infection of green frog (Rana clamitans) tadpoles and metamorphs by parasitic flatworms (Echinostoma spp.) through the alteration of landscapes surrounding ponds and concomitant changes in water quality. Using sixteen small ponds along a forest-suburban land use gradient, we assessed how the extent of suburban land use surrounding ponds influenced echinostome infection in both primary snail and secondary frog hosts. Our results show that the degree of suburbanization and concurrent chemical loading are positively associated with the presence and burden of echinostome infection in both host populations. This work contributes to a broader understanding of how land use mediates wildlife parasitism and shows how human activities at the household scale can have similar consequences for wildlife health as seemingly more intensive land uses like agriculture or urbanization.

Characterization of Echinostoma revolutum and Echinostoma robustum from ducks in Bangladesh based on morphology, nuclear ribosomal ITS2 and mitochondrial nad1 sequences.

Precise discrimination of Echinostoma species within the 'revolutum' group is quite difficult because of their morphological similarities. The objective of this study was to precisely characterize the echinostomes of ducks from Bangladesh based on both morphological and molecular characteristics. Two Echinostoma species were identified: E. revolutum and E. robustum. In the phylogenetic trees (ITS2 and nad1), E. revolutum and E. robustum belonged to their respective Eurasian clade, which is distinct from the American clade. These results suggest that both species have two distinct and geographically separated lineages, Eurasian and American. Our molecular and morphological data combined with previously published data supports the synonymy of E. robustum, E. miyagawai, and E. friedi previously based on either molecular or morphological evidence. This study thus improves our understanding of species diversity of the 'revolutum' group, particularly in Asia.

Echinostoma revolutum: Development of a high performance DNA-specific primer to demonstrate the epidemiological situations of their intermediate hosts.

Echinostomiasis caused by the Echinostoma group, in particular E. revolutum are a significant problem for both humans and other animals. This group has a large number of morphological similarities that are difficult and time-consuming to identify. The present study aimed to develop high-performance tools for the detection of the prevalence of E. revolutum and to reveal the prevalence of E. revolutum infections in intermediate snail hosts in Lopburi province, Thailand. The snail specimens were collected by stratified sampling method and examined to collect trematodes in the larval stage. The specific primer was manually designed and based on 18 s rDNA and verified the specificity and sensitivity for use as an identification tool to compare with classical method, constructed by epidemic mapping. The overall prevalence value of E. revolutum was found to be 16.26%. Tha Luang district had the highest prevalence (70.14%), followed by Chai Badan, Phatthana Nikhom, Tha Wung, Ban Mi, Khok Samrong, Nong Muang and Sa Bot at 42%, 25.14%, 2.52%, 1.73%, 2%, 1.33% and 0.40%, respectively. With regard to the specific primer, it can amplify both cercarial and metacercarial DNA (90 pg/µl.) and discriminated E. revolutum from its hosts, other trematodes and other echinostome larvae with no cross-reactions. Therefore, the developed specific primer can be used as a species-specific identification tool with a high degree of sensitivity and specificity. Consequently, this data is important for monitoring the outbreak of E. revolutum. It can be applied for initiating surveillance programs of snail-borne diseases in both medical and veterinary studies.

Endocrine and immune responses of larval amphibians to trematode exposure.

In nature, multiple waves of exposure to the same parasite are likely, making it important to understand how initial exposure or infection affects subsequent host infections, including the underlying physiological pathways involved. We tested whether experimental exposure to trematodes (Echinostoma trivolvis or Ribeiroia ondatrae) affected the stress hormone corticosterone (known to influence immunocompetence) in larvae representing five anuran species. We also examined the leukocyte profiles of seven host species after single exposure to R. ondatrae (including four species at multiple time points) and determined if parasite success differed between individuals given one or two challenges. We found strong interspecific variation among anuran species in their corticosterone levels and leukocyte profiles, and fewer R. ondatrae established in tadpoles previously challenged, consistent with defense "priming." However, exposure to either trematode had only weak effects on our measured responses. Tadpoles exposed to E. trivolvis had decreased corticosterone levels relative to controls, whereas those exposed to R. ondatrae exhibited no change. Similarly, R. ondatrae exposure did not lead to appreciable changes in host leukocyte profiles, even after multiple challenges. Prior exposure thus influenced host susceptibility to trematodes, but was not obviously associated with shifts in leukocyte counts or corticosterone, in contrast to work with microparasites.

Infection Status of Isthmiophora hortensis Metacercariae in Dark Sleepers, Odontobutis Species, from Some Water Systems of the Republic of Korea.

Present study was performed to survey on infection status of Isthmiophora hortensis (formerly Echinostoma hortense) metacercariae (IhMc) in dark sleepers, Odontobutis spp., from some water systems of the Republic of Korea. A total of 237 Odontobutis spp. was collected in the water systems of 5 rivers, i.e., Mangyeong-gang (gang means river), Geum-gang, Tamjin-gang, Seomjin-gang, and Nakdong-gang. They were all examined with artificial digestion method for 5 years (2013-2017). A total of 137 (57.8%) Odontobutis spp. were infected with 14.8 IhMc in average. The prevalence was the highest in Nakdong-gang areas (62.9%) and followed by in Mangyeong-gang (57.1%), Geum-gang (56.3%), Tamjin-gang (54.8%), and Seomjin-gang (53.9%) areas. Metacercarial densities were 28.1 (Geum-gang), 13.9 (Mangyeong-gang), 13.3 (Nakdong-gang), 13.1 (Tamjin-gang), and 2.3 (Seomjin-gang) per infected fish. Especially, in case of Yugucheon (cheon means stream), a branch of Geum-gang, IhMc were detected in all fish (100%) examined and their density was about 48 per fish. By the present study, it was confirmed that the infection status of IhMc is more or less different by the surveyed areas and the dark sleepers, Odontobutis spp., are suitable fish hosts of I. hortensis.

Exposure to a cyanobacterial toxin increases larval amphibian susceptibility to parasitism.

Anthropogenic activities are promoting the proliferation of aquatic primary producers in freshwater habitats, including cyanobacteria. Among various problems stemming from eutrophication, cyanobacterial blooms can be toxic due to the production of secondary compounds, including microcystins such as microcystin-LR (MC-LR); however, it is unknown whether cyanotoxins can affect the susceptibility of aquatic vertebrates such as fish and larval amphibians to parasites or pathogens even though infectious diseases can significantly affect natural populations. Here, we examined how exposure to environmentally relevant concentrations of MC-LRs affected the resistance of larval amphibians (northern leopard frog, Rana pipiens) to infection by a helminth parasite (the trematode Echinostoma sp.), and whether this was manifested by reductions in host anti-parasite behavior. Exposure to a relatively high (82 µg L-1) concentration of MC-LR caused over 70% mortality, and tadpoles that survived exposure to the low MC-LR (11 µg L-1) treatment had significantly higher infection intensities than those in the control; however, anti-parasite behavior was not affected by treatment. Our results indicate that MC-LR can have both direct and indirect negative effects on larval amphibians by increasing their mortality and susceptibility to parasitism, which may have implications for other aquatic vertebrates in eutrophic habitats dominated by cyanobacteria as well.

The distribution of echinostome parasites in ponds and implications for larval anuran survival.

Parasites can influence host population dynamics, community composition and evolution. Prediction of these effects, however, requires an understanding of the influence of ecological context on parasite distributions and the consequences of infection for host fitness. We address these issues with an amphibian - trematode (Digenea: Echinostomatidae) host-parasite system. We initially performed a field survey of trematode infection in first (snail) and second (larval green frog, Rana clamitans) intermediate hosts over 5 years across a landscape of 23 ponds in southeastern Michigan. We then combined this study with a tadpole enclosure experiment in eight ponds. We found echinostomes in all ponds during the survey, although infection levels in both snails and amphibians differed across ponds and years. Echinostome prevalence (proportion of hosts infected) in snails also changed seasonally depending on host species, and abundance (parasites per host) in tadpoles depended on host size and prevalence in snails. The enclosure experiment demonstrated that infection varied at sites within ponds, and tadpole survival was lower in enclosures with higher echinostome abundance. The observed effects enhance our ability to predict when and where host-parasite interactions will occur and the potential fitness consequences of infection, with implications for population and community dynamics, evolution and conservation.

Host food resource supplementation increases echinostome infection in larval anurans.

Host-parasite interactions are often influenced by environmental factors through multiple mechanisms. For example, changes in host food resources may affect multiple host traits (e.g., body size, behavior, immunocompetence), which may increase or decrease infection levels and the impact of parasites on host fitness. We often lack an understanding of which traits are most important for parasite transmission and fitness effects, posing challenges to predicting consequences of changing environmental conditions (e.g., eutrophication). Here, I examined the effects of food resources and host traits experimentally in a larval frog (Rana clamitans Latreille, 1801)-trematode parasite (Echinostoma revolutum Looss, 1899) system. I hypothesized that higher food resources reduce parasite infection and parasite effects on host growth and survival, due to increased host investment in parasite defenses, which I tested in a laboratory experiment. Contrary to my hypothesis, the results indicated that increased food levels enhanced infection in hosts, while the effect of parasites on survival did not depend on host food resources. A potential explanation for the positive effect of food level on infection was size-dependent infection rates (i.e., higher food levels increased infection through increased host growth), which is supported by a positive relationship between host body size and infection. These findings emphasize the complex relationship between host food resources and parasitism and the importance of environmental context and host traits (i.e., body size) in mediating interactions with parasites. The results also have relevance for conservation in light of rising anthropogenic impacts on aquatic systems and recent amphibian declines.

Auto-infection by Echinostoma spp. cercariae in Helisoma anceps.

Auto-infection is a life history strategy used by many parasitic organisms, including digenetic trematodes. The process of autoinfection most frequently involves the transfer of a life cycle stage of the parasite from one site to another inside the same host, usually accompanied by morphological transformation. Moreover, among trematodes, the stage being transferred may also move from one host to another in completing the life cycle, i.e., an indirect cycle. Echinostoma spp. parasites offer the opportunity to study auto-infection because they utilize gastropods as both first and second intermediate hosts. Rejection of a null model predicting independent infection of first and second intermediate larval stages coupled with the presence of rediae being the best predictor of metacercariae prevalence and intensity suggests that auto-infection by Echinostoma spp. cercariae is occurring in their molluscan hosts. Shell length was also found to be a significant predictor of metacercariae intensity in the snails hosts, but this is most likely attributed to larger snails being more commonly infected with Echinostoma spp. rediae as opposed to an increased likelihood of cercariae infection. Auto-infection as a life history strategy increases transmission success of the parasite, but may also have negative consequences for the parasite that necessitate auto-infection coupled with the release of cercariae to maximize transmission success and host survival.

Three echinostome species from wild birds in the Republic of Korea.

Three echinostome species, i.e., Patagifer bilobus, Petasiger neocomense, and Saakotrema metatestis, are newly recorded in the trematode fauna of the Republic of Korea. They were recovered from 3 species of migratory birds (Platalea minor, Podiceps cristatus, and Egretta garzetta), which were donated by the Wildlife Center of Chungbuk (WCC) and the Conservation Genome Resource Bank for Korean Wildlife (CGRB). Only 1 P. bilobus specimen was recovered from the intestine of a black-faced spoonbill (P. minor), and characterized by the bilobed head crown with a deep dorsal incision and 54 collar spines. Twenty P. neocomense were recovered from the intestine of a great crested grebe (P. cristatus), and they had a well-developed head crown with 19 spines and 2 testes obliquely located at the posterior middle of the body. Total 70 S. metatestis were collected from the bursa of Fabricius of 1 little egret (E. garzetta). It is characterized by stout tegumental spines covered in the entire leaf-shaped body, posterior extension of the uterus, presence of the uroproct and a well-developed head crown with 12 pairs of collar spines on each side. By the present study, these 3 echinostome species are newly added to the trematode fauna in Korea.

Epidemiology and molecular genotyping of echinostome metacercariae in Filopaludina snails in Lamphun Province, Thailand.

OBJECTIVE: To analyze the prevalence of echinostome metacercariae in Filopaludina dorliaris (F. dorliaris) and Filopaludina martensi martensi (F. martensi martensi) and genotype variation of echinostome metacercariae by using random amplified polymorphic DNA (RAPD) analysis. METHODS: Filopaludina sp. snails were collected from eight localities of Lamphun Province, Northern Thailand and examined for echinostome metacercariae. RAPD-PCR was used to analyze genotype variation of echinostome metacercariae. RESULTS: A total of 3 226 F. dorliaris and F. martensi martensi snails were collected from eight localities. The overall prevalences of echinostome metacercariae in F. dorliaris and F. martensi martensi were 40.89% and 36.27%, while the intensity of infection was 20.37 and 12.04, respectively. The dendrogram constructed base on RAPD profiles, 4 well supported domains were generated; (i) group of metacercariae from Ban Hong, Mae Ta, Meaung, Pa Sang, Toong Hua Chang, and Weang Nong that were clustered in the group of E. revolutum, (ii) Ban Thi, (iii) Lee, and (iv) 3 adults of an out group. CONCLUSIONS: This research demonstrated RAPD profiling has been a useful tool to detect DNA polymorphisms to determine genetic relationship between echinostome metacercariae in Lamphun Province, Northern Thailand.

Echinostoma hortense infection with enteritis diagnosed by upper gastrointestinal endoscopy in a dog.

An 8-year-old male Shiba dog presented with chronic vomiting and diarrhea. Upper gastrointestinal endoscopy revealed severe enteritis and infection of the duodenal mucosa with Echinostoma hortense. We performed therapy for parasites and enteritis. The therapy was successful for deworming and temporarily improved the symptoms, but the dog died soon thereafter. To the authors' knowledge, this is the first case report of an antemortem diagnosis of E. hortense infection in a dog.

Evaluation of praziquantel effects on Echinostoma paraensei ultrastructure.

Echinostomiasis is a food-borne, intestinal, zoonotic, snail-mediated helminthiasis caused by digenean trematodes of the family Echinostomatidae with seven species of the genus Echinostoma infecting humans or domestic and wildlife animals. Echinostoma paraensei is a peristomic 37-collar-spined echinostome belonging to the "revolutum group". Praziquantel (PZQ) is the drug of choice for treatment and control of human schistosomiasis and food-borne trematodiasis. In the present study we used scanning and transmission electron microscopy to further elucidate the trematocidal effect of PZQ on adult E. paraensei and confirmed that this trematode is a suitable model to study anthelmintic drugs. Hamsters infected with E. paraensei were treated with a single dose of 30 mg kg(-1) of PZQ. The worms were recovered 15, 30, 90 and 180 min after drug administration. There was a significant decrease in worm burden in the small intestine in the hamster-E. paraensei model at the intervals of 30, 90 and 180 min after the treatment. The worms displayed damage of the peristomic collar with internalization of the spines and erosion of the tegument of the circumoral head-collar of spines. Ultrastructural analysis demonstrated an intense vacuolization of the tegument, appearance of autophagic vacuoles and swelling of the basal infolds of the tegumental syncytium. There was no change in the morphology of cells from the excretory system of adult E. paraensei, however, there was an apparent decrease of stores of glycogen particles in parenchymal cells in PZQ-treated worms. Our results demonstrated that PZQ promotes surface and ultrastructural damage of the tegument of adult E. paraensei supporting the idea that this trematode may constitute a good model to investigate drug effects mechanisms in vitro and in vivo.

The combined influence of trematode parasites and predatory salamanders on wood frog (Rana sylvatica) tadpoles.

Predators can have important impacts on host-parasite dynamics. For many directly transmitted parasites, predators can reduce transmission by removing the most heavily infected individuals from the population. Less is known about how predators might influence parasite dynamics in systems where the parasite relies on vectors or multiple host species to complete their life cycles. Digenetic trematodes are parasitic flatworms with complex life cycles typically involving three host species. They are common parasites in freshwater systems containing aquatic snails, which serve as obligate first intermediate hosts, and multiple trematode species use amphibians as second intermediate hosts. We experimentally examined the impact of predatory salamanders (Ambystoma jeffersonianum) and trematode parasites (Echinostoma trivolvis and Ribeiroia ondatrae) on short-term survival of wood frog tadpoles (Rana sylvatica) in 150-L outdoor pools. Two trematode species were used in experiments because field surveys indicated the presence of both species at our primary study site. Parasites and predators both significantly reduced tadpole survival in outdoor pools; after 6 days, tadpole survival was reduced from 100% in control pools to a mean of 46% in pools containing just parasites and a mean of 49% in pools containing just predators. In pools containing both infected snails and predators, tadpole survival was further reduced to a mean of 5%, a clear risk-enhancement or synergism. These dramatic results suggest that predators may alter transmission dynamics of trematodes in natural systems, and that a complete understanding of host-parasite interactions requires studying these interactions within the ecological framework of community interactions.

Genetic characterization of Echinostoma revolutum and Echinoparyphium recurvatum (Trematoda: Echinostomatidae) in Thailand and phylogenetic relationships with other isolates inferred by ITS1 sequence.

Echinostomatidae are common, widely distributed intestinal parasites causing significant disease in both animals and humans worldwide. In spite of their importance, the taxonomy of these echinostomes is still controversial. The taxonomic status of two species, Echinostoma revolutum and Echinoparyphium recurvatum, which commonly infect poultry and other birds, as well as human, is problematical. Previous phylogenetic analyses of Southeast Asian strains indicate that these species cluster as sister taxa. In the present study, the first internal transcribed spacer (ITS1) sequence was used for genetic characterization and to examine the phylogenetic relationships between an isolate from Thailand with other isolates available from GenBank database. Interspecies differences in ITS1 sequence between E. revolutum and E. recurvatum were detected at 6 (3%) of the 203 alignment positions. Of these, nucleotide deletion at positions 25, 26, and 27, pyrimidine transition at 50, 189, and pyrimidine transversion at 118 were observed. Phylogenetic analysis revealed that E. recurvatum from Thailand clustered as a sister taxa with E. revolutum and not with other members of the genus Echinoparyphium. Interestingly, this result confirms a previous report based on allozyme electrophoresis and mitochondrial DNA that E. revolutum and E. recurvatum in Southeast Asia are sister species. Hence, the taxonomic status of E. recurvatum in Thailand, as well as in Southeast Asian countries needs to be confirmed and revised using more comprehensive analyses based on morphology and other molecular techniques.

Echinostome-induced mortality varies across amphibian species in the field.

Echinostomes are receiving increased attention because of their emerging parasite status in landscapes associated with human development and their ability to infect and kill many North American larval amphibians. While laboratory experiments have shown that echinostomes can cause extensive mortality in their amphibian hosts, their effect on tadpoles in the field is less clear. I conducted a controlled-infection field-enclosure experiment in 4 ponds to compare the effects of echinostomes on green frog (Rana clamitans) and gray tree frog (Hyla versicolor) tadpoles in the field. I measured tadpole growth, development, mortality, and infection intensity. Echinostome infection resulted in high mortality in green frog tadpoles and less mortality in gray tree frogs. However, metacercariae encystment rates were higher in gray tree frog tadpoles than in green frog tadpoles. The effect of echinostomes on mortality varies across amphibian species, with the result that some species may experience more extensive echinostome-induced mortality than others. Mortality as a result of echinostome infection in green frog tadpoles was similar to mortality observed in predation experiments.