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Articles • Rev. Bras. entomol. 70 (3) • 2026 • https://doi.org/10.1590/1806-9665-RBENT-2026-0023 linkcopy

Open-access Culturable gut bacterial microbiota of Catharsius dung beetles: Isolation, characterization, and in vitro effects of albendazole and ivermectin

AuthorshipSCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

This study investigated the culturable gut-associated bacterial microbiota of Catharsius javanus and Catharsius molossus and evaluated the potential non-target in vitro effects of albendazole and ivermectin using disc diffusion assays. Dung beetles were collected by hand-picking from three dung-dominated agro-rural pasture fields in District Gujrat, Pakistan, namely Chopala, Bhaddar and Chak. Gut-associated culturable bacteria were isolated under laboratory conditions, and isolates were identified through 16S rRNA gene amplification and sequencing. A total of ten culturable bacterial taxa belonging to the genera Bacillus, Enterococcus, Pseudomonas, and Proteus were identified. Host-associated differences were observed, as two bacterial species were recovered only from C. javanus, three only from C. molossus, and five from both host species. Albendazole produced stronger and more consistent inhibitory effects than ivermectin, while susceptibility varied among bacterial taxa. Overall, the study suggests that veterinary antiparasitic drugs may negatively affect selected culturable gut-associated bacterial taxa of dung beetles under in vitro conditions. These results highlighted the need for further studies on their ecological consequences for dung beetle gut microbiota and dung-associated microbial processes.

Keywords:
Veterinary pharmaceuticals; Microbial community; Ecotoxicology; Albendazole; Ivermectin

1. Introduction

The culturable gut microbiota associated with insects has become an increasingly important area of research in insect physiology, microbiology and ecology (Berasategui et al., 2016; Wang et al., 2020, 2025). The gut-associated bacteria in insects may contribute to host nutrition, digestion, detoxification, and immune defense (Jing et al., 2020; Yasika and Shivakumar, 2025). Additionally, these microbial communities may help insects adapt to changing environmental conditions (Berasategui et al., 2016). These microbial communities may also be involved in the production of enzymes, vitamins, pheromone-related compounds, and other metabolites that influence insect development, survival, and ecological performance (Ozdal et al., 2016). However, insect gut microbial communities are not fixed; their composition may vary according to host species, diet, developmental stage, habitat, and environmental conditions (Shukla et al., 2016; Ebert et al., 2021; Jácome-Hernández et al., 2023; Wang et al., 2025).

Dung beetles (Coleoptera: Scarabaeidae) are ecologically important insects, with more than 6,000 described species belonging to approximately 250 genera worldwide (Espinoza and Noriega, 2018; Lopes et al., 2023). These species provide essential ecosystem services, including dung removal, dung decomposition, soil nutrient cycling, soil aeration, seed dispersal, and the suppression of dung-breeding parasites and flies (Nichols et al., 2008; Chen et al., 2024). Because dung is a nutrient-rich but microbially complex and chemically variable resource, dung beetles are expected to maintain close associations with gut microorganisms that assist in digestion, nutrient acquisition, and adaptation to dung-based habitats (Pérez-Cobas et al., 2015; Thiyonila et al., 2018; Wang et al., 2020; Zhang et al., 2022). Previous studies have shown that gut-associated bacterial communities in dung beetles and other insects may be shaped by both host-related and environmental factors. Host species, feeding behavior, dung type, developmental stage, and local environmental conditions can all influence bacterial composition.

In some insects, including dung-associated beetles, microbial transmission between parents and offspring may also contribute to the persistence of particular bacterial taxa across generations (Rohner and Moczek, 2021; Poulin et al., 2023; Natta et al., 2025). Moreover, these communities may be influenced by dung type and local environmental conditions (Rivera-Duarte et al., 2025). Therefore, comparative studies of closely related dung beetle species collected from similar habitats can provide useful insight into whether culturable gut-associated bacteria are shared among hosts or show host-associated differences.

Veterinary antiparasitic drugs such as albendazole and ivermectin are widely used to control livestock parasites. After administration, these compounds or their metabolites may be excreted in dung, where they can affect non-target dung-dwelling organisms and dung-associated microbial processes (Sommer and Bibby, 2002; Ruhinda et al., 2025). Previous studies have reported lethal and sublethal effects of antiparasitic drug residues on dung fauna, including insects living in dung pats (Sommer et al., 1992; Tovar et al., 2023).

Such effects may reduce dung removal, alter dung degradation, and disrupt ecological functions performed by dung beetles and associated organisms (Tovar et al., 2023; Villar and Schaeffer, 2023). In addition, these residues may influence dung-associated microbial communities and related ecological processes (Edmonds et al., 2018; Belete et al., 2025; Saleem et al., 2025). However, direct experimental assessments of their effects on dung beetle gut bacterial isolates are scarce.

However, direct experimental evidence of the effects of these drugs on dung beetle gut-associated bacterial isolates remains limited. Most available studies on dung beetle gut microbiota have focused on broader microbial community composition (Ebert et al., 2021), whereas comparatively little attention has been given to culturable gut-associated bacteria and their susceptibility to veterinary antiparasitic drugs. Culture-dependent approaches do not represent the complete microbiota, but they are useful for isolating viable bacterial taxa that can be experimentally tested under controlled laboratory conditions (Suárez-Moo et al., 2020). Such studies are important for understanding whether commonly used veterinary drugs may exert non-target inhibitory effects on bacterial isolates associated with dung beetle guts (Sakhawat et al., 2026). In the present study, we investigated the culturable gut-associated bacterial microbiota of adults of two dung beetle species, Catharsius javanus and Catharsius molossus, collected from cattle dung in pasture sites in Gujrat District, Pakistan. By focusing on two congeneric dung beetle species, we aimed to determine whether closely related hosts harbor similar or distinct culturable gut bacterial taxa. In addition, we evaluated the in vitro non-target inhibitory effects of albendazole and ivermectin on the recovered bacterial isolates using the disc diffusion method. This study provides baseline information on the culturable gut-associated bacteria of Catharsius dung beetles and their susceptibility to selected veterinary antiparasitic drugs.

2. Materials and methods

2.1. Collection of dung beetles

The selected sites were located in Gujrat District (32.5731° N, 74.0789° E), Punjab, Pakistan represented dung-dominated agro-rural pasture landscapes with intensive livestock rearing, particularly cattle and buffalo farming (Fig. 1). The continuous availability of dung from these animals provides an important resource for sustaining dung beetle activity. Adult dung beetles belonging to the genus Catharsius, including Catharsius molossus and Catharsius javanus, were collected from three localities: Chopala (32.639444° N, 74.321944° E), Bhaddar (32.783611° N, 73.972778° E), and Chak (32.814167° N, 74.086389° E). Dung beetles are known to move between dung pats within the same pasture; however, movement between pastures generally decreases as the distance between them increases (Roslin, 2000).

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Figure 1
Map showing the sampling locations of Catharsius javanus and Catharsius molossus in Gujrat District, Pakistan.

2.2. Gut extraction and microbial isolation

Dung beetles were collected by handpicking from the study sites and randomly selected individual dung beetles for dissection (n=3) in each replicate. Gut tissues from Catharsius specimens were aseptically dissected under a laminar airflow cabinet using a CSM6 dissection microscope. Dissected gut samples were transferred into sterile 1.5 mL Eppendorf tubes and processed under aseptic conditions to minimize external contamination. For each 10 mg of gut tissue, 100 μL of preheated (60°C) 2% CTAB extraction buffer was added. The buffer consisted of 1 M Tris-HCl (pH 8.0), 1.4 M NaCl, 0.5 M EDTA (pH 8.0), and distilled water. The gut samples were centrifuged at 10,000 rpm for 5 min. The supernatant obtained after processing was subjected to serial dilution, and appropriate dilutions were spread on nutrient agar plates to isolate individual bacterial colonies. The plates were incubated at 37°C for 24 h. After incubation, colonies showing different morphological characteristics were carefully selected and streaked onto fresh nutrient agar plates to obtain pure cultures. The purified colonies were then transferred into Falcon tubes containing nutrient broth and incubated for 24 h in a shaker incubator. After bacterial growth, genomic DNA was extracted, purified, and stored at ≤ −20°C until further molecular analysis (Sanders, 2012). Based on colony morphology and molecular identification, ten distinct bacterial isolates were recovered from the gut tissues of Catharsius molossus and Catharsius javanus.

2.3. Molecular characterization of bacterial isolates

Molecular identification of bacterial isolates was performed by amplifying the 16S rRNA gene using universal primers. The forward primer was 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and the reverse primer was 1492R (5′-TACGGCTACCTTGTTACGACTT-3′). The amplified PCR products were visualized using horizontal agarose gel electrophoresis to confirm successful amplification of the target gene region.

2.4. Preparation of ivermectin and albendazole stock solutions

Ivermectin stock solution was prepared by dissolving the compound in absolute ethanol to obtain a 10% (w/v) solution. Briefly, ivermectin was dissolved in 80 mL of absolute ethanol with vortex mixing, and the final volume was adjusted to 100 mL, giving a stock concentration of 100 mg/mL. Albendazole stock solution was prepared in the same way by dissolving 10 g of albendazole in 80 mL of absolute ethanol, followed by volume adjustment to 100 mL to achieve a final concentration of 100 mg/mL.

Both stock solutions were stored at 4 °C in the dark until use. On the day of the experiment, working solutions of 10, 20, and 30 ppm were freshly prepared by diluting the stock solutions with sterile distilled water. These concentrations were selected to represent low-to-moderate exposure levels commonly used in antimicrobial sensitivity screening assays.

2.5. Antibacterial activity assay

The bacterial isolates were cultured on Mueller Hinton agar plates and incubated at 37°C for 24 h (Al-Aboghaubar et al., 2025). Antibacterial activity was evaluated using the disc diffusion method. Sterile paper discs of 6 mm in diameter were aseptically placed on the inoculated agar plates. Each disc was impregnated with 10, 20, or 30 ppm of albendazole or ivermectin. The plates were then incubated at 37°C for 24 h. After incubation, the zones of inhibition were measured in millimeters using a Vernier caliper. Measurements were taken along two perpendicular axes, and the mean value was recorded for each treatment. The experiment was arranged in a completely randomized design with three replicates.

2.6. Sequence and BLAST analysis

The sequencing results of the 16S rRNA gene, with accession number: HC01348097, generated by the Center of Excellence in Molecular Biology, were edited using Chromas Lite software. We only used high-quality sequence regions with sharp, clear, well-defined chromatogram peaks for further analysis. Edited sequences were compared using the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI) to assess sequence similarity with previously reported bacterial taxa. For phylogenetic analysis, 16S rRNA gene sequences of ten closely related reference bacterial species were obtained from NCBI. Multiple sequence alignment was performed using MEGA 12 software and the aligned sequences were saved in MEGA format.

Phylogenetic trees were constructed using the Tamura–Nei model through both neighbor-joining and maximum likelihood methods (Tamura and Nei, 1993). The resulting phylogenetic trees were used to illustrate the evolutionary relationships between the newly identified bacterial isolates and closely related previously reported taxa (Kumar et al., 2024).

2.7. Statistical analysis

The Shapiro–Wilk test was used to evaluate the normality of inhibition-zone diameter data, and Levene’s test was used for the evaluation of homogeneity of variances. Data normality was visually assessed using Q–Q plots and histograms before statistical analysis. The effects of drug type, drug concentration and bacterial species on inhibition zone diameter were evaluated using analysis of variance (ANOVA). When significant differences were detected at p < 0.05, Tukey’s honest significant difference (HSD) post hoc test was applied to determine pairwise differences among treatment groups. All statistical analyses were performed using R software version 4.5.2. Statistical interpretations were limited to the tested experimental conditions, and the absence of significant concentration-dependent effects was interpreted cautiously because of the limited concentration range and replication level.

3. Results

3.1. Collection of adult dung beetles

In our surveys, a total of 221 individuals belonging to Catharsius javanus and C. molossus were collected from three study sites, namely, Bhaddar, Chak, and Chopala. A relatively higher abundance of C. javanus was observed (116 individuals; 52.49%), whereas C. molossus showed a comparatively lower abundance (105 individuals; 47.51%). The highest abundance was recorded at Bhaddar with (96 individuals; 43.44%) followed by Chopala (65 individuals; 29.41%) and Chak (60 individuals; 27.15%).

3.2. Characterization of culturable gut-associated bacteria

The following results describe the culturable fraction of the gut-associated bacterial microbiota isolated from C. javanus and C. molossus under laboratory conditions. A total of ten distinct bacterial strains were isolated from the gut contents of both beetle species using nutrient agar medium. Colony morphology differed among isolates in terms of shape, color, surface texture and moisture content. These morphological differences were first used as a criterion for differentiating bacterial isolates before molecular identification (Table 1).

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Table 1
Colony characteristics of bacterial species isolated from Catharsius javanus and Catharsius molossus.

The dominant isolates belonged to the genera Bacillus, Proteus, Pseudomonas and Enterococcus. Conversely, metabolically versatile Gram-negative taxa, such as Proteus and Pseudomonas, were recovered from the gut microbiota of both beetle species. The two Catharsius species harbored distinct host-associated bacterial assemblages. Only two bacterial species were found exclusively in C. javanus, namely, Enterococcus faecalis and Bacillus arachidis and three species were found exclusively in C. molossus i.e., Bacillus cereus, Bacillus licheniformis and Proteus mirabilis. Five bacterial species, Bacillus haynesii, Bacillus paralicheniformis, Proteus penneri, Pseudomonas aeruginosa and Pseudomonas fluorescens were shared by both host species. These common taxa accounted for 50% of the total recovered isolates and may indicate the presence of a core culturable gut-associated bacterial community in the Catharsius species sampled (see Table 1).

The bacterial isolation from the two dung beetle hosts revealed both host-specific and shared culturable bacterial taxa in C. javanus and C. molossus. Only two bacterial species were isolated exclusively from C. javanus: Enterococcus faecalis and Bacillus arachidis. On the other hand, C. molossus carried three bacterial species that were not found in C. javanus: Bacillus cereus, Bacillus licheniformis and Proteus mirabilis. Five bacterial species were shared by both host species: Bacillus haynesii, Bacillus paralicheniformis, Proteus penneri, Pseudomonas aeruginosa and Pseudomonas fluorescens. The presence of these common bacterial taxa suggests the presence of a core culturable gut-associated bacterial community in both dung beetle species. The detection of exclusive taxa for each host suggests the potential for host-associated variation in gut bacterial composition (Table 2; Fig. 2).

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Table 2
Culturable gut-associated bacterial species identified in Catharsius javanus and Catharsius molossus.
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Figure 2
Venn diagram showing the distribution of culturable bacterial species associated with Catharsius javanus and Catharsius molossus.

3.3. Molecular identification by 16S rRNA gene amplification

The gut contents of the dung beetles were dissected, homogenized and cultured to obtain culturable bacterial strains which were then molecularly identified using PCR amplification of the 16S rRNA gene. All 10 isolates were successfully amplified, producing PCR products of approximately 1500 bp, consistent with the size of the bacterial 16S rRNA gene, were produced for all of them. All isolates showed distinct, well-defined bands on agarose gel electrophoresis, indicating successful amplification (Fig. 3). The results showed that the extracted genomic DNA was suitable for further sequencing and phylogenetic analysis.

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Figure 3
Agarose gel electrophoresis showing amplified 16S rRNA gene fragments (~1500 bp) from ten bacterial strains isolated from dung beetle gut samples.

3.4. Phylogenetic relationships of gut-associated culturable bacterial isolates

Sequence-based identification showed that the bacterial isolates shared a high level of sequence similarity (99–100%) with reference strains available in GenBank. Identified species included Proteus penneri, Proteus mirabilis, Bacillus cereus, Bacillus licheniformis, Bacillus paralicheniformis, Bacillus arachidis, Bacillus haynesii, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Enterococcus faecalis. Phylogenetic analysis based on partial 16S rRNA gene sequences further confirmed the close evolutionary relationships between the isolates and their corresponding reference species. All isolates clustered with their respective taxa with high bootstrap support, validating the molecular identification results. Maximum likelihood phylogenetic trees constructed using the Tamura–Nei model demonstrated that all isolates clustered with their respective taxa with high bootstrap support, thereby validating the molecular identification results. Overall, the trees consistently placed the isolates within the genera Bacillus, Proteus, Pseudomonas, and Enterococcus (Fig. 4).

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Figure 4
Phylogenetic tree based on partial 16S rRNA gene sequences showing the relationships among the isolates and closely related reference strains retrieved from GenBank.

3.5. Antibacterial activity of albendazole and ivermectin

Albendazole and ivermectin were tested for their antibacterial activity against the isolated gut-associated bacteria at concentrations of 10, 20 and 30 ppm using the agar disc diffusion method. Prior to the use of parametric statistical tests, the data were evaluated and the following assumptions were tested. The Shapiro–Wilk test confirmed that the residuals were normally distributed (W = 0.98, p = 0.63), and Levene's test showed that there was no difference in variance among the treatment groups (F5, 54 = 0.28, p = 0.63). The results showed that the data satisfied the requirements for parametric analysis. The main effect of drug type was significant for inhibition zone diameter (F1, 118 = 7.08, p = 0.009), suggesting that the two drugs exhibited different antibacterial activities. Bacterial species also had a significant effect (F9, 118 = 2.76, p = 0.0057), indicating species-specific differences in the susceptibility of bacterial species to the tested antiparasitic drugs. Drug concentration, however, did not have a significant effect, and there were no significant interactions among drug type, concentration, and bacterial species (p > 0.05). This suggests that neither concentration-dependent nor interactive drug effects were major factors influencing the antibacterial response, but rather that drug type and bacterial species were the main factors. The mean inhibition-zone diameters ranged between 17 and 25 mm and were similar among the various bacterial species and treatments (Table 3). In general, the inhibition zone was bigger in albendazole than for ivermectin for most of the bacterial taxa, indicating a more potent in vitro inhibitory effect. The heatmap visualization also revealed different species-specific patterns of susceptibility with Bacillus paralicheniformis being comparatively more susceptible and Enterococcus faecalis more resistant against both drugs (Fig. 5). These differences in inhibition zones, however, need to be interpreted as a relative indication of the in vitro sensitivity of bacterial taxa and not as a direct indication of in vivo antibacterial susceptibility.

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Table 3
Antibacterial activity of albendazole and ivermectin against culturable gut-associated bacterial species isolated from Catharsius javanus and Catharsius molossus.
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Figure 5
Heatmap illustrating the antibacterial activity of albendazole and ivermectin against bacterial species isolated from Catharsius molossus and Catharsius javanus.

4. Discussion

Dung beetles are ecologically important insects that provide numerous ecosystem services in terrestrial ecosystems. Both adult and larval stages of coprophagous dung beetles primarily feed on mammalian dung (Chen et al., 2024). These beetles obtain essential nutrients from herbivore dung, which contains plant-derived components such as cellulose, hemicellulose, and pectin that support their growth, development, and survival (Holter and Scholtz, 2007; Holter, 2016). Adult dung beetles use their mouthparts to selectively ingest smaller dung particles, thereby reducing fiber intake while consuming a microbe-rich diet (Hanski and Cambefort, 2014; Holter, 2016). In contrast, dung beetle larvae develop within brood balls that are relatively drier, coarser, and more cellulose-rich. Because larvae lack the hardened mouthparts that adults use to sieve dung particles, they may depend more heavily on gut-associated microbes for the degradation of complex plant-derived materials. In many dung beetle species, parents have been observed to deposit secretions, often referred to as a maternal gift, inside the brood ball where eggs are laid (Estes et al., 2013; Shukla et al., 2016). Larvae acquire beneficial microbes by consuming these secretions, which may assist in the digestion of more complex and fibrous food materials (Parker et al., 2019). Therefore, brood success may largely depend on beneficial microbial communities that facilitate the digestion of dung-derived organic matter (Chen et al., 2024).

Previously, the toxic threshold levels of tiamulin, olaquindox, metronidazole, and the anthelmintic ivermectin were evaluated in bacteria associated with springtails and enchytraeids. The potential risk of ivermectin to non-target species was expected at higher concentrations. Although the effects of anthelmintic drugs on dung-dwelling hexapods have been documented, available evidence remains limited (Sommer et al., 1992; Holter et al., 1993), highlighting the need for further investigation. A comparison of bacterial species recovered from dung beetles in our study and those previously recovered from other species is presented in Table 4.

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Table 4
Comparison of bacterial taxa recovered in the present study with previous reports from dung beetle gut microbiota.

In our study, we explored culturable gut-associated bacterial strains isolated from C. javanus and C. molossus. A total of ten culturable gut-associated bacterial strains representing both Gram-positive and Gram-negative taxa were recovered from the gut. The isolates were predominantly represented by members of the genus Bacillus, along with recurrent representatives of Pseudomonas and Proteus, indicating that spore-forming, metabolically versatile, and environmentally resilient bacteria constitute a major component of the culturable gut microbiota of Catharsius species (Raymond, 2017). Bacillus and Pseudomonas are widely distributed in natural environments and represent two of the most extensively studied bacterial genera in soil ecosystems.

Catharsius javanus uniquely harbored E. faecalis and B. arachidis, whereas C. molossus contained B. cereus, B. licheniformis, and P. mirabilis. Five bacterial species were shared between the two hosts, suggesting the presence of a core culturable microbiota within the genus Catharsius. These results indicate that although a common bacterial community is present, host-specific gut environments, dietary exposure, and ecological factors may contribute to the occurrence of unique bacterial strains (Natta et al., 2025). This diversity is consistent with previous studies of various dung-beetles, which have shown that these insect harbor complex and multi-functional gut microbiomes, presumably due to their microbe-rich and diverse diet (Beer et al., 2026). Bacterial community structure may be influenced by host species identity, which, together with environmental acquisition of bacteria from dung substrates, may contribute to bacterial community structure, as shown by the presence of both shared and host-specific bacterial lineages among closely related Catharsius species (Chen et al., 2024). Although the 16S rRNA gene was employed for taxonomic classification, species-level identification should be treated with care as the 16S rRNA gene may lack discriminatory power in some cases and cannot provide adequate resolution for closely related species, especially Bacillus spp. The clustering of isolates into multiple well-supported clades representing Bacillus, Proteus, Pseudomonas, and Enterococcus suggests that the dung beetle gut harbors a taxonomically diverse and phylogenetically structured bacterial community.

The observed intrageneric variation within phylogenetic clades also suggests possible strain-level diversification, which may enhance functional redundancy, adaptability, and resilience of the gut microbiome associated with dung beetles (Natta et al., 2025). Such phylogenetically structured diversity may contribute to dung digestion, nutrient assimilation, and host adaptation to environmental stressors. However, it is important to note that this study focused exclusively on culturable gut-associated bacteria, which represent only a subset of the total gut microbiota and may bias community composition toward fast-growing, easily cultivable, and environmentally tolerant taxa. Host phylogeny and diet are often cited as two major factors contributing to the diversity and structure of insect gut microbiome (Yun et al., 2014; Huang et al., 2021).

The present investigation integrates bacterial diversity analysis, antibacterial bioassays, and multivariate statistical approaches to provide insight into Catharsius-associated culturable gut bacterial communities and the potential non-target antibacterial effects of commonly used antiparasitic drugs. Despite clear differences among drug types and bacterial species, no statistically significant dose-dependent effect was detected within the tested concentration range. This implies that, within the tested concentration range, drug concentration may not have been the main factor affecting the antibacterial response, although a concentration effect cannot be excluded. These finding highlights that even comparatively low levels of antiparasitic drug residues can have measurable antibacterial activity, corroborating previous reports of microbial sensitivity to low-dose exposure to pharmaceuticals (Amer et al., 2021; Tovar et al., 2023). The prevalence of Bacillus spp. and the frequent occurrence of Pseudomonas spp. and Proteus spp. are consistent with previous studies on the microbiota of insects. The high abundance of Bacillus spp. and the frequent detection of Pseudomonas spp. and Proteus spp. are consistent with earlier studies on insect gut microbiota (Li et al., 2022). These taxa reflect the metabolic versatility, stress resistance, and capacity of these bacterial groups to survive in harsh environments (Engel and Moran, 2013; Turenne et al., 2015). The features probably enable these taxa to occur across habitats with varying nutrient levels in dung and to withstand various chemical stresses (Castro‐Arrazola et al., 2023). Disc diffusion assays revealed that albendazole and ivermectin exhibited detectable and quantifiable antibacterial activity against the culturable gut-associated bacterial isolates. Albendazole showed superior antibacterial activity with larger inhibition zone and stronger intensity observed in the heatmap visualization (Al-Aboghaubar et al., 2025). Ivermectin, on the other hand, exhibited greater variability in antibacterial activity, which may be attributed to variations in bacterial membrane permeability, efflux activity or intrinsic resistance properties of the bacteria (Baena-Díaz et al., 2018). The broader anti-bacterial activity of albendazole aligns with previous studies showing that some anthelmintic medications can have effects beyond their activity against helminths. Including antibacterial activity that may affect a range of bacterial taxa (Goyal, 2015; Chadha et al., 2024). The disc diffusion method offers a standardized screening method for evaluating the antibacterial activity but does not adequately mimic the physicochemical conditions of the dung beetle gut or field exposure conditions. The lack of a significant concentration-dependent response in the tested range of 10–30 ppm does not indicate biological insensitivity to increasing drug concentrations but may be due to limited statistical power, threshold-related effects, or drug-specific response patterns (Hajji et al., 2024). Moreover, the two anthelmintics, albendazole and ivermectin, are poorly soluble in water, so ethanol was employed as a solvent. The absence of a solvent control is an important limitation, which should be investigated in future studies as the concentrations of solvents in the working solutions were very low. Finally, while direct functional outcomes such as altered digestion or reduced host performance were not measured, the observed inhibition of culturable gut-associated bacteria indicates a potential mechanistic pathway through which antiparasitic residues may influence host–microbiome interactions in dung beetles.

5. Conclusion

This study demonstrates that albendazole and ivermectin exert measurable in vitro antibacterial effects on culturable gut-associated bacteria of Catharsius dung beetles, with albendazole showing comparatively stronger activity across taxa. A shared core culturable microbiota alongside host-specific bacterial assemblages was identified between C. javanus and C. molossus. Although limited to culture-dependent and in vitro conditions, these findings highlight the sensitivity of dung beetle-associated bacteria to veterinary pharmaceuticals and emphasize the need for future in vivo and culture-independent investigations to evaluate functional and ecological consequences.

Acknowledgements

The authors gratefully acknowledge the support of Dr. Ghulam Murtaza for providing access to the laboratory equipment.

Data statement

The entire dataset supporting the results of this study was published with the article.

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Edited by

  • Associate Editor:
    Sérvio Pontes Ribeiro

Publication Dates

  • Publication in this collection
    07 Sept 2026
  • Date of issue
    2026

History

  • Received
    05 Mar 2026
  • Accepted
    02 July 2026
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