Parasitic helminths of alien invasive anurans in Butuan City, Northeastern Mindanao, Philippines

Site 1, located in Brgy. Libertad (8°56′55.0″N and 125° 30′ 44.9″E) is an urbanized barangay with numerous commercial buildings, subdivisions, and a few agricultural areas. Site 2 is situated in Brgy. Tungao (8°46′47.6″N and 125°30′40.0″E), a small rural barangay that serves as an agroforestry site with vegetation that includes falcata (Paraserianthes falcataria), rubber tree (Havea brasiliensis), coconut (Cocos nucifera), rambutan (Nephelium lappaceum), and lanzones (Lancium parasiticum). Site 3, Brgy. Masao (9°00′10.2″N and 125°29′23.0″E) is a coastal area with human settlement, beach resorts, and an adjacent river system (Torralba et al., 2022). Within each barangay, extensive and opportunistic sampling was employed in collecting invasive anurans from 7:00 to 9:00 P.M., using handling equipment such as gloves and wooden sticks, then placed inside a net bag (DENR-BMB 2017; Jabon et al., 2019). Available taxonomic keys, published articles, and other online sources were utilized for identifying the captured IAS (Solania & Fernandez-Gamalinda, 2018; Diesmos et al., 2015).

Fig. 1.

The collected samples were sourced from three barangays in Butuan City, Agusan del Norte, Philippines. Butuan City is a coastal, highly urbanized area in the Caraga region. As of 2020, Butuan City has a population of 372,910 and covers an area of 81,662 hectares. It serves as the region's administrative, commercial, and industrial center and is located in the northeastern part of the Agusan Valley, Mindanao (Mapa, 2021).

The methods of Akani et al. (2011) and Imasuen et al. (2012) were utilized to identify the parasites obtained from the collected IAS. Individuals were first pitted to immobilize. It is a technique used to destroy the brain and is relatively painless to the frog (Back et al., 1915). All anurans dissected were processed humanely. The samples were then eviscerated by opening a longitudinal incision from its snout to the vent, and internal organs were collected. The heart, lungs, liver, stomach, intestine, and body cavities were sliced open in the longitudinal section to examine endoparasites. The parasitic helminths were taken from samples and kept in a 0.96 % NaCl solution before being cleaned in PBS and preserved in 70 % ethanol. Before being stored in 70 % ethanol, collected parasitic helminths were placed and given a brief bath in warm water to soothe and bolster the worms. In order to allow the specimens to discharge eggs that could otherwise cover some organs before they are preserved in 70 % ethanol, trematodes were additionally relaxed in warm water. Parasitic helminths were collected and kept in vials with the appropriate labels. For identification, helminth parasites were put on slides. A trinocular stereo zoom microscope (Leica, China) and a compound microscope (Nikon, Tokyo) were used to examine all processed permanent slides. Stage, ocular micrometers, and an optic camera (OptixCam, OCS-SK2-5.2X, China; ToupView software) were used to measure the specimens. Typical reference sources were used to identify the parasites (Khalil et al., 1994; Imasuen et al., 2012).

Collected data on endo-parasites were tested using SPSS v. 20.0 software, applying a 95 % confidence level. Differences in prevalence among age, sex, and habitat of the collected invasive anuran species were analyzed using the chi-square test of independence. Parasites' mean intensity among invasive anuran species was also computed. The Shapiro–Wilk test was used to determine the normality of the data collected in the study. The Kruskal-Wallis test was used to compare the intensity of parasite infection among anuran species and across habitat types of selected barangays. Pearson correlation test was used to associate the parasite's infection intensity with the host's weight and length. Mann–Whitney U-test was used to compare the parasite intensity between sex and ages of invasive anuran (Estaño et al., 2020).

The following formula were also used in this study: Prevalence=# of infected samplestotal number of samples×100Mean Intensity=# of parasite in anuranstotal number of infected anuran samples \matrix{{{\rm Prevalence} = {{\# \,of\,infected\,samples} \over {total\,number\,of\,samples}} \times 100} \hfill \cr {{\rm Mean\,Intensity} = {{\# \,of\,parasite\,in\,anurans} \over {total\,number\,of\,infected\,anuran\,samples}}} \hfill \cr}

Three invasive anuran species were obtained from the three selected barangays of Butuan city (Table 1), namely the cane toad (Rhinella marina, Linnaeus 1758), Chinese tiger frog (Hoplobatrachus rugulosus, Weigmann 1834), and Banded Bullfrog (Kaloula pulchra, Gary 1831). The chi-square test of independence revealed a significant relationship between species and age (P = 0.002) among habitat types.

In the Philippines, invasive species like R. marina, H. rugulosus, and K. pulchra have proliferated rapidly in all habitat types. The distribution of these species in the study area was due to patchy environmental conditions, dispersal along environmental corridors, anthropogenic activities, and water bodies in all habitat types. R. marina is the commonly found species reported to compete with native frogs in breeding sites (Joshi, 2006). It is now widely distributed from Luzon to Mindanao. Climate is one of the factors that affect anuran activity. Rainfall and temperature influence the reproductive activity and the abundance and species richness of the anurans due to the quality and availability of breeding sites (Garcia, 2019; Luna-Gomez et al., 2017).

Rhinella marina and Hoplobatrachus rugulosus were the only observed species with parasite infection, and a total of 247 parasites were recovered from their organs. However, no recorded parasite infection in the examined samples of K. pulchra is supported by the findings of Zhang et al. (2020). This suggests that K. pulchra may be insensitive to parasite infection. Among the identified parasite species, Spirometra sp. was the most prevalent 17.58 % (95 % CI 11.12–26.67), followed by Echinostoma sp. 16.5 % (95 % CI 10.25–25.43), Rhabdias bufonis 14.3 % (95 % CI 8.543–22.92), Cosmocerca sp. 6.6 % (95 % CI 3.057–13.65), and Strongyloides stercoralis 3.30 % (95 % CI 1.12–9.24), respectively.

The most prevalent parasite among all the collected parasites from various habitat types was found to be Spirometra sp. This finding is consistent with a recent report from the Hainan Province of China (Fu et al., 2022). The presence of Spirometra sp. in all habitat types in this study is attributed to the presence of water bodies where the parasites deposit eggs inhabited by copepods, including plankton. Consequently, copepods become infected with Spirometra sp. plerocercoid larvae, which are then consumed as part of the anuran diet (Box et al., 2021). The second most prevalent parasite recovered from the examined samples was Echinostoma sp. High rates of Echinostoma infection have been reported among Filipinos, which is believed to be caused by the consumption of secondary hosts, such as freshwater snails and fish, known to contain metacercariae (Belizario et al., 2007).

The prevalence of Echinostoma sp. in this study was attributed to the snails found in the agroforest and freshwater habitats near the residential area. Rhabdias bufonis, the third most prevalent parasite detected, has also been reported in several studies to have a prevalence among Bufonidae family members (Morsy et al., 2018). The prevalence of R. bufonis in both the agroforest and residential areas is due to the availability of snails, the intermediate host of the parasites that serve as food items of invasive anurans in these areas. Cosmocerca sp., the fourth most prevalent parasite, was found to have a high intensity and prevalence in agroforests. Cosmocerca sp. is a bioindicator and bioaccumulator of pesticides that inhabits moist soil and dry areas with a direct life cycle through skin penetration of its host (Okeagu et al., 2022 & Saad et al., 2009). Strongyloides stercoralis, the least prevalent parasite recovered from invasive anuran species, is a soil-transmitted parasite that has been shown to affect people's health worldwide (Buonfrate et al., 2020). The first recorded case of Strongyloides is in anurans (Petterson-Kane et al., 2001). In this study, the recorded S. stercoralis was manifested in anurans captured in an agricultural area due to the chemicals used in farming, which aided its free-living life cycle (Hendrix et al., 1987).

The parasitological survey involved collecting and examining 91 specimens of invasive anuran species, as summarized in Table 2. The results showed an overall prevalence of 46.2 % of parasitic helminths, with an average intensity of 5.9 ± 1.8 worms per host. Among the invasive species of anurans, H. rugulosus harbors the highest prevalence with 70 % (95 % CI 39.68–89.22) and a mean intensity of 9.14 ± 0.81, and R. marina 43.75 % (95 % CI 33.41–54.66) with a mean intensity of 5.23 ± 0.12. Statistical analysis showed a significant difference in prevalence among invasive anuran species (P = 0.001). H. rugulosus is an edible frog that plays an economic role in several Asian countries. However, intensive farming causes poor environmental conditions and causes health problems. A study from Thailand revealed that all stages of H. rugulosus (91.72 %) were affected by parasites (Phadee & Julawong, 2019). Similarly, a recent study from Metro Manila, Philippines, reported a parasite infection prevalence of 46 % in R. marina (Duco et al., 2020). The dispersal of invasive anurans also contributes to their susceptibility to parasitic infection, as frequent movement compromises their immune system and exposes them to a wider range of pathogens (Brown & Shine, 2014).

In terms of habitat type comparison, the highest prevalence was observed in residential areas with 56.7 % (95 % CI 39.2–72.62) and mean intensity of 4.59 ± 0.17, followed by agroforests with 46.7 % (95 % CI 30.23–63.86) and mean intensity of 6 ± 0.20, and then agriculture 35.5 % (95 % CI 21.12–53.05) and mean intensity of 7.72±0.47. However, statistical analysis revealed no significant difference in prevalence and mean intensity at p=0.990 and p=0.214, respectively, among the three habitat types. Residential development has been identified as a leading cause of land-use change, which poses significant implications for biodiversity conservation (Pejchar et al., 2015). In the urban areas of the Philippines, high prevalence of gastrointestinal parasites was reported in anurans (Ybanez et al., 2018). The process of urbanization also reduces the number of hosts and increases the vector population of anurans (Aisen et al., 2021).

In contrast, agroforestry is a land management strategy that involves the integration of woody vegetation and livestock to diversify production systems (Brown et al., 2013). In Brazil, a prevalence of 52.14 % helminth parasites has been reported in cocoa agroforestry (Kersul et al., 2020). In this study, agroforest had a recorded 46.7 % (95 % CI 30.23–63.86) parasite infection due to the high biodiversity in the area, where there is close interaction between anurans, reptiles, and rodents. Agroforestry helps to maintain bio-diversity by creating diverse habitats and reducing environmental disruptions caused by agricultural activities (Rosenstock et al., 2019). However, agricultural practices that involve pesticides and herbicides can alter the environment and contribute to the development of free-living parasite forms due to their immunosuppressive and inhibitory effects (Aisien et al., 2021). In the Philippines, farm areas in Luzon have recorded a prevalence of 40 % to 72 % soil-transmitted parasites. Also, the use of manure as fertilizer and wash water has been found to be significantly associated with soil parasite contamination (Paller & Babia-Abion, 2019; De Guia & Flores, 2019).

Mann-Whitney U-test showed that the recorded parasitic helminth infection between sexes was insignificant (p= 0.911). The high prevalence of the parasitic helminths recovered in male hosts with 49 %, (95 % CI 33.01–64.43) compared to females with 45 % (95 % CI 32.39–57.59). The highest infection rate may be due to their higher dispersal activity. However, the female host had a higher mean intensity (6.76 ± 0.18) than male (4.5 ± 0.20) invasive anurans. This report agrees with the study of Kia et al. (2018), where a 92 % prevalence rate of gastrointestinal parasites from male Hoplobatrachus sp. and 50 % in females were reported. Another reason is that male frogs are more malnourished than females, especially during the dry season. It results in high activity, making them susceptible to parasitic burden (Kia et al., 2018). The test also showed that the distribution of parasites is the same among ages (p=0.096). As the age of the host increases, its vulnerability against infection also increases. It increases the burden of parasite infection, especially in males, due to their shorter life expectancy than females. In juveniles, the low prevalence of infection is due to their less exposure to pathogens and acquired immune response as they grow and develop (Bradley et al., 2019). In this study, adult hosts have a 48.5 % (95 % CI 33.01–64.43) prevalence rate higher than juvenile hosts with a 33.3 % (95 % CI 15.6–50.86) prevalence rate. The results of age influence on the adult showed a higher prevalence and mean intensity compared to juvenile. The higher infection is attributed to the exposure and vulnerability of adult hosts to parasitic helminths. The high infection in adult invasive anurans has more prolonged exposure to acquired parasitic helminths infective stage in the environment compared to juveniles living for a short time than adults. Adult invasive anurans have a more extensive niche availability in most parasites due to their behavior and ability to travel, seeking food accessibility (Tujan et al., 2016; Estaño et al., 2020).

The statistical analysis using Pearson's correlation test revealed no significant correlation between the prevalence of parasite infection and the weight (r = 0.024, P = 0.821) and length (r = −0.031, P = 0.771) of the host in various habitats. However, a weak positive correlation was observed between the body weight of the host and Cosmocerca sp. (r= 0.224, p= 0.033), and a negative weak correlation was found between the length of the host and Strongyloides stercoralis (r = −0.247, p= 0.018). The recorded correlation of Cosmocerca sp. and S. stercoralis to the weight and length of the host is that the larger the host, the more space and resources it can provide to parasites (Poulin, 1997). Nematodes are also the most common anuran parasites, and it was associated with a larger host that harbors the richest parasite fauna (Campiao et al., 2015). The correlation between invasive anuran's body weight, body length, and parasitic helminth intensity will increase as the body length and weight of the invasive anuran hosts increases. The findings of this survey concur with the study of parasitic nematodes in rats by Castillo and Paller (2018) and Estaño et al. (2020) that large-bodied hosts provide more niches and nutrients for parasitic helminths. The occurrence of parasitic helminths in invasive anurans might exhibit a high possibility of anurans to human transmission of parasitic helminths that threaten public health. The existence of these invasive anuran species threatens both the native wildlife and other animals with its parasite infection. Areas with high anthropogenic activities exhibit high prevalence and infection. Therefore, authorities must implement strategies to control its fast-growing population, spread awareness to the locals about these invasive anuran species and their parasites, and improve their sanitation practices. Due to the lack of published information on the parasitic helminths in invasive anurans in the Philippines, monitoring and surveys should be done in other areas to assess the potential impact of parasitic helminths on public health and veterinary importance.