Assessment of different methodologies for evaluating the virulence of biological agents against fire ants (Solenopsis spp.) under laboratory conditions

ABSTRACT

The genus Solenopsis, commonly known as fire ants, includes 196 species. These ants are considered pests due to the damage they cause in urban areas and agricultural systems. Their painful stingers hinder manual harvesting, and they feed on crops and engage in mutualistic interactions with aphids. As a result, there is considerable interest in identifying biological control agents and developing effective laboratory methods for evaluating their virulence. Such efforts are hindered by the fact that most available methodologies were originally designed for chemical insecticides. This study aimed to evaluate six different laboratory methodologies for testing the virulence of biological agents against Solenopsis spp. The tested approaches were: spraying water into trays with approximately 35 worker ants; pipetting water into containers with 30 worker ants; spraying water into containers with 30 worker ants; using a Potter spray tower to apply water, and treat containers with 30 workers; pipetting water onto distinct developmental stages of ants in Petri dishes; and applying water to microcolonies in Petri dishes. Additionally, various concentrations of culture media were assessed to determine their suitability for bacterial assays. Among the methods tested, the last two mentioned approaches presented fewer logistical challenges and were more likely to generate reliable data for fire ant control. The 25% Luria-Bertani medium was associated with the lowest worker mortality and is recommended as a negative control in bacterial experiments.

Keywords
laboratory biossay; Solenopsis spp.; biological control; virulence testing; culture media

INTRODUCTION

The genus Solenopsis, originally from South America, comprises 196 valid species and 22 subspecies (Bolton, 2024), with 161 of them occurring in the Neotropical region (Baccaro et al., 2015). This group includes ants commonly known in Brazil as lava-pés or formigas-de-fogo, and internationally referred to as “fire ants” or “red imported fire ants”. These ants are highly aggressive, especially when their colonies are disturbed or during foraging. Their sting can cause a range of reactions in humans, from localized pain to anaphylactic shock in sensitized individuals, because of their venom (Deshazo et al., 1984).

Fire ants are omnivorous and opportunistic, feeding on a wide range of plant and animal matter, as well as various household food sources (Campos et al., 2017). They also consume honeydew, a sugary exudate secreted by aphids, which reinforces their role as agricultural pests due to the mutualistic relationship that promotes aphid survival and crop damage (Morales; Beal, 2006).

Some fire ant species were accidentally introduced from South America to other parts of the world, particularly to the United States of America, through timber shipments transported by sea (Pitts et al., 2005). Among them, Solenopsis invicta Buren is the most problematic, causing serious health concerns and substantial agricultural losses in every country where it has become established. It is currently regarded as one of the most significant invasive insect species worldwide (Henshaw et al., 2005).

There are a few registered agricultural products specifically approved for the Solenopsis species control. Available control methods are mostly limited to disinfectant pest control products, either restricted for use by specialized pest management companies or freely available over-the-counter formulations registered with the Brazilian Health Regulatory Agency (ANVISA) for urban pest control purposes (Brasil, 2009).

Fire ant control is not well established yet, although numerous recommendations for managing these insects are readily available online. Pinto et al. (2019) conducted a survey of information from websites, videos, and scientific publications in Brazil regarding control strategies for fire ants. Among the recommended approaches, homemade methods were the most frequently cited (65%; n = 39), followed by chemical methods (31.67%; n = 19), and biological control (3.33%; n = 2).

Given the scarcity of studies evaluating biological agents against Solenopsis spp., it was necessary to test different methodologies to identify the most appropriate approach for assessing virulence under laboratory conditions.

MATERIAL AND METHODS

Collection of fire ant colonies in the field and maintenance in the laboratory

Solenopsis colonies were collected using a shovel in the outdoor area of Instituto Biológico, located in São Paulo, SP, Brazil. The colonies were placed in 20-L plastic buckets and transported to the laboratory. Worker ants, along with brood and the queen, were separated from the soil using a water displacement technique, in which water was slowly dripped onto the soil, causing the ants to float to the surface (Jouvenaz et al., 1980). To control the water flow, an intravenous set was connected to a 5-L plastic bottle, allowing a steady drip during the separation process.

To facilitate the capture of worker ants and queens, a small wooden box (10 × 6 cm) with a lid was used. A string was attached to one end of the box, while the opposite end was positioned on the soil where the ants were located. As the area was flooded, ants climbed to the water and soil surface and entered the box, which simplified their collection.

Artificial rearing was performed in polyethylene trays measuring 33 cm × 30 cm, with the inner walls coated with a 1:1 mixture of solid and liquid petroleum jelly to prevent ants from escaping. Disposable Petri dishes were lined with plaster of Paris, and their lids were covered with red cellophane to block light while still allowing visual monitoring of the ants. Each dish was placed individually in a rearing tray.

For laboratory maintenance, water, a sugar solution, and Tenebrio molitor (Coleoptera) larvae were provided to the ants ad libitum.

Evaluation of different methodologies for testing fire ants under laboratory conditions

Methodology 1: Tray method

Thirty-five medium worker ants were placed in polypropylene trays measuring 32 cm × 22 cm. The inner walls of the trays were coated with solid petroleum jelly to prevent escape. Inside each tray, a 9-cm Petri dish was placed, containing two T. molitor larvae, a diet composed of dehydrated beef liver, pineapple cake, and honey (1:1:1 ratio), and water supplied, ad libitum, in a test tube with cotton. The food was supplied only once, at the beginning of the experiment, and remained available throughout the entire seven-day period. Additionally, 1.5 mL of water was manually sprayed into each tray daily. Ten replicates were performed, and ant mortality was assessed daily by counting and removing dead individuals (Fig. 1).

Figure 1
Diagram of methodology 1 in trays.

Methodology 2: Filter paper in a container

Ten 10-cm diameter plastic containers were prepared; each one lined with filter paper. On the first day, two T. molitor larvae, the same diet as described above, and water (provided in a 2-cm-diameter cotton-topped cap) were placed inside the containers. Thirty medium worker ants were then introduced into each container, and 1.5 mL of water was pipetted onto the filter paper at the bottom. Each container was sealed with voile fabric to allow air exchange. Dead ants were counted and removed every 24 hours over seven days to assess mortality rates (Fig. 2).

Figure 2
Diagram of methodology 2 in containers with filter paper.

Methodology 3: Sprayed containers

Ten plastic containers (10 cm in diameter), each one lined with filter paper and supplied only with water via a test tube plugged with cotton (based on previous results indicating that ants did not consume the larvae or diet), were prepared. Thirty worker ants were introduced into each container, and 1.5 mL of water was manually sprayed over the ants. Each container was sealed with voile fabric (Fig. 3). Dead ants were removed every 24 hours over seven days to assess mortality.

Figure 3
Diagram of methodology 3 in containers by spraying.

Methodology 4: Potter tower

Ten plastic containers (10 cm in diameter) were prepared with petroleum jelly on their rims, lined with filter paper, and supplied with water. Thirty worker ants were introduced into each container, and 1.5 mL of water was sprayed onto the filter paper using a Potter spray tower (Fig. 4). Dead ants were counted and removed every 24 hours over seven days.

Figure 4
Diagram of methodology 4 using the Potter tower.

Methodology 5: Petri dish by life stage

Each experimental unit consisted of a 9-cm-diameter Petri dish lined with Whatman No. 1 filter paper, onto which 1.5 mL of water was pipetted. Ten ants were placed in each dish, grouped according to developmental stage (larvae, pupae, workers, and alates). Water was not provided in cotton-topped containers because previous methodologies demonstrated that the inoculated water sufficiently increased environmental humidity, which was adequate for the ants’ hydration needs. The dishes were sealed with plastic film to prevent ants from escaping. Each treatment included 10 replicates (Fig. 5). Mortality was assessed visually by counting dead individuals over seven days after water was pipetted.

Figure 5
Diagram of methodology 5 in Petri dishes by developmental stages.

Methodology 6: Microcolonies

Each experimental unit consisted of a 9-cm-diameter Petri dish lined with Whatman No. 1 filter onto which 1.5 mL of water was pipetted. Each dish contained a microcolony composed of 10 worker ants, five pupae, five larvae, and one alate (Fig. 6). Water was not provided in cotton-topped containers because previous methodologies demonstrated that the inoculated water sufficiently increased environmental humidity, which was adequate for the ants’ hydration needs. The dishes were sealed with plastic film to prevent escape, and each treatment was replicated 10 times. Mortality was visually assessed by counting dead individuals for up to seven days after water was pipetted.

Figure 6
Diagram of methodology 6 with microcolonies.

Evaluation of different culture media on fire ant workers under laboratory conditions

Fire ant workers were exposed to different culture media, Luria-Bertani (LB), tryptic soy broth, nutrient broth, and brain heart infusion, at three concentrations (25, 50, and 100%). Each experimental unit consisted of a 9-cm-diameter Petri dish lined with Whatman No. 1 filter paper, onto which 1.5 mL of the respective treatment was pipetted. Ten worker ants were introduced into each dish. The dishes were sealed with plastic film to prevent ants from escaping, and each treatment was replicated 10 times. Water was used as the control. Mortality was visually assessed daily over seven days by counting the number of dead individuals.

RESULTS AND DISCUSSION

In methodology 1 (tray method), most ants did not come into direct contact with water droplets, likely due to the large surface area of the tray and the increased mobility it afforded the specimens. A similar approach was employed by Zarzuela et al. (2012) to evaluate the control of Monomorium floricola using entomopathogenic nematodes. However, their setup involved 2,000–3,000 worker ants, at least five queens, and approximately 3 cm2 of brood (eggs, larvae, pupae) placed in a Petri dish positioned at the center of a Teflon-coated tray. This configuration encouraged the ants to remain inside the dish, increasing their exposure to the treatment. In contrast, methodology 1 in the present study involved only worker ants, which dispersed freely across the tray, reducing the likelihood of consistent contact with the applied treatment.

In methodology 2 (filter paper containers), the ants did not feed throughout the seven-day trial, likely due to the absence of larvae and a queen, which may have reduced trophallactic and foraging behavior. Fungal contamination of both the diet and T. molitor larvae was observed after a few days, further compromising food quality and attractiveness. Ants were also frequently observed climbing toward the voile fabric, exhibiting escape behavior and thereby limiting contact with the treatment. Moreover, the voile allowed for faster water evaporation, which likely reduced the duration of exposure to the applied treatment.

In methodology 3 (sprayed containers), similar escape behavior was observed, with ants climbing onto the voile fabric shortly after spraying and often avoiding contact with the filter paper for most of the seven days. Jouvenaz; Martin (1992) reported comparable behavior in container-based assays and addressed this issue by adding “escape chambers” to simulate more natural relocation dynamics, thereby improving the reliability of exposure assessments.

In methodology 4 (Potter tower), moisture was retained in the containers due to the presence of lids, although fungal contamination became evident after the fourth day. The petroleum jelly barrier effectively reduced climbing behavior, keeping the ants in closer proximity to the treated surface. The use of the Potter spray tower improved the likelihood of direct contact between the ants and water droplets, and such contact was observed. However, ants also exhibited self-grooming behavior, actively attempting to remove particles from their bodies, which has reduced treatment efficacy.

In methodology 5 (stage in Petri dishes), some difficulty was found during the placement of ants due to their agitation, but the confined space of the Petri dishes ensured consistent contact with the water treatment. No fungal contamination was observed throughout the seven days, and the plastic film seal effectively prevented escape. Pinto et al. (2019) employed a similar Petri dish setup with Solenopsis saevissima, sealing the dishes with adhesive tape after applying a plant-based compound. In their study, mortality was visually assessed after 24 hours post-treatment without reopening the dishes.

In methodology 6 (microcolonies), worker ants exhibited protective and grooming behaviors toward larvae, pupae, and alates following water application. Similar observations were reported by Jouvenaz et al. (1990), who tested Solenopsis sp. In glass culture tubes containing moistened sandy soil and microcolonies composed of 25–30 workers, larvae, and a queen. In their study, no handling difficulties were noted, and they observed up to 58% queen mortality, along with the high mortality of workers and immature stages. Workers displayed self-grooming and protective behaviors following treatment. Likewise, Drees et al. (1992) documented intense grooming activity in fire ants during field experiments with nematodes, noticing that workers actively removed nematodes from immatures, alates, and themselves.

Analysis of ant mortality across the six methodologies (Fig. 7) revealed the lowest rates in methodology 3. However, in subsequent trials using bacterial and nematode treatments (data not shown), mortality remained comparable to that of the control groups, suggesting that droplets may have dried too quickly and/or that ants effectively avoided contact with the treated surfaces.

Figure 7
Mortality (%) of Solenopsis spp. worker ants exposed to different methodologies (1, 2, 3, 4, 5, and 6).

In the tests using culture media, the 25%-LB medium resulted in the lowest mortality among worker ants (Fig. 8). No prior studies comparing different culture media for biological control assays were found in the literature. This experiment was necessary due to the high mortality observed with the undiluted media, which may be attributed to the strong odor or specific compounds present in their composition.

Figure 8
Mortality (%) of Solenopsis spp. worker ants were exposed to different concentrations (25, 50, and 100%) of four culture media (Luria-Bertani, tryptic soy broth, nutrient broth, and brain heart infusion) under laboratory conditions.

CONCLUSIONS

Among all methodologies tested, methodologies 5 and 6, using isolated developmental stages and microcolonies in Petri dishes, proved to be the most practical and consistent, with fewer logistical constraints and greater potential to yield realistic and reliable data for evaluating biological control strategies against the fire ants. Regarding the culture media, the 25%-LB medium resulted in the lowest mortality among worker ants and is therefore recommended as a suitable negative control for future bacterial assays.

ACKNOWLEDGEMENTS

Not applicable.

• Peer Review History: Double-blind Peer Review.
• FUNDING
  Fundação de Amparo à Pesquisa do Estado de São Paulo
  Grant no.: 2022/14952-2

AVAILABILITY OF DATA AND MATERIAL

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Edited by

• Associate Editor:
  Silvia Galleti https://orcid.org/0000-0002-0745-5716

Publication Dates

• Publication in this collection
  15 Sept 2025
• Date of issue
  2025

History

• Received
  10 Nov 2023
• Accepted
  01 July 2025