Experimental modulation of ants’ external microbiome revealed symbiont-mediated survival and behavioral modifications in workers of Solenopsis invicta

Experimental modulation of ants’ external microbiome revealed symbiont-mediated survival and behavioral modifications in workers of Solenopsis invicta | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Experimental modulation of ants’ external microbiome revealed symbiont-mediated survival and behavioral modifications in workers of Solenopsis invicta Bamisope Steve BAMISILE, Junaid Ali SIDDIQUI, Lei NIE, Atif IDREES, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6990545/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Feb, 2026 Read the published version in International Journal of Tropical Insect Science → Version 1 posted 12 You are reading this latest preprint version Abstract A wide range of interactions between insects and their associated microbial communities have been documented, with profound implications for host characteristics such as development, biology, and behavior. While much emphasis has been placed on bacterial endosymbionts, the mutual interactions between insects and their external symbionts are often overlooked. In this study, we investigated the potential of the external microbiome to mediate survival and behavioral changes in the workers of red imported fire ants (RIFA). Using culture-based methods, the bacterial species present on the cuticle of the ants were isolated and identified. Experimental manipulation of ant exosymbionts was achieved through the treatment of workers with bacterial monocultures, antibiotics, or a combination of both. Artificial modification of ant cuticle bacterial symbionts revealed significant changes in survival rates and behavioral patterns. The removal of ants’ cuticle exosymbionts induced about 89% cumulative mortality within 10 days of treatment, significantly higher than that observed in other treatments and the control group. Similarly, artificial manipulation of ant cuticle bacterial symbionts impaired the ants’ ability to be recognized by their nestmates, as worker ants with an altered cuticle experienced a higher rejection rate compared to those with an intact external microbiome. In addition, foraging activities were affected, including the workers’ ability to kill prey, search for food, and the weight of food carried over a given duration. These results revealed that cuticular bacteria influence both survival and certain social behaviors in RIFA. Understanding the diversity and potential use of these exosymbiotic bacteria would provide insights into promising biological strategies for RIFA management. Formicidae social insects physiology mortality cuticle bacteria invasive species Figures Figure 1 Figure 2 Figure 3 Figure 4 Key Message Effects of cuticle bacteria modification on the survival and social behavior of red fire ants were examined. Removal of exosymbionts caused up to 89% mortality within 10 days. Ants with altered symbionts faced higher rejection by their nestmates. Foraging behaviors were impaired by bacterial manipulation. Findings suggest biocontrol potential via cuticular microbiome. Introduction The red imported fire ant Solenopsis invicta Buren has earned its reputation as a notorious invasive species due to its ability to cause harm to humans, crops, livestock, and wildlife through painful bites and stings (Kafle et al., 2011 ; Nie et al., 2019 ). These highly aggressive ants are of huge economic importance and have been documented to be native of South America. The extensive spread of these noxious ants in various parts of the world has been a cause for serious concerns. The red imported fire ant (RIFA), being a globally invasive pest, has presented a significant challenge to China's environment since the first discovery in Wuchuan, Guangdong Province in 2004 (Li et al., 2016 ; Nie et al., 2019 ). Since that period, RIFA had spread to a total of 12,680 hectares of land in the country, with an expansion rate of 26.5–48.1 km/year (Lu, 2014 ). RIFA has since become prevalent in most Southern Provinces of China and continues to expand its range. The introduction of the fire ants in China has resulted in a major decline of arthropods in tropical and subtropical regions of the country. Surveys conducted in China have discovered that wherever RIFA occur, they caused a severe reduction in the abundance and diversity of native ant species as they have successfully colonized and established in both human-altered and unaltered habitats. This devastation is most evident in orchards, lawns, abandoned lands, grasslands, and residential areas (Lei et al., 2019 ; Li et al., 2016 ). Aggressive invasive ant species like S. invicta have led to a drastic reduction in arthropod diversity and the local distribution of resident species, with ant species richness decreasing by over a third in some habitats (Jun et al., 2012 ). Apart from this, the presence of RIFA in an area can have notable negative effects on the diversity index and evenness index of ant communities within a radius of three meters of its mounds. To combat this, it is essential to obtain detailed information on the biology and ecology of these fire ants. In response to the spread of S. invicta , China has taken the initiative of investing in numerous research establishments to understand the ecology and biology of fire ants (Lei et al., 2019 ). Living organisms, including social ants, commonly come in contact with environmental microbes, and several scientific studies have explored the various roles microbial organisms play in their hosts, including influencing different functions in their immune, digestive, circulatory, endocrine, and nervous systems (McFall-Ngai et al., 2013 ). In the past, the mutual relationships between insect hosts and environmental bacteria have garnered much less attention in the field of behavioral ecology; however, emerging studies have provided evidence to suggest that host-associated microbial communities could offer far more advantages than hitherto presumed (Ezenwa et al., 2012 ; Grenham et al., 2011 ). These findings shed light on the possibility that the composition of these microbial communities could offer considerable advantages to insect hosts. Insects have therefore been observed to demonstrate various levels of reliance on symbiotic bacteria for their primary functions. Nevertheless, the majority of the currently available studies are limited to pathogenic or beneficial bacteria inhabiting the gut of insects (Bamisile et al., 2023 ; Parks et al., 2018 ). In addition to the well-studied bacterial endosymbionts inhabiting various internal organs of insects, scientific findings have shown that external bacteria present on the cuticle of ants can as well influence basic insect functions, including survival, nestmate recognition, foraging, and oviposition (Douglas, 2015 ). These symbiotic bacteria are fundamental for the development and existence of host organisms, aiding in the degradation of food, energy production, vitamin synthesis, and regulation of immune reactions (Hamby and Becher, 2016 ; Łukasik et al., 2013 ). Thus, research has repeatedly highlighted the several ways in which bacteria on the ant’s cuticle can affect the life of the hosts, including beneficially acting as a protective component against pathogens. For RIFA, numerous studies have been conducted to unravel the underlying mechanisms behind their huge invasion success (Chen et al., 2014 ; Hu et al., 2018 ; Wilder et al., 2011 ; Yang et al., 2009 ). There are suggestions that their competitive ability is highly dependent on several important factors, such as colony size, resource finding, monopolizing capabilities, and the level of aggression towards non-nestmates (Holway et al., 2002 ). In addition, McFall-Ngai et al. ( 2013 ) had suggested that in social animals, the most important ecological interactions involve internal, epidermal, and environmental microbes, and not those that involve nestmates, competitors, or predators. To assess the costs and benefits of sociality for any specific social group, it is necessary to further explore the relationships between individuals and the microorganisms specific to their environment. Owing to the ubiquity and diversity of bacterial communities, there is a need to expand our understanding of insect–bacterial interactions, as it would help to broaden our general knowledge of their biology, adaptability, and social responses. In the current study, we tested the hypothesis that artificial modification of external bacterial symbionts of the RIFA would affect their survival and social behavior, including feeding, foraging aggressiveness, and nestmate recognition. It was predicted that RIFA workers with an altered external microbiome would be rejected by their nestmates if the external microbiome plays a role in nestmate recognition. We experimentally modulated the external microbes on the cuticles of RIFA workers via the topical application of bacterial monocultures and/or antibiotics. This study helps to understand the potential roles of the exosymbiotic microbiome in enabling the host to survive and recognize its nestmates. This could also serve as a background for future studies to understand their roles in the host defense against biotic stressors. The current study adds to the available data on the importance of insect-associated external symbionts in the survival and social behavior of the hosts. Materials and Methods Field collection and laboratory rearing of S. invicta : Fire ant colonies were collected from lawns across different counties in Guangdong province of China. The social form of RIFA used in the current study was polygene. Mound excavation was done with the help of shovels, digging at approximately 10–15 cm below the ground surface, and transferring the mound soils into plastic boxes with a lid (43 × 32 × 25 cm). Between each digging, the shovels were disinfected with 70% ethanol and allowed to dry. Insect boxes were transported to the laboratory, and the removal of ant colonies from the mounds was achieved via the traditional dripping-flooding method. Thereafter, the extracted colonies were transferred into clean insect boxes (40 × 28 × 12 cm), with the walls coated with Fluon to prevent the ants from escaping. The ant populations were fed with water, 10% w/w sugar water, and larvae of mealworm Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae). The colonies were reared in the laboratory for two weeks before the commencement of the experiment. Laboratory conditions during the period were maintained at 24 ± 2°C, 75% relative humidity, and 14:10 h L:D. Solid and Liquid media preparation: Luria–Bertani (LB) broth deployed for bacteria isolation from ant body consists of yeast extract (5 g), sodium chloride (10 g), tryptone (10 g), and 1 L of sterile distilled water. For LB solid media, 15 g of agar was added and supplemented with 50 mg/L nystatin (Sangon Biotech, Shanghai, China), to prevent fungal growth on bacteria culture plates. Isolation of cuticle bacteria from S. invicta workers: From each colony, about 12 medium-sized workers of S. invicta were randomly selected. The ants were transferred into sterile 15 mL conical tubes and temporarily placed in a fridge to immobilize them. Cuticle bacteria were collected by gently rubbing the surface of ants with sterile damp cotton swab. Thereafter, the cotton swabs were placed in fresh 15 mL conical tubes containing 5 mL of sterile LB broth and vortexed for 3 min at 1000 × g. Afterwards, from the stock suspension, three serial dilutions were made, while 100 µL of the third serial dilution was evenly spread on LB agar plates in triplicate using sterile disposable cell spreader (Changde Bkmam Biotech, Changde, China). The inoculated plates were kept and allowed to dry under aerobic conditions before being transferred into a BOD incubator (BS-1E, China), and incubated at 25°C for 72 h. Conventional colony forming unit (CFU) assay was conducted to identify the emerging bacteria. Single colonies with distinct morphological features were re-streaked multiple times on freshly prepared LB agar plates (approximately 3–4 times) using sterile disposable inoculating loops (Biologix Group Ltd. Jinan, China). Preparation of bacterial liquid monocultures: Following multiple sub-cultures until bacterial monocultures were obtained, the individual distinct colonies were inoculated into LB broth contained in 50 ml round-bottom centrifuge tubes using sterile micropipette tips. The tubes were shaken overnight on a rotary shaker (ZD-85; GTCS, Shanghai, China) at 200 rpm and 30°C. The homogenized suspensions were immediately used for DNA extraction, or preserved on 25% sterile glycerol stocks and store at -80°C for future use. DNA extraction, molecular and phylogenetic characterization of bacterial isolates: For DNA extraction, TIANamp Bacterial DNA kit supplied by the manufacturer (Tiangen Biotech, Beijing, China) was used. The full-length of the bacterial small subunit 16S rRNA gene was amplified using primer pairs (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R: 5′-ACGGCTAACTTGTTACGACT-3′). The PCR reaction mix and thermocycling conditions were similar to those described by Cheng et al. ( 2019 ) and Bamisile et al. ( 2023 ). Bidirectional sequencing of bacterial isolates was conducted by Sangon Biotech, Guangzhou, China. Manual editing was done for the obtained bacterial sequence traces using BioEdit v 7.1.9., while reference sequences were downloaded from the GenBank database of National Center for Biotechnology Information (NCBI) ( http://www.ncbi.nlm.nih.gov/ , accessed on 12 March 2023). The reference sequences were used to manually edit and align the target sequences, while phylogenetic analysis using Neighbor-Joining method analysis based on Kimura 2-parameter model was performed via MEGA v. 11. The robustness of branches was estimated by bootstrap analysis with 1000 repeated samples from the data. Culturing Axenic, Gnotobiotic, and Symbiotic workers of S. invicta : From the characterized bacterial strains (Supplementary Table 1), four strains ( Vagococcus fluvialis D121, Myroides odoratus Z442, Providencia rettgeri Z511, and Pseudomonas monteilii G911) were selected based on their relative abundance for further analysis. The bacterial strains and four antibiotic treatments were selected for the artificial manipulation of the RIFA external microbiome. Three treatment groups of RIFA workers (axenic, gnotobiotic, and symbiotic) were achieved through the topical application of antibiotics, microbial monocultures, or both. The axenic ants include treatment with either of penicillin 100 µg/ml, streptomycin 100 µg/ml, gentamicin 150 µg/ml, or tetracycline 50 µg/ml. The symbiotic ants were nestmates treated with any of the four selected bacterial strains. Meanwhile, to create a single-species bacterial microbiome on the ants’ cuticle (gnotobiotic ants), ants were first treated with gentamicin 150 µg/ml, prior to exposure to each of the four bacterial strains. On the other hand, the control group consisted of ants treated with either of sterile distilled water (SDW) or sterile LB broth (SLB), and untreated nestmates (NM) (Supplementary Table 2). For all treatment and control groups, ants were treated by dipping in 0.5 ml of suspensions or monocultures for 3 s. Excess liquids were removed from the body of treated ants using absorbent papers. The entire experiment was set up in a completely randomized design with 12 combinations comprising three groups and four treatments each, and the control group. Assessment of the efficacy of ants’ external microbiome modulation procedure: To verify that our treatment had the intended effects on the external microbiome of the treated ants, we utilized the methodology outlined by Ren et al. ( 2007 ) to culture the external microbiome of the ants. We compared the number of culturable microbes obtained by counting the number of microbial colony-forming units (CFUs) grown on LB agar plates to evaluate the effectiveness of the treatments. A higher number of CFUs implies that there are more external microbes on the surface of the ant. Effects of external microbiome modulation on the survival of RIFA workers: This study underlines the rate of survival of worker ants in response to systemic manipulation of their external microbiomes via topical application of either antibiotics or microbial cultures. To determine whether the treatments had any effect on survival, 30 ants from all treatment and control groups were kept in plates and fed with sugar water only for 10 days, while conducting daily checks to determine their survival rates. We examined individual ants regularly and noted whether they were alive or dead. Workers were considered dead if they showed no reaction to gentle touching with tweezers. Each trial was replicated three times. Behavioral assays: Nestmate recognition: The hypothesis being tested is that the microbial composition of the ant's cuticle can mediate the recognition of nestmates. The treatment and control ants were prepared as previously discussed. Following the procedures of Gordon et al. ( 2005 ) and Dosmann et al. ( 2016 ), workers in each treatment groups were marked and returned to locations near the artificial nest, where they could interact with resident nestmates. Individual sampled ants received one of twelve experimental or three control treatments, each represented by a corresponding colour for ease of identification during behavioral observations. For the untreated control nestmates (NM), the ants were transferred from their colony into separate artificial nests, where they were kept in isolation for about 24 h prior to the commencement of the experiment. For each experimental trial, the observations were blind and conducted by a human observer. The total observation time was 10 min, during which rejection or acceptance of the treated or control ants was scored on a scale of 1–4 as follows: Level 1: No sign of rejection was observed: introduced ants were successfully reunited with nestmates, exhibited no change in direction, or simply moved away upon contact with nestmates. Level 2: scored when it took approximately 3 min to join the nestmate without visible rejection or when ants made antennal contact that lasted for more than one second. Level 3: The introduced ant was completely isolated from the rest with no reaction from the nestmates in the period under observation or when upon contact, ants opened their mandibles or turned their gasters upwards or towards their heads. Level 4: Total rejection: ascribed when the introduced ant was seized and carried out of the nest area by resident ants. Numeric scores were assigned to the corresponding interaction levels and used to calculate the mean aggression scores. Foraging aggressiveness: Prey killing rate : As the red fire ants, similar to several other social insects, are known to hunt for their prey, attacking them using their upper jaws for grappling or by stinging. Further experimental trials were conducted to examine the effects of antibiotic/microbial treatments on the ability of RIFA workers to attack and kill their prey. The foraging aggressiveness indices were compared upon the introduction of instar larvae of mealworm (body length = 2.0-2.5 cm; weight = 0.20 g) into the same petri plates (diameter = 5 cm, height = 1.3 cm, with the inner walls coated with Fluon to prevent ant escape) as 30 workers of S. invicta from individual treatment and control groups (body length = 4.0 ± 0.5 mm, head width = 0.8 ± 0.2 mm). The study was adapted to a similar behavior assay of Cheng et al. ( 2019 ), with slight modifications. Here, the aggressiveness index was based on the number of interacting workers per time interval (5, 10, 15, 20, 25, and 30 min post larvae introduction into the plates), as well as the speed of kill, that is, the duration of time taken for the ants to kill the mealworm larvae following several attacks. The larvae were assumed to be dead when no movement was recorded in response to further attacks by the ants. For further confirmation, the larvae were touched with forceps. As it was difficult to ascertain the exact time of larval death, the time range was denoted as follows; (1: 0–5 min, 2: 5–10 min, 3: 10–15 min, 4: 15–20 min, 5: 20–25 min, and 6: 25–30 min). We deployed the scoring scale introduced by Rice and Silverman ( 2013 ) as adapted for fire ants by Cheng et al. ( 2019 ). In this study, the interactions among treatment and control groups were scored on the scale of 1–6; where 1 was assigned the highest possible value (highest number of interactions and fastest speed of kill, respectively), while 6 was assigned the lowest possible value (lowest number of interactions or when no death was recorded in the period under observation). Each trial was replicated three times. Food searching To expand on the previous findings, additional bioassays were conducted to examine the effects of artificial manipulation of the ant external microbiome on other foraging activities, such as workers’ ability to search for food and the rate of food carrying. In this study, 60 workers ants from each treatment and control stock were kept in plates (12 cm × 8.5 cm × 4.2 cm; 0.3 L), and to this plate containing the ants was another plate (12 cm × 8.5 cm × 4.2 cm; 0.3 L) joined to the former using a duct, where ham sausage (weight = 0.50–0.60 g) was placed (Supplementary Fig. 1). The actual weights of the sausages were estimated and recorded at the time of placement. Here, the time taken for the first ant to locate the sausage, the number of ants that successfully located the food after 30 min, and the mean net weight of sausage taken by the ants to be eaten over a period of 48 h were recorded. The observation period was 240 h, during which the total weight of sausages taken by ants was estimated every 48 h. The sausage was replaced every 48 h to maintain its attractiveness. For the first bioassay, all dead ants were carefully removed every other day but were not replaced, while for the second bioassay, all dead ants were replaced every 48 h. All survival and behavioral bioassays were replicated three times (at least) for each treatment and control, whereas individual bioassays were conducted twice. Statistical analysis: Where applicable, the collected data were subjected to normality and homogeneity tests of variances using Shapiro-Wilk Normality test (at 0.05 significance level) prior to any data analysis. To equalize the variances, cumulative mortality percentage values were subjected to log transformation, while the square roots of the scores assigned in the foraging aggression and nestmate recognition trials were used for analysis. The foraging aggression indices among treatment and control ants were determine using the formula introduced by Rice and Silverman ( 2013 ) with slight modification as follows: Here, δi denotes the interaction score, fi is the number of interacting ants per individual observation, and T represents the sum of all interactions among ants. As the CFU counts were non-parametric, the data were analyzed using the Kruskal–Wallis test, followed by Dunn’s pairwise comparisons. The cumulative mortality percentage and food carrying rate data were subjected to a repeated-measures one-way ANOVA with assessment time as a repeated factor, while other data analyses were performed using a one-way ANOVA. When a significant F test was obtained at P < 0.05, multiple comparisons were performed using Tukey’s Honestly Significant Difference (HSD) tests. All statistical analyses were performed using IBM SPSS statistical software v22.0 (SPSS Inc., Chicago, IL, USA) and Statistix 10.0 (Analytical Software, Tallahassee, FL, USA). Results Isolation and characterization of cuticle bacteria from S. invicta workers: Morphological Characterization To identify key symbiotic bacteria in S. invicta workers, cultivable bacterial strains were isolated and identified using a conventional Colony Forming Unit (CFU) assay. Phenotypic characterization of the bacterial isolates was conducted based on evidence of their phenotypic distinctiveness. Although LB agar is a nutrient-rich medium, it has been confirmed that some environmental bacteria cannot be readily grown using this method; therefore, it is assumed that only a subset of the ants’ cuticular bacterial community was successfully cultured using this isolation method. Molecular characterization Following the phylogenetic placement of the bacterial isolates, 16 bacterial taxa were successfully characterized from the cuticle of RIFA workers. The details of all identified bacterial strains are presented in Supplementary table 1 , and four bacterial strains were selected based on relative abundance for behavioral bioassays (Table 1 ). Table 1 Bacterial strains characterized from RIFA cuticle for behavioral and survival bioassays Assigned isolate number Bacterial strains Source of RIFA colony D121 Vagococcus fluvialis Dongguan Z442 Myroides odoratus Zhuhai Z511 Providencia rettgeri Zhuhai G911 Pseudomonas monteilii Guangzhou Experimental validation of ants’ external microbiome modulation following topical application of antibiotics or bacterial cultures: The number of culturable bacteria obtained from the individual samples, measured by counting the number of microbial CFUs that grew on LB agar plates, was used to indicate the effectiveness of the antibiotics/microbial homogenates treatments. A higher quantity of CFUs suggests a greater quantity of external bacteria present on the ant. Therefore, the significantly smaller number of CFUs observed on plates containing homogenates from antibiotic-treated ants (axenic group) validates the success of artificially modifying the external microbiome of the ants (Fig. 1 ). The number of CFUs in the axenic group was substantially lower than those in the other treatment and control groups ( F 14, 30 = 9.08, P < 0.001). The effects of external microbiome artificial modulation on RIFA workers survival: The survival rate across the treatment and control groups was observed over a period of 10 days. The rate was similar across treatments and control within 48 h of treatment (data not shown). Meanwhile, within 4 days of treatment, the percentage cumulative mortality recorded across treatments and control was significantly different ( F 14, 28 = 2.26, P = 0.0296). The highest cumulative mortality (27.8%) was recorded in the ants treated with tetracycline (50 µg/ml). The lowest cumulative mortality at this stage was recorded among the SDW control ants (1.1%), the symbiotic ants exposed to V. fluvialis D121 strain (1.1%), and the untreated nestmates (NM) at 2.2% (Fig. 2 a). Similarly, at 7 days post-treatment, the highest cumulative mortality was recorded in Tetra-treated ants at 60% and Strep-treated at 36.7%, while 4.5, 26.7, and 28.9% were recorded for NM, SDW, and SLB control ants, respectively. Cumulative percent mortality ranges from 14.5–26.7% across the gnotobiotic treatment group ( F 14, 28 = 4.90, P < 0.001; Fig. 2 b). At 10 days post-treatment, cumulative percent mortality was generally highest across the axenic treatment group, where 44.4%, 61.1%, 73.3%, and 88.9% were recorded in Gent, Strep, Pen, and Tetra-treated ants, respectively. In comparison, a significantly lower percentage mortality was recorded among the symbiotic group, where 32.2% was recorded for ants treated with both V. fluvialis D121 and M. odoratus Z442 strains. Meanwhile 41.1 and 44.4% were recorded for P. rettgeri Z511 and P. monteilii G911 treated ants ( F 14, 28 = 7.96, P < 0.001; Fig. 2 c). In general, treatment of RIFA ants with tetracycline 50 µg/ml induced the highest rate of mortality in RIFA workers, as evident in the highest cumulative percentage mortality recorded across each assessment period: 4, 7, and 10 days post-treatment, which was significantly higher than that in other treatment and control groups ( F 14, 112 = 13.09, P < 0.001). Effects of external bacteria manipulation on nestmates recognition: The hypothesis that the microbial composition of the ant's cuticle mediates recognition by nestmates was evaluated. We propose that the augmented cuticular microbes may produce certain odor or alter the cuticular hydrocarbons that attract or repel nestmates. The findings of the study revealed erratic responses of nestmates to treated and untreated ants. We recorded the highest percentage of rejection across the microbial-treated ants (i.e., the symbiotic and gnotobiotic ants), which was significantly higher than that across the antibiotic treatment group and control ( F 14, 135 = 6.21, P < 0.001). Interestingly, the ants that received no antibiotic nor microbial treatments, except for a two seconds dipping in sterile LB were rejected more than other control and antibiotics treated ants (Fig. 3 ). Throughout the period of observation, no visible sign of rejection was recorded for the untreated ants (NM) that were isolated for 24 h before returning to interact with their nestmates, except for a couple of occasions where a returning ant took about 3–5 min to reunite with the nestmates. Effects of external bacteria manipulation on workers’ foraging aggressiveness: Prey killing In a study conducted to assess the effects of artificial modulation of bacterial exosymbionts of RIFA on worker foraging aggressiveness, the study revealed differences in the aggressive index across treatment and control groups. The number of interacting ants per individual observation varied across treatments and control. The highest mean number of interactions per individual observation (14.7) was recorded at 10 m post larvae introduction in gnotobiotic ants treated with M. odoratus Z442 strain. The highest mean interaction over the entire observation period (10.2) was also recorded among the gnotobiotic ants treated with M. odoratus Z442 strain. Although the data collected were generally inconsistent across individual treatment and control groups, the fastest speed of kill was also recorded among the gnotobiotic ants, with the highest rate recorded among the ants treated with M. odoratus Z442 strain, where 100% of the introduced larvae died within 5 m of placement in Petri dishes. In contrast, no larval death was recorded across axenic Pen-treated, and SLB control ants. Overall, the significantly highest aggressiveness indices of 4.30, 3.82, and 3.13 were recorded in the gnotobiotic M. odoratus Z442 strain treated ants at 10 ( F 14, 30 = 8.47, P < 0.001), 5 ( F 14, 30 = 11.32, P < 0.001), and 15 min after larval introduction ( F 14, 30 = 6.56, P < 0.001), respectively. The lowest aggressive index (0.08) was recorded in the SDW control ants at 15 min post larvae introduction. Generally, across all treatment and control groups, the mean number of interacting ants decreased with an increase in time after larval introduction (Table 2 ). Table 2 The aggressive index of axenic, gnotobiotic, symbiotic and the control workers of S. invicta upon the introduction of instar larvae of mealworm, T. molitor Duration Control Axenic Symbiotic Gnotobiotic Sterile LB Nestmates Sterile water Streptomycin Penicillin Tetracycline Gentamycin V. fluvialis D121 M. odoratus Z442 P. rettgeri Z511 P. monteilii G911 V. fluvialis D121 M. odoratus Z442 P. rettgeri Z511 P. monteilii G911 5min - - 0.71 ± 0.07 bc 1.38 ± 0.11 bc - 0.48 ± 0.12bc 2.52 ± 0.24 ab 0.18 ± 0.00 c 0.21 ± 0.05 c 1.53 ± 0.05 bc 0.48 ± 0.06 bc 1.90 ± 0.48 abc 3.82 ± 0.28 a 1.94 ± 0.45 abc 1.53 ± 0.22 bc 10min - - 0.54 ± 0.04 bc 1.67 ± 0.11 abc - 1.07 ± 0.13 abc 2.18 ± 0.39 ab 0.50 ± 0.11 bc 0.51 ± 0.13 bc 1.09 ± 0.14 abc 0.36 ± 0.05 bc 2.38 ± 0.28 ab 4.30 ± 0.28 a 3.04 ± 0.87 ab 1.16 ± 0.21 abc 15min - - 0.08 ± 0.02 b 1.05 ± 0.04 ab - 0.48 ± 0.00 ab 2.35 ± 0.65 ab 0.35 ± 0.08 b 0.31 ± 0.11 b 1.04 ± 0.14 ab 0.52 ± 0.10 ab 1.90 ± 0.40 ab 3.13 ± 0.33 a 1.58 ± 0.30 ab 1.11 ± 0.22 ab 20min - - 0.42 ± 0.04 cd 0.57 ± 0.00 abcd - 0.75 ± 0.15 bcd 1.34 ± 0.28 abcd 0.32 ± 0.10 cd 0.51 ± 0.17 bcd 0.65 ± 0.09 bcd 0.27 ± 0.07 cd 2.50 ± 0.26 abc 3.13 ± 0.40 a 2.67 ± 0.49 ab 0.63 ± 0.04 bcd 25min - - 0.25 ± 0.03 ab 0.57 ± 0.00 ab - 0.75 ± 0.15 ab 1.34 ± 0.28 ab 0.38 ± 0.06 ab 0.31 ± 0.11 ab 0.93 ± 0.07 ab 0.21 ± 0.05 ab 1.54 ± 0.06 ab 1.76 ± 0.28 a 1.94 ± 0.41 a 0.89 ± 0.02 ab 30min - - - 0.76 ± 0.08 ab - 0.48 ± 0.13 abc 1.26 ± 0.31 ab 0.26 ± 0.09 abc 0.15 ± 0.07 bc 0.76 ± 0.03 abc 0.15 ± 0.05 bc 1.78 ± 0.17 a 1.86 ± 0.41 a 1.82 ± 0.36 a 0.68 ± 0.02 abc Means aggressive index (± S.E.) with different letters within the same row indicate significant differences among treatments and control at P < 0.05 (Tukey’s HSD test after One-way ANOVA). In the columns marked ( - ); the aggressive index was not computed in trials where no larvae death was recorded after 30 min post larvae introduction, or for any point of observation where no worker was interacting with the larvae. Food searching and rate of food carrying The ability of worker ants to locate their food and the number of workers actively searching for food at 30 min post food placement varies across treatment and control groups. The symbiotic ants, less the ones treated with P. monteilii G911 strain, took lesser time to locate the food in comparison with other treatments and control ants ( F 14, 30 = 2.59, P = 0.0139). The average time taken for the first ant to locate the sausage was 3.38 min, recorded in the symbiotic ants treated with P. rettgeri Z511 strain; which was the fastest rate recorded across all treatments and control (Fig. 4 a). Following 30 min of observation, the number of ants that successfully located the food was similar across all treatments and control ( F 14, 30 = 0.79, P = 0.6692; Fig. 4 b). The assessment of food carrying rate across all treatment and control groups revealed relative similarities at 48 h post sausage placement ( F 14, 28 = 1.3, P = 0.2664). Thereafter, we recorded significant differences among treatments and control for individual assessment time, 96 h; ( F 14, 28 = 2.38, P = 0.0246), 144 h; ( F 14, 28 = 6.54, P < 0.001), 192 h; ( F 14, 28 = 7.15, P < 0.001), and 240 h of observation; ( F 14, 28 = 5.39, P < 0.001; Fig. 4 c). Here, we observed a decline in the rate of food carrying across all treatments and control with an increase in observation period ( F 4, 56 = 172.82, P < 0.001). In addition, from these results, we established a correlation between the number of surviving ants and the weight of food carried. Here, the significantly low food-carrying rate that was recorded among the axenic Tetra-treated ants and the SLB-control ants was predicted to be partly due to the high mortality rate recorded among these two groups in comparison to other treatment and control groups (data not shown). Bearing this outcome in mind, a second trial was conducted in which dead ants from individual treatments and control were removed and replaced with new ones from the stock at each assessment date. The outcome revealed a similar trend to the first trial, where, aside the Pen-treated ants, the lowest average weight of food carried was recorded across axenic ants, although not statistically different from the average weight recorded in the control and other treatment groups ( F 14, 28 = 1.61, P = 0.0802; Fig. 4 d). Similar to the first trial, a decline in the rate of food carrying across all treatments and control with an increase in observation period was observed ( F 4, 56 = 43.2, P < 0.001). Generally, within 48 h of observation, the mean weight of sausage carried among the axenic ants’ ranges from 0.37-0.50g, 0.44-0.63g for the gnotobiotic ants, while 0.53-0.60g and 0.32-0.40g were recorded for the symbiotic and control ants, respectively. Following 240 h of observation, average weight of sausage carried ranges from 0.21-0.25g, 0.22-0.38g, 0.25-0.30g, and 0.26-0.45g for the control, axenic, symbiotic, and gnotobiotic ants, respectively. Discussion The epidermis of insects serves as a protective layer against invading pathogens and can be naturally colonized by bacteria (Douglas, 2015 ). Pathogenic and protective microbes live on the surface of insect cuticles and can mediate direct or indirect changes in host physiology and other biochemical processes (Davis et al., 2013 ; Keiser et al., 2016 ; McFall-Ngai et al., 2013 ). The current study tested the hypothesis that the external microbial composition of RIFA influences survival rate and certain social behaviors, including worker aggressiveness, nestmate recognition, and foraging activities. Artificial modulation of the external microbiomes of ants via topical application of antibiotics or microbial monocultures was successful. Data from the validation test revealed significantly greater CFU counts in the symbiotic and gnotobiotic ants than in the axenic and untreated control ants. The successful removal of cuticle bacterial symbionts mediated a significant reduction in the survival rate of workers. Higher mortality rates were recorded across the treatments in the axenic group. Specifically, the highest mortality rate was recorded in ants treated with tetracycline. Meanwhile, the symbiotic ants revealed a corresponding induced survival rate that gradually decreased with an increase in the number of days after topical application of bacterial monocultures. Consequently, the results of the survival bioassay showed that successful artificial modification of the cuticle bacterial symbionts of RIFA directly affects the survival rate. This outcome is related to a previous study that suggested that the exosymbiotic microbiome of insects can influence their health status and life history (Ezenwa et al., 2012 ). In addition, dissimilarities in survival rates between control and microbial treated red harvester ants ( Pogonomyrmex barbatus ) were reported by Dosmann et al. ( 2016 ). When an organism comes in contact with bacteria, either pathogenic or benign, its health status, behavior, and other social functions are affected (Ben-Yosef et al., 2008 ; Freitak et al., 2009 ; Freitak et al., 2007 ; Sharon et al., 2010 ). The diversity of microbial communities within each member of a social group is related to individual experiences such as diet, as well as the interactions with the microbiota of nestmates. In addition, the effects on the host can extend over a very long period of time (Wallace et al., 2010 ). The evolution of nestmate recognition has been of great interest to the scientific community. Social insects strongly rely on odours to differentiate between within- and between-colony members. In the current study, cuticle microbes influenced the ability of ants to recognize their nestmates. We observed the highest rate of rejection among the microbial-treated ants, whereas for the antibiotics-treated ants, the rate of rejection was relatively low, albeit not statistically different in comparison to the control ants. The results revealed that ants with augmented cuticular microbiota were more likely to be rejected by their nestmates. Although, it appears that the treated ants are less likely to be rejected by their nestmates due to the absence or artificial removal of a familiar odour. Rather, ants are more likely to be rejected because of the presence of a foreign odour or artificial introduction of novel cuticle microbes. This finding is consistent with that of Dosmann et al. ( 2016 ) in red harvester ants. In a few other related studies, it has been suggested that the rejection of microbial-treated insects could be related to a social immunity response, whereby nestmates perceive insects with augmented cuticular microbiomes as sick nestmates that need to be isolated (Cremer et al., 2007 ; Dosmann et al., 2016 ). Similar findings have been reported in other economic insects, including leaf-cutting ants (Richard et al., 2007 ), termites (Matsuura, 2001 ), and Drosophila melanogaster (Lizé et al., 2014 ). Our findings, in addition to the aforementioned studies, retrace the important roles of external microbes of insects in nestmate recognition. Aside the microbial composition on insect cuticles, other factors have been suggested could be involved. The most commonly mentioned are cuticular chemical hydrocarbons (CHCs), which have been described as important nestmate discriminators in several social insects. CHCs are the predominant compounds responsible for intraspecific recognition within a colony; however, other volatile factors are thought to contribute to the signature odours of individual colonies (Sturgis and Gordon, 2012 ; van Zweden and d’Ettorre, 2010 ; Vander Meer and Morel, 1998). To understand the role of external microbes in RIFA foraging activities, workers’ aggressiveness upon contact with prey, the ability to search for food, and the rate of food carrying were examined. The values obtained for the aggressive index varied across control and treatments, where M. odoratus Z442 strain, tested on the gnotobiotic ants, caused the fastest kill as well as the highest number of interactions, resulting in the highest aggressive index. With respect to the rate of food searching, the symbiotic ants generally needed less time to locate the food in comparison to the other treatment and control groups. However, within 30 min of sausage placement, the number of ants that successfully located the food was similar across treatments and control. The initial period of observation also revealed similarities in the average weight of sausage taken across treatments and control. With an increase in the observation time, a significant reduction was recorded among the antibiotic-treated ants, specifically in ants treated with tetracycline. Across this group, the decreasing number of surviving ants might be partly responsible for the decline in their foraging activities, as the percentage of mortality recorded was generally higher across the antibiotic-treated ants throughout the experiment. Tellingly, a second trial, in which dead ants were replaced, confirmed this assumption. Here, a reduction in foraging activity across antibiotic-treated ants, which was not statistically different from other treatments and control, was recorded. Several factors directly influence ant foraging activities. For instance, the volatiles produced by microorganisms on insect cuticle surfaces may have a direct effect on many aspects of insect social behavior, including foraging activities (Davis et al., 2013 ). In other insects, cuticular microbiota are also known to play major roles. For instance, Parks et al. ( 2018 ) reported a 10-fold decrease in the foraging aggressiveness of spiders toward a prey artificially placed in their web following topical application of bacterial monocultures of Dermacoccus nishinomiyaensis and Staphylococcus saprophyticus . The ability of RIFA to successfully invade new areas is determined by a variety of factors, namely, their aggressive behavior, competitive ability, and fighting capability of both single individuals and multiple colonies (Chen et al., 2014 ; Lai et al., 2015 ). Such characteristics can result in the successful defeat of another group, even when the invaders are numerically outnumbered, as seen in social animal battles (Traniello and Beshers, 1991 ). According to Obin and Meer ( 1989 ), species with higher levels of aggression are more likely to gain competitive advantage in interspecific encounters. In conclusion, the examined cuticular bacterial species suggested existing interactions between them and their ant host. Furthermore, the topical application of bacterial homogenates to ants significantly improved their survival and foraging aggressiveness. These outcomes indicate the importance of microbial exosymbionts in the ecology and behavior of social insects and suggest that further research is necessary. Although this study has shed light on the beneficial effects of cuticular bacterial symbionts of RIFA, the specific mechanisms underlying these beneficial effects are yet to be fully explored. It is crucial for future research to focus on this area and uncover the mechanisms of action of these symbionts in enhancing host survival and altering ant social behavior. Declarations Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This study received financial support through the Research Fund for International Young Scientists (32150410344), a research grant received from National Natural Science Foundation of China (NSFC). Author Contribution BSB and YX conceived and designed research. BSB, JAS, and LN conducted the experiments. AI and BSB analyzed the data. BSB and JAS prepared the first manuscript draft and was revised by YX and CJ. The funding for the study was obtained by BSB and YX. All authors read and approved the manuscript. Acknowledgement We are grateful to all of our colleagues at the Red Imported Fire Ants Research Centre of South China Agricultural University, Guangzhou, China, for their assistance in samples collection, culture media preparation, and procurement of experimental items used for the study. Data Availability Data supporting the results can be found in NCBI’s Genbank database following this link - http://www.ncbi.nlm.nih.gov/- accessed on February 16, 2025. References Bamisile, B.S., Nie, L., Siddiqui, J.A., Ramos Aguila, L.C., Akutse, K.S., Jia, C., and Xu, Y. (2023). Assessment of mound soils bacterial community of the red imported fire ant, Solenopsis invicta across Guangdong province of China. Sustainability 15 , 1350. Ben‐Yosef, M., Jurkevitch, E., and Yuval, B. (2008). Effect of bacteria on nutritional status and reproductive success of the Mediterranean fruit fly Ceratitis capitata. Physiological entomology 33 , 145-154. Chen, J., Rashid, T., and Feng, G. (2014). A comparative study between Solenopsis invicta and Solenopsis richteri on tolerance to heat and desiccation stresses. PLoS One 9 , e96842. Cheng, D., Chen, S., Huang, Y., Pierce, N.E., Riegler, M., Yang, F., Zeng, L., Lu, Y., Liang, G., and Xu, Y. (2019). Symbiotic microbiota may reflect host adaptation by resident to invasive ant species. PLoS pathogens 15 , e1007942. Cremer, S., Armitage, S.A., and Schmid-Hempel, P. (2007). Social immunity. Current biology 17 , R693-R702. Davis, T.S., Crippen, T.L., Hofstetter, R.W., and Tomberlin, J.K. (2013). Microbial volatile emissions as insect semiochemicals. Journal of chemical ecology 39 , 840-859. Dosmann, A., Bahet, N., and Gordon, D.M. (2016). Experimental modulation of external microbiome affects nestmate recognition in harvester ants (Pogonomyrmex barbatus). PeerJ 4 , e1566. Douglas, A.E. (2015). Multiorganismal insects: diversity and function of resident microorganisms. Annual review of entomology 60 , 17-34. Ezenwa, V.O., Gerardo, N.M., Inouye, D.W., Medina, M., and Xavier, J.B. (2012). Animal behavior and the microbiome. Science 338 , 198-199. Freitak, D., Heckel, D.G., and Vogel, H. (2009). Bacterial feeding induces changes in immune-related gene expression and has trans-generational impacts in the cabbage looper (Trichoplusia ni). Frontiers in zoology 6 , 1-11. Freitak, D., Wheat, C.W., Heckel, D.G., and Vogel, H. (2007). Immune system responses and fitness costs associated with consumption of bacteria in larvae of Trichoplusia ni. Bmc Biology 5 , 1-13. Gordon, D., Chu, J., Lillie, A., Tissot, M., and Pinter, N. (2005). Variation in the transition from inside to outside work in the red harvester ant Pogonomyrmex barbatus. Insectes Sociaux 52 , 212-217. Grenham, S., Clarke, G., Cryan, J.F., and Dinan, T.G. (2011). Brain–gut–microbe communication in health and disease. Frontiers in physiology 2 , 16175. Hamby, K.A., and Becher, P.G. (2016). Current knowledge of interactions between Drosophila suzukii and microbes, and their potential utility for pest management. Journal of Pest Science 89 , 621-630. Holway, D.A., Lach, L., Suarez, A.V., Tsutsui, N.D., and Case, T.J. (2002). The causes and consequences of ant invasions. Annual review of ecology and systematics 33 , 181-233. Hu, L., Balusu, R., Zhang, W.-Q., Ajayi, O., Lu, Y.-Y., Zeng, R.-S., Fadamiro, H., and Chen, L. (2018). Intra-and inter-specific variation in alarm pheromone produced by Solenopsis fire ants. Bulletin of entomological research 108 , 667-673. Jun, H., Yi-Juan, X., Yong-Yue, L., and Ling, Z. (2012). Effects of Solenopsis invicta invasion on the diversity of spider communities in a corn field. Yingyong Shengtai Xuebao 23 . Kafle, L., Wu, W.J., Kao, S.S., and Shih, C.J. (2011). Efficacy of Beauveria bassiana against the red imported fire ant, Solenopsis invicta (Hymenoptera: Formicidae), in Taiwan. Pest management science 67 , 1434-1438. Keiser, C.N., Shearer, T.A., DeMarco, A.E., Brittingham, H.A., Knutson, K.A., Kuo, C., Zhao, K., and Pruitt, J.N. (2016). Cuticular bacteria appear detrimental to social spiders in mixed but not monoculture exposure. Current Zoology 62 , 377-384. Lai, L.-C., Hua, K.-H., and Wu, W.-J. (2015). Intraspecific and interspecific aggressive interactions between two species of fire ants, Solenopsis geminata and S. invicta (Hymenoptera: Formicidae), in Taiwan. Journal of Asia-Pacific Entomology 18 , 93-98. Lei, W., XU, Y.-j., Ling, Z., and LU, Y.-y. (2019). Impact of the red imported fire ant Solenopsis invicta Buren on biodiversity in South China: A review. Journal of Integrative Agriculture 18 , 788-796. Li, J., Guo, Q., Lin, M., Jiang, L., Ye, J., Chen, D., Li, Z., Dai, J., and Han, S. (2016). Evaluation of a new entomopathogenic strain of Beauveria bassiana and a new field delivery method against Solenopsis invicta. Plos One 11 , e0158325. Lizé, A., McKay, R., and Lewis, Z. (2014). Kin recognition in Drosophila: the importance of ecology and gut microbiota. The ISME journal 8 , 469-477. Lu, Y. (2014). Long‐distance spreading speed and trend prediction of red imported fire ant, Solenopsis invicta Buren, in mainland China. Guangdong Agricultural Science 41 , 70. Łukasik, P., van Asch, M., Guo, H., Ferrari, J., and Charles J. Godfray, H. (2013). Unrelated facultative endosymbionts protect aphids against a fungal pathogen. Ecology letters 16 , 214-218. Matsuura, K. (2001). Nestmate recognition mediated by intestinal bacteria in a termite, Reticulitermes speratus. Oikos 92 , 20-26. McFall-Ngai, M., Hadfield, M.G., Bosch, T.C., Carey, H.V., Domazet-Lošo, T., Douglas, A.E., Dubilier, N., Eberl, G., Fukami, T., and Gilbert, S.F. (2013). Animals in a bacterial world, a new imperative for the life sciences. Proceedings of the National Academy of Sciences 110 , 3229-3236. Nie, L., Ni, M., Ning, D., Ran, H., Hassan, B., and Xu, Y. (2019). Comparing mechanisms of competition among introduced and resident ants in China: from behavior to trophic position (Hymenoptera: Formicidae). Myrmecological News 29 . Obin, M.S., and Meer, R.K.V. (1989). Between-and within-species recognition among imported fire ants and their hybrids (Hymenoptera: Formicidae): Application to hybrid zone dynamics. Annals of the Entomological Society of America 82 , 649-652. Parks, O.B., Kothamasu, K.S., Ziemba, M.J., Benner, M., Cristinziano, M., Kantz, S., Leger, D., Li, J., Patel, D., and Rabuse, W. (2018). Exposure to cuticular bacteria can alter host behavior in a funnel-weaving spider. Current zoology 64 , 721-726. Ren, C., Webster, P., Finkel, S.E., and Tower, J. (2007). Increased internal and external bacterial load during Drosophila aging without life-span trade-off. Cell metabolism 6 , 144-152. Rice, E.S., and Silverman, J. (2013). Submissive behaviour and habituation facilitate entry into habitat occupied by an invasive ant. Animal Behaviour 86 , 497-506. Richard, F.-J., Poulsen, M., Hefetz, A., Errard, C., Nash, D.R., and Boomsma, J.J. (2007). The origin of the chemical profiles of fungal symbionts and their significance for nestmate recognition in Acromyrmex leaf-cutting ants. Behavioral Ecology and Sociobiology 61 , 1637-1649. Sharon, G., Segal, D., Ringo, J.M., Hefetz, A., Zilber-Rosenberg, I., and Rosenberg, E. (2010). Commensal bacteria play a role in mating preference of Drosophila melanogaster. Proceedings of the National Academy of Sciences 107 , 20051-20056. Sturgis, S.J., and Gordon, D.M. (2012). Nestmate recognition in ants (Hymenoptera: Formicidae): a review. Myrmecological news 16 , 101-110. Traniello, J.F., and Beshers, S.N. (1991). Maximization of foraging efficiency and resource defense by group retrieval in the ant Formica schaufussi. Behavioral Ecology and Sociobiology 29 , 283-289. van Zweden, J.S., and d’Ettorre, P. (2010). Nestmate recognition in social insects and the role of hydrocarbons. Insect hydrocarbons: biology, biochemistry and chemical ecology 11 , 222-243. Vander Meer, R., and Morel, L. (1998). Nestmate recognition in ants. Pheromone communication in social insects. Vander Meer RK, Breed MD, Winston ML & Espelie KE, 79-103. Wallace, J.R., Gordon, M.C., Hartsell, L., Mosi, L., Benbow, M.E., Merritt, R.W., and Small, P.L. (2010). Interaction of Mycobacterium ulcerans with mosquito species: implications for transmission and trophic relationships. Applied and environmental microbiology 76 , 6215-6222. Wilder, S.M., Holway, D.A., Suarez, A.V., LeBrun, E.G., and Eubanks, M.D. (2011). Intercontinental differences in resource use reveal the importance of mutualisms in fire ant invasions. Proceedings of the National Academy of Sciences 108 , 20639-20644. Yang, C.C.S., Shoemaker, D.D., Wu, J.C., Lin, Y.K., Lin, C.C., Wu, W.J., and Shih, C.J. (2009). Successful establishment of the invasive fire ant Solenopsis invicta in Taiwan: insights into interactions of alternate social forms. Diversity and Distributions 15 , 709-719. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 09 Feb, 2026 Read the published version in International Journal of Tropical Insect Science → Version 1 posted Editorial decision: Revision requested 28 Aug, 2025 Reviews received at journal 23 Aug, 2025 Reviews received at journal 21 Aug, 2025 Reviewers agreed at journal 11 Aug, 2025 Reviewers agreed at journal 11 Aug, 2025 Reviews received at journal 10 Aug, 2025 Reviewers agreed at journal 06 Aug, 2025 Reviewers agreed at journal 05 Aug, 2025 Reviewers invited by journal 05 Aug, 2025 Editor assigned by journal 01 Jul, 2025 Submission checks completed at journal 01 Jul, 2025 First submitted to journal 27 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6990545","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":497276211,"identity":"3896d9f7-33f2-4611-90e9-d94405e36613","order_by":0,"name":"Bamisope Steve BAMISILE","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYBACAyA+gGAYWDDwg3gJBcRrkWCQbABpMcCvBZkhwWBwAEUcE5izn048+KPijhyEUSAhZ3x+deKHBwYM8vxiB7BqsezJ3XCY58wzYwjDQMLY7MbbzRJAhxnOnJ2A3WEHgCoZ2w4nbgAxgH5J3Hbj7AaQlgSD2zi0nH+74eDPtsP1G0CMHwYS9ZtnnN38A6+WG7kbDvC2HU4AM4AOSzDg792G1xbLGW/BfjHcCWYYSBjOuMG7zSIByMDlF3P+3M0fgSEmD2H8sZHn7z+7+eaPCiBDGrsWKDiAxJYAq5TApxxdC/8BHIpGwSgYBaNgpAIAJ79uEp2rSpwAAAAASUVORK5CYII=","orcid":"","institution":"South China Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Bamisope","middleName":"Steve","lastName":"BAMISILE","suffix":""},{"id":497276212,"identity":"ff1591b1-6caa-44e2-9925-1eac59b0fcd4","order_by":1,"name":"Junaid Ali SIDDIQUI","email":"","orcid":"","institution":"The Hong Kong University","correspondingAuthor":false,"prefix":"","firstName":"Junaid","middleName":"Ali","lastName":"SIDDIQUI","suffix":""},{"id":497276213,"identity":"247fe813-9209-45c3-851c-04ca8ebd88a6","order_by":2,"name":"Lei NIE","email":"","orcid":"","institution":"South China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"NIE","suffix":""},{"id":497276214,"identity":"b42081ee-5844-4c64-912e-aaefb23baefe","order_by":3,"name":"Atif IDREES","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Atif","middleName":"","lastName":"IDREES","suffix":""},{"id":497276215,"identity":"9099d50e-a5bd-4036-8676-4bf08f7b52ac","order_by":4,"name":"Chunsheng JIA","email":"","orcid":"","institution":"Shaoguan University","correspondingAuthor":false,"prefix":"","firstName":"Chunsheng","middleName":"","lastName":"JIA","suffix":""},{"id":497276217,"identity":"e64f64ff-657c-4afc-93c6-cd069b6d7c02","order_by":5,"name":"Yijuan XU","email":"","orcid":"","institution":"South China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yijuan","middleName":"","lastName":"XU","suffix":""}],"badges":[],"createdAt":"2025-06-27 10:08:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6990545/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6990545/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s42690-026-01768-9","type":"published","date":"2026-02-09T15:59:11+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88588166,"identity":"64df8d98-d8d9-400f-8c19-74f2e3111945","added_by":"auto","created_at":"2025-08-08 05:02:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":44437,"visible":true,"origin":"","legend":"\u003cp\u003eThe total number of bacterial colony-forming units (CFU) cultured from the cuticle of RIFA workers following artificial modification of the cuticle bacteria via exposure of nestmates to topical application of antibiotics and bacterial monocultures to generate axenic, symbiotic, and gnotobiotic workers of \u003cem\u003eS. invicta\u003c/em\u003e, respectively. Bacterial CFU was plotted on log scale. Bars (± S.E.) with different letters indicate significant differences among treatments at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. (Dunn’s pairwise comparisons following Kruskal–Wallis test)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6990545/v1/facb2277e987aba3aa7fb589.png"},{"id":88589110,"identity":"3e8b468b-75c7-448b-8eaa-7c62460dcf3d","added_by":"auto","created_at":"2025-08-08 05:10:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42324,"visible":true,"origin":"","legend":"\u003cp\u003eCumulative percentage mortality of RIFA workers at 4 [a], 7 [b], and 10 days post exposure [c] to topical application of antibiotics, bacterial monocultures, or both. The control ants were treated with sterile distilled water (SDW), while sterile LB broth (SLB) and the untreated nestmates were used as positive control. Bars (± S.E.) with different letters indicate significant differences among treatments at \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05. (Tukey’s HSD test after repeated-measures One-way ANOVA)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6990545/v1/e85cf6097ba19b24fe4a10ac.png"},{"id":88588168,"identity":"19f24420-6a7f-4f49-b393-369719f8a0e4","added_by":"auto","created_at":"2025-08-08 05:02:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":42390,"visible":true,"origin":"","legend":"\u003cp\u003eNestmates recognition; group aggression scores of RIFA nestmates following interactions with colony members exposed to antibiotics, bacterial monocultures, or both. Each bar represents mean of aggression scores ± S.E. Bars with different letters indicate significant differences among treatments at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. (Tukey’s HSD test after One-way ANOVA)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6990545/v1/abba9bc552d28e8f2356d7a1.png"},{"id":88588180,"identity":"8ea1eea9-3902-4dae-9feb-b7b66d0c0707","added_by":"auto","created_at":"2025-08-08 05:02:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":119289,"visible":true,"origin":"","legend":"\u003cp\u003eWorkers’ foraging activities; the average time taken by RIFA workers to locate sausage [a], mean number of ants that located the sausage within 30 min of placement [b], and the cumulative mean weight of sausage carried over 240 h in experiment 1 [c] and experiment 2 [d]. Bars (± S.E.) with different letters indicate significant differences among treatments at \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 (Tukey’s HSD test after One-way ANOVA [a] and repeated-measures One-way ANOVA [c])\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6990545/v1/5b61f36994b31172f898d23b.png"},{"id":102785902,"identity":"e09f5b20-c529-450b-a3c5-ac83390e7c9f","added_by":"auto","created_at":"2026-02-16 16:10:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1040966,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6990545/v1/c704e989-ac35-44b4-ad8d-eb6fe6fa59ca.pdf"},{"id":88588171,"identity":"338aebf6-75ec-4f43-adcd-61c45d1b264d","added_by":"auto","created_at":"2025-08-08 05:02:51","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":199114,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6990545/v1/fe90a152b31895a13e4ea683.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eExperimental modulation of ants’ external microbiome revealed symbiont-mediated survival and behavioral modifications in workers of Solenopsis invicta\u003c/p\u003e","fulltext":[{"header":"Key Message","content":"\u003cul\u003e\n \u003cli\u003eEffects of cuticle bacteria modification on the survival and social behavior of red fire ants were examined.\u003c/li\u003e\n \u003cli\u003eRemoval of exosymbionts caused up to 89% mortality within 10 days.\u003c/li\u003e\n \u003cli\u003eAnts with altered symbionts faced higher rejection by their nestmates.\u003c/li\u003e\n \u003cli\u003eForaging behaviors were impaired by bacterial manipulation.\u003c/li\u003e\n \u003cli\u003eFindings suggest biocontrol potential via cuticular microbiome.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe red imported fire ant \u003cem\u003eSolenopsis invicta\u003c/em\u003e Buren has earned its reputation as a notorious invasive species due to its ability to cause harm to humans, crops, livestock, and wildlife through painful bites and stings (Kafle et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Nie et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These highly aggressive ants are of huge economic importance and have been documented to be native of South America. The extensive spread of these noxious ants in various parts of the world has been a cause for serious concerns. The red imported fire ant (RIFA), being a globally invasive pest, has presented a significant challenge to China's environment since the first discovery in Wuchuan, Guangdong Province in 2004 (Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Nie et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Since that period, RIFA had spread to a total of 12,680 hectares of land in the country, with an expansion rate of 26.5\u0026ndash;48.1 km/year (Lu, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). RIFA has since become prevalent in most Southern Provinces of China and continues to expand its range.\u003c/p\u003e\u003cp\u003eThe introduction of the fire ants in China has resulted in a major decline of arthropods in tropical and subtropical regions of the country. Surveys conducted in China have discovered that wherever RIFA occur, they caused a severe reduction in the abundance and diversity of native ant species as they have successfully colonized and established in both human-altered and unaltered habitats. This devastation is most evident in orchards, lawns, abandoned lands, grasslands, and residential areas (Lei et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Aggressive invasive ant species like \u003cem\u003eS. invicta\u003c/em\u003e have led to a drastic reduction in arthropod diversity and the local distribution of resident species, with ant species richness decreasing by over a third in some habitats (Jun et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Apart from this, the presence of RIFA in an area can have notable negative effects on the diversity index and evenness index of ant communities within a radius of three meters of its mounds. To combat this, it is essential to obtain detailed information on the biology and ecology of these fire ants. In response to the spread of \u003cem\u003eS. invicta\u003c/em\u003e, China has taken the initiative of investing in numerous research establishments to understand the ecology and biology of fire ants (Lei et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLiving organisms, including social ants, commonly come in contact with environmental microbes, and several scientific studies have explored the various roles microbial organisms play in their hosts, including influencing different functions in their immune, digestive, circulatory, endocrine, and nervous systems (McFall-Ngai et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In the past, the mutual relationships between insect hosts and environmental bacteria have garnered much less attention in the field of behavioral ecology; however, emerging studies have provided evidence to suggest that host-associated microbial communities could offer far more advantages than hitherto presumed (Ezenwa et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Grenham et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These findings shed light on the possibility that the composition of these microbial communities could offer considerable advantages to insect hosts. Insects have therefore been observed to demonstrate various levels of reliance on symbiotic bacteria for their primary functions. Nevertheless, the majority of the currently available studies are limited to pathogenic or beneficial bacteria inhabiting the gut of insects (Bamisile et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Parks et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In addition to the well-studied bacterial endosymbionts inhabiting various internal organs of insects, scientific findings have shown that external bacteria present on the cuticle of ants can as well influence basic insect functions, including survival, nestmate recognition, foraging, and oviposition (Douglas, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThese symbiotic bacteria are fundamental for the development and existence of host organisms, aiding in the degradation of food, energy production, vitamin synthesis, and regulation of immune reactions (Hamby and Becher, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Łukasik et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Thus, research has repeatedly highlighted the several ways in which bacteria on the ant\u0026rsquo;s cuticle can affect the life of the hosts, including beneficially acting as a protective component against pathogens.\u003c/p\u003e\u003cp\u003eFor RIFA, numerous studies have been conducted to unravel the underlying mechanisms behind their huge invasion success (Chen et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wilder et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). There are suggestions that their competitive ability is highly dependent on several important factors, such as colony size, resource finding, monopolizing capabilities, and the level of aggression towards non-nestmates (Holway et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In addition, McFall-Ngai et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) had suggested that in social animals, the most important ecological interactions involve internal, epidermal, and environmental microbes, and not those that involve nestmates, competitors, or predators. To assess the costs and benefits of sociality for any specific social group, it is necessary to further explore the relationships between individuals and the microorganisms specific to their environment. Owing to the ubiquity and diversity of bacterial communities, there is a need to expand our understanding of insect\u0026ndash;bacterial interactions, as it would help to broaden our general knowledge of their biology, adaptability, and social responses.\u003c/p\u003e\u003cp\u003eIn the current study, we tested the hypothesis that artificial modification of external bacterial symbionts of the RIFA would affect their survival and social behavior, including feeding, foraging aggressiveness, and nestmate recognition. It was predicted that RIFA workers with an altered external microbiome would be rejected by their nestmates if the external microbiome plays a role in nestmate recognition. We experimentally modulated the external microbes on the cuticles of RIFA workers via the topical application of bacterial monocultures and/or antibiotics. This study helps to understand the potential roles of the exosymbiotic microbiome in enabling the host to survive and recognize its nestmates. This could also serve as a background for future studies to understand their roles in the host defense against biotic stressors. The current study adds to the available data on the importance of insect-associated external symbionts in the survival and social behavior of the hosts.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eField collection and laboratory rearing of \u003cem\u003eS. invicta\u003c/em\u003e:\u003c/p\u003e\u003cp\u003eFire ant colonies were collected from lawns across different counties in Guangdong province of China. The social form of RIFA used in the current study was polygene. Mound excavation was done with the help of shovels, digging at approximately 10\u0026ndash;15 cm below the ground surface, and transferring the mound soils into plastic boxes with a lid (43 \u0026times; 32 \u0026times; 25 cm). Between each digging, the shovels were disinfected with 70% ethanol and allowed to dry. Insect boxes were transported to the laboratory, and the removal of ant colonies from the mounds was achieved via the traditional dripping-flooding method. Thereafter, the extracted colonies were transferred into clean insect boxes (40 \u0026times; 28 \u0026times; 12 cm), with the walls coated with Fluon to prevent the ants from escaping. The ant populations were fed with water, 10% w/w sugar water, and larvae of mealworm \u003cem\u003eTenebrio molitor\u003c/em\u003e Linnaeus (Coleoptera: Tenebrionidae). The colonies were reared in the laboratory for two weeks before the commencement of the experiment. Laboratory conditions during the period were maintained at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 75% relative humidity, and 14:10 h L:D.\u003c/p\u003e\u003cp\u003eSolid and Liquid media preparation:\u003c/p\u003e\u003cp\u003eLuria\u0026ndash;Bertani (LB) broth deployed for bacteria isolation from ant body consists of yeast extract (5 g), sodium chloride (10 g), tryptone (10 g), and 1 L of sterile distilled water. For LB solid media, 15 g of agar was added and supplemented with 50 mg/L nystatin (Sangon Biotech, Shanghai, China), to prevent fungal growth on bacteria culture plates.\u003c/p\u003e\u003cp\u003eIsolation of cuticle bacteria from \u003cem\u003eS. invicta\u003c/em\u003e workers:\u003c/p\u003e\u003cp\u003eFrom each colony, about 12 medium-sized workers of \u003cem\u003eS. invicta\u003c/em\u003e were randomly selected. The ants were transferred into sterile 15 mL conical tubes and temporarily placed in a fridge to immobilize them. Cuticle bacteria were collected by gently rubbing the surface of ants with sterile damp cotton swab. Thereafter, the cotton swabs were placed in fresh 15 mL conical tubes containing 5 mL of sterile LB broth and vortexed for 3 min at 1000 \u0026times; g. Afterwards, from the stock suspension, three serial dilutions were made, while 100 \u0026micro;L of the third serial dilution was evenly spread on LB agar plates in triplicate using sterile disposable cell spreader (Changde Bkmam Biotech, Changde, China). The inoculated plates were kept and allowed to dry under aerobic conditions before being transferred into a BOD incubator (BS-1E, China), and incubated at 25\u0026deg;C for 72 h. Conventional colony forming unit (CFU) assay was conducted to identify the emerging bacteria. Single colonies with distinct morphological features were re-streaked multiple times on freshly prepared LB agar plates (approximately 3\u0026ndash;4 times) using sterile disposable inoculating loops (Biologix Group Ltd. Jinan, China).\u003c/p\u003e\u003cp\u003ePreparation of bacterial liquid monocultures:\u003c/p\u003e\u003cp\u003eFollowing multiple sub-cultures until bacterial monocultures were obtained, the individual distinct colonies were inoculated into LB broth contained in 50 ml round-bottom centrifuge tubes using sterile micropipette tips. The tubes were shaken overnight on a rotary shaker (ZD-85; GTCS, Shanghai, China) at 200 rpm and 30\u0026deg;C. The homogenized suspensions were immediately used for DNA extraction, or preserved on 25% sterile glycerol stocks and store at -80\u0026deg;C for future use.\u003c/p\u003e\u003cp\u003eDNA extraction, molecular and phylogenetic characterization of bacterial isolates:\u003c/p\u003e\u003cp\u003eFor DNA extraction, TIANamp Bacterial DNA kit supplied by the manufacturer (Tiangen Biotech, Beijing, China) was used. The full-length of the bacterial small subunit 16S rRNA gene was amplified using primer pairs (27F: 5\u0026prime;-AGAGTTTGATCCTGGCTCAG-3\u0026prime; and 1492R: 5\u0026prime;-ACGGCTAACTTGTTACGACT-3\u0026prime;). The PCR reaction mix and thermocycling conditions were similar to those described by Cheng et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and Bamisile et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Bidirectional sequencing of bacterial isolates was conducted by Sangon Biotech, Guangzhou, China. Manual editing was done for the obtained bacterial sequence traces using BioEdit v 7.1.9., while reference sequences were downloaded from the GenBank database of National Center for Biotechnology Information (NCBI) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on 12 March 2023). The reference sequences were used to manually edit and align the target sequences, while phylogenetic analysis using Neighbor-Joining method analysis based on Kimura 2-parameter model was performed via MEGA v. 11. The robustness of branches was estimated by bootstrap analysis with 1000 repeated samples from the data.\u003c/p\u003e\u003cp\u003eCulturing Axenic, Gnotobiotic, and Symbiotic workers of \u003cem\u003eS. invicta\u003c/em\u003e:\u003c/p\u003e\u003cp\u003eFrom the characterized bacterial strains (Supplementary Table\u0026nbsp;1), four strains (\u003cem\u003eVagococcus fluvialis\u003c/em\u003e D121, \u003cem\u003eMyroides odoratus\u003c/em\u003e Z442, \u003cem\u003eProvidencia rettgeri\u003c/em\u003e Z511, and \u003cem\u003ePseudomonas monteilii\u003c/em\u003e G911) were selected based on their relative abundance for further analysis. The bacterial strains and four antibiotic treatments were selected for the artificial manipulation of the RIFA external microbiome. Three treatment groups of RIFA workers (axenic, gnotobiotic, and symbiotic) were achieved through the topical application of antibiotics, microbial monocultures, or both. The axenic ants include treatment with either of penicillin 100 \u0026micro;g/ml, streptomycin 100 \u0026micro;g/ml, gentamicin 150 \u0026micro;g/ml, or tetracycline 50 \u0026micro;g/ml. The symbiotic ants were nestmates treated with any of the four selected bacterial strains. Meanwhile, to create a single-species bacterial microbiome on the ants\u0026rsquo; cuticle (gnotobiotic ants), ants were first treated with gentamicin 150 \u0026micro;g/ml, prior to exposure to each of the four bacterial strains. On the other hand, the control group consisted of ants treated with either of sterile distilled water (SDW) or sterile LB broth (SLB), and untreated nestmates (NM) (Supplementary Table\u0026nbsp;2). For all treatment and control groups, ants were treated by dipping in 0.5 ml of suspensions or monocultures for 3 s. Excess liquids were removed from the body of treated ants using absorbent papers. The entire experiment was set up in a completely randomized design with 12 combinations comprising three groups and four treatments each, and the control group.\u003c/p\u003e\u003cp\u003eAssessment of the efficacy of ants\u0026rsquo; external microbiome modulation procedure:\u003c/p\u003e\u003cp\u003eTo verify that our treatment had the intended effects on the external microbiome of the treated ants, we utilized the methodology outlined by Ren et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) to culture the external microbiome of the ants. We compared the number of culturable microbes obtained by counting the number of microbial colony-forming units (CFUs) grown on LB agar plates to evaluate the effectiveness of the treatments. A higher number of CFUs implies that there are more external microbes on the surface of the ant.\u003c/p\u003e\u003cp\u003eEffects of external microbiome modulation on the survival of RIFA workers:\u003c/p\u003e\u003cp\u003eThis study underlines the rate of survival of worker ants in response to systemic manipulation of their external microbiomes via topical application of either antibiotics or microbial cultures. To determine whether the treatments had any effect on survival, 30 ants from all treatment and control groups were kept in plates and fed with sugar water only for 10 days, while conducting daily checks to determine their survival rates. We examined individual ants regularly and noted whether they were alive or dead. Workers were considered dead if they showed no reaction to gentle touching with tweezers. Each trial was replicated three times.\u003c/p\u003e\u003cp\u003eBehavioral assays:\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eNestmate recognition:\u003c/h2\u003e\u003cp\u003eThe hypothesis being tested is that the microbial composition of the ant's cuticle can mediate the recognition of nestmates. The treatment and control ants were prepared as previously discussed. Following the procedures of Gordon et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and Dosmann et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), workers in each treatment groups were marked and returned to locations near the artificial nest, where they could interact with resident nestmates. Individual sampled ants received one of twelve experimental or three control treatments, each represented by a corresponding colour for ease of identification during behavioral observations. For the untreated control nestmates (NM), the ants were transferred from their colony into separate artificial nests, where they were kept in isolation for about 24 h prior to the commencement of the experiment. For each experimental trial, the observations were blind and conducted by a human observer. The total observation time was 10 min, during which rejection or acceptance of the treated or control ants was scored on a scale of 1\u0026ndash;4 as follows: Level 1: No sign of rejection was observed: introduced ants were successfully reunited with nestmates, exhibited no change in direction, or simply moved away upon contact with nestmates. Level 2: scored when it took approximately 3 min to join the nestmate without visible rejection or when ants made antennal contact that lasted for more than one second. Level 3: The introduced ant was completely isolated from the rest with no reaction from the nestmates in the period under observation or when upon contact, ants opened their mandibles or turned their gasters upwards or towards their heads. Level 4: Total rejection: ascribed when the introduced ant was seized and carried out of the nest area by resident ants. Numeric scores were assigned to the corresponding interaction levels and used to calculate the mean aggression scores.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eForaging aggressiveness:\u003c/h3\u003e\n\u003cp\u003e\u003cb\u003ePrey killing rate\u003c/b\u003e: As the red fire ants, similar to several other social insects, are known to hunt for their prey, attacking them using their upper jaws for grappling or by stinging. Further experimental trials were conducted to examine the effects of antibiotic/microbial treatments on the ability of RIFA workers to attack and kill their prey. The foraging aggressiveness indices were compared upon the introduction of instar larvae of mealworm (body length\u0026thinsp;=\u0026thinsp;2.0-2.5 cm; weight\u0026thinsp;=\u0026thinsp;0.20 g) into the same petri plates (diameter\u0026thinsp;=\u0026thinsp;5 cm, height\u0026thinsp;=\u0026thinsp;1.3 cm, with the inner walls coated with Fluon to prevent ant escape) as 30 workers of \u003cem\u003eS. invicta\u003c/em\u003e from individual treatment and control groups (body length\u0026thinsp;=\u0026thinsp;4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mm, head width\u0026thinsp;=\u0026thinsp;0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mm). The study was adapted to a similar behavior assay of Cheng et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), with slight modifications. Here, the aggressiveness index was based on the number of interacting workers per time interval (5, 10, 15, 20, 25, and 30 min post larvae introduction into the plates), as well as the speed of kill, that is, the duration of time taken for the ants to kill the mealworm larvae following several attacks. The larvae were assumed to be dead when no movement was recorded in response to further attacks by the ants. For further confirmation, the larvae were touched with forceps. As it was difficult to ascertain the exact time of larval death, the time range was denoted as follows; (1: 0\u0026ndash;5 min, 2: 5\u0026ndash;10 min, 3: 10\u0026ndash;15 min, 4: 15\u0026ndash;20 min, 5: 20\u0026ndash;25 min, and 6: 25\u0026ndash;30 min). We deployed the scoring scale introduced by Rice and Silverman (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) as adapted for fire ants by Cheng et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, the interactions among treatment and control groups were scored on the scale of 1\u0026ndash;6; where 1 was assigned the highest possible value (highest number of interactions and fastest speed of kill, respectively), while 6 was assigned the lowest possible value (lowest number of interactions or when no death was recorded in the period under observation). Each trial was replicated three times.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFood searching\u003c/strong\u003e\u003cp\u003eTo expand on the previous findings, additional bioassays were conducted to examine the effects of artificial manipulation of the ant external microbiome on other foraging activities, such as workers\u0026rsquo; ability to search for food and the rate of food carrying. In this study, 60 workers ants from each treatment and control stock were kept in plates (12 cm \u003cem\u003e\u0026times;\u003c/em\u003e 8.5 cm \u003cem\u003e\u0026times;\u003c/em\u003e 4.2 cm; 0.3 L), and to this plate containing the ants was another plate (12 cm \u003cem\u003e\u0026times;\u003c/em\u003e 8.5 cm \u003cem\u003e\u0026times;\u003c/em\u003e 4.2 cm; 0.3 L) joined to the former using a duct, where ham sausage (weight\u0026thinsp;=\u0026thinsp;0.50\u0026ndash;0.60 g) was placed (Supplementary Fig.\u0026nbsp;1). The actual weights of the sausages were estimated and recorded at the time of placement. Here, the time taken for the first ant to locate the sausage, the number of ants that successfully located the food after 30 min, and the mean net weight of sausage taken by the ants to be eaten over a period of 48 h were recorded. The observation period was 240 h, during which the total weight of sausages taken by ants was estimated every 48 h. The sausage was replaced every 48 h to maintain its attractiveness. For the first bioassay, all dead ants were carefully removed every other day but were not replaced, while for the second bioassay, all dead ants were replaced every 48 h. All survival and behavioral bioassays were replicated three times (at least) for each treatment and control, whereas individual bioassays were conducted twice.\u003c/p\u003e\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis:\u003c/h2\u003e\u003cp\u003eWhere applicable, the collected data were subjected to normality and homogeneity tests of variances using Shapiro-Wilk Normality test (at 0.05 significance level) prior to any data analysis. To equalize the variances, cumulative mortality percentage values were subjected to log transformation, while the square roots of the scores assigned in the foraging aggression and nestmate recognition trials were used for analysis. The foraging aggression indices among treatment and control ants were determine using the formula introduced by Rice and Silverman (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) with slight modification as follows:\u003c/p\u003e\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"72\" height=\"44\"\u003e\u003c/p\u003e\u003cp\u003eHere, δi denotes the interaction score, fi is the number of interacting ants per individual observation, and T represents the sum of all interactions among ants. As the CFU counts were non-parametric, the data were analyzed using the Kruskal\u0026ndash;Wallis test, followed by Dunn\u0026rsquo;s pairwise comparisons. The cumulative mortality percentage and food carrying rate data were subjected to a repeated-measures one-way ANOVA with assessment time as a repeated factor, while other data analyses were performed using a one-way ANOVA. When a significant F test was obtained at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, multiple comparisons were performed using Tukey\u0026rsquo;s Honestly Significant Difference (HSD) tests. All statistical analyses were performed using IBM SPSS statistical software v22.0 (SPSS Inc., Chicago, IL, USA) and Statistix 10.0 (Analytical Software, Tallahassee, FL, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eIsolation and characterization of cuticle bacteria from \u003cem\u003eS. invicta\u003c/em\u003e workers:\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMorphological Characterization\u003c/strong\u003e\u003cp\u003eTo identify key symbiotic bacteria in \u003cem\u003eS. invicta\u003c/em\u003e workers, cultivable bacterial strains were isolated and identified using a conventional Colony Forming Unit (CFU) assay. Phenotypic characterization of the bacterial isolates was conducted based on evidence of their phenotypic distinctiveness. Although LB agar is a nutrient-rich medium, it has been confirmed that some environmental bacteria cannot be readily grown using this method; therefore, it is assumed that only a subset of the ants\u0026rsquo; cuticular bacterial community was successfully cultured using this isolation method.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMolecular characterization\u003c/strong\u003e\u003cp\u003eFollowing the phylogenetic placement of the bacterial isolates, 16 bacterial taxa were successfully characterized from the cuticle of RIFA workers. The details of all identified bacterial strains are presented in Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and four bacterial strains were selected based on relative abundance for behavioral bioassays (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBacterial strains characterized from RIFA cuticle for behavioral and survival bioassays\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAssigned isolate number\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBacterial strains\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSource of RIFA colony\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD121\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eVagococcus fluvialis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDongguan\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZ442\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMyroides odoratus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eZhuhai\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZ511\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eProvidencia rettgeri\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eZhuhai\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eG911\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas monteilii\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGuangzhou\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eExperimental validation of ants\u0026rsquo; external microbiome modulation following topical application of antibiotics or bacterial cultures:\u003c/p\u003e\u003cp\u003eThe number of culturable bacteria obtained from the individual samples, measured by counting the number of microbial CFUs that grew on LB agar plates, was used to indicate the effectiveness of the antibiotics/microbial homogenates treatments. A higher quantity of CFUs suggests a greater quantity of external bacteria present on the ant. Therefore, the significantly smaller number of CFUs observed on plates containing homogenates from antibiotic-treated ants (axenic group) validates the success of artificially modifying the external microbiome of the ants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The number of CFUs in the axenic group was substantially lower than those in the other treatment and control groups (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 30\u003c/sub\u003e = 9.08, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe effects of external microbiome artificial modulation on RIFA workers survival:\u003c/p\u003e\u003cp\u003eThe survival rate across the treatment and control groups was observed over a period of 10 days. The rate was similar across treatments and control within 48 h of treatment (data not shown). Meanwhile, within 4 days of treatment, the percentage cumulative mortality recorded across treatments and control was significantly different (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 28\u003c/sub\u003e = 2.26, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0296). The highest cumulative mortality (27.8%) was recorded in the ants treated with tetracycline (50 \u0026micro;g/ml). The lowest cumulative mortality at this stage was recorded among the SDW control ants (1.1%), the symbiotic ants exposed to \u003cem\u003eV. fluvialis\u003c/em\u003e D121 strain (1.1%), and the untreated nestmates (NM) at 2.2% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Similarly, at 7 days post-treatment, the highest cumulative mortality was recorded in Tetra-treated ants at 60% and Strep-treated at 36.7%, while 4.5, 26.7, and 28.9% were recorded for NM, SDW, and SLB control ants, respectively. Cumulative percent mortality ranges from 14.5\u0026ndash;26.7% across the gnotobiotic treatment group (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 28\u003c/sub\u003e = 4.90, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). At 10 days post-treatment, cumulative percent mortality was generally highest across the axenic treatment group, where 44.4%, 61.1%, 73.3%, and 88.9% were recorded in Gent, Strep, Pen, and Tetra-treated ants, respectively. In comparison, a significantly lower percentage mortality was recorded among the symbiotic group, where 32.2% was recorded for ants treated with both \u003cem\u003eV. fluvialis\u003c/em\u003e D121 and \u003cem\u003eM. odoratus\u003c/em\u003e Z442 strains. Meanwhile 41.1 and 44.4% were recorded for \u003cem\u003eP. rettgeri\u003c/em\u003e Z511 and \u003cem\u003eP. monteilii\u003c/em\u003e G911 treated ants (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 28\u003c/sub\u003e = 7.96, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In general, treatment of RIFA ants with tetracycline 50 \u0026micro;g/ml induced the highest rate of mortality in RIFA workers, as evident in the highest cumulative percentage mortality recorded across each assessment period: 4, 7, and 10 days post-treatment, which was significantly higher than that in other treatment and control groups (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 112\u003c/sub\u003e = 13.09, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eEffects of external bacteria manipulation on nestmates recognition:\u003c/p\u003e\u003cp\u003eThe hypothesis that the microbial composition of the ant's cuticle mediates recognition by nestmates was evaluated. We propose that the augmented cuticular microbes may produce certain odor or alter the cuticular hydrocarbons that attract or repel nestmates. The findings of the study revealed erratic responses of nestmates to treated and untreated ants. We recorded the highest percentage of rejection across the microbial-treated ants (i.e., the symbiotic and gnotobiotic ants), which was significantly higher than that across the antibiotic treatment group and control (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 135\u003c/sub\u003e = 6.21, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Interestingly, the ants that received no antibiotic nor microbial treatments, except for a two seconds dipping in sterile LB were rejected more than other control and antibiotics treated ants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Throughout the period of observation, no visible sign of rejection was recorded for the untreated ants (NM) that were isolated for 24 h before returning to interact with their nestmates, except for a couple of occasions where a returning ant took about 3\u0026ndash;5 min to reunite with the nestmates.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eEffects of external bacteria manipulation on workers\u0026rsquo; foraging aggressiveness:\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePrey killing\u003c/strong\u003e\u003cp\u003eIn a study conducted to assess the effects of artificial modulation of bacterial exosymbionts of RIFA on worker foraging aggressiveness, the study revealed differences in the aggressive index across treatment and control groups. The number of interacting ants per individual observation varied across treatments and control. The highest mean number of interactions per individual observation (14.7) was recorded at 10 m post larvae introduction in gnotobiotic ants treated with \u003cem\u003eM. odoratus\u003c/em\u003e Z442 strain. The highest mean interaction over the entire observation period (10.2) was also recorded among the gnotobiotic ants treated with \u003cem\u003eM. odoratus\u003c/em\u003e Z442 strain. Although the data collected were generally inconsistent across individual treatment and control groups, the fastest speed of kill was also recorded among the gnotobiotic ants, with the highest rate recorded among the ants treated with \u003cem\u003eM. odoratus\u003c/em\u003e Z442 strain, where 100% of the introduced larvae died within 5 m of placement in Petri dishes. In contrast, no larval death was recorded across axenic Pen-treated, and SLB control ants. Overall, the significantly highest aggressiveness indices of 4.30, 3.82, and 3.13 were recorded in the gnotobiotic \u003cem\u003eM. odoratus\u003c/em\u003e Z442 strain treated ants at 10 (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 30\u003c/sub\u003e = 8.47, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), 5 (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 30\u003c/sub\u003e = 11.32, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and 15 min after larval introduction (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 30\u003c/sub\u003e = 6.56, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), respectively. The lowest aggressive index (0.08) was recorded in the SDW control ants at 15 min post larvae introduction. Generally, across all treatment and control groups, the mean number of interacting ants decreased with an increase in time after larval introduction (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe aggressive index of axenic, gnotobiotic, symbiotic and the control workers of \u003cem\u003eS. invicta\u003c/em\u003e upon the introduction of instar larvae of mealworm, \u003cem\u003eT. molitor\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"16\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDuration\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c8\" namest=\"c5\"\u003e\u003cp\u003eAxenic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c12\" namest=\"c9\"\u003e\u003cp\u003eSymbiotic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c16\" namest=\"c13\"\u003e\u003cp\u003eGnotobiotic\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSterile LB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNestmates\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSterile water\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStreptomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePenicillin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTetracycline\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eGentamycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cem\u003eV. fluvialis\u003c/em\u003e D121\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cem\u003eM. odoratus\u003c/em\u003e Z442\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cem\u003eP. rettgeri\u003c/em\u003e Z511\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cem\u003eP. monteilii\u003c/em\u003e G911\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u003cem\u003eV. fluvialis\u003c/em\u003e D121\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e\u003cem\u003eM. odoratus\u003c/em\u003e Z442\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e\u003cem\u003eP. rettgeri\u003c/em\u003e Z511\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e\u003cem\u003eP. monteilii\u003c/em\u003e G911\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e5min\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48 abc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e3.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e1.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 abc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 bc\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e10min\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 abc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 abc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 abc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e2.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e4.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e3.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 abc\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e15min\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e3.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 ab\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e20min\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 cd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 abcd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003cp\u003ebcd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 abcd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 cd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 bcd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 bcd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 cd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 abc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e3.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e2.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 bcd\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e25min\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e1.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 ab\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e30min\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 abc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 abc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 abc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e1.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 abc\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eMeans aggressive index (\u0026plusmn;\u0026thinsp;S.E.) with different letters within the same row indicate significant differences among treatments and control at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Tukey\u0026rsquo;s HSD test after One-way ANOVA). In the columns marked (\u003cb\u003e-\u003c/b\u003e); the aggressive index was not computed in trials where no larvae death was recorded after 30 min post larvae introduction, or for any point of observation where no worker was interacting with the larvae.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFood searching and rate of food carrying\u003c/strong\u003e\u003cp\u003eThe ability of worker ants to locate their food and the number of workers actively searching for food at 30 min post food placement varies across treatment and control groups. The symbiotic ants, less the ones treated with \u003cem\u003eP. monteilii\u003c/em\u003e G911 strain, took lesser time to locate the food in comparison with other treatments and control ants (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 30\u003c/sub\u003e = 2.59, \u003cem\u003eP\u003c/em\u003e = 0.0139). The average time taken for the first ant to locate the sausage was 3.38 min, recorded in the symbiotic ants treated with \u003cem\u003eP. rettgeri\u003c/em\u003e Z511 strain; which was the fastest rate recorded across all treatments and control (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Following 30 min of observation, the number of ants that successfully located the food was similar across all treatments and control (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 30\u003c/sub\u003e = 0.79, \u003cem\u003eP\u003c/em\u003e = 0.6692; Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The assessment of food carrying rate across all treatment and control groups revealed relative similarities at 48 h post sausage placement (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 28\u003c/sub\u003e = 1.3, \u003cem\u003eP\u003c/em\u003e = 0.2664). Thereafter, we recorded significant differences among treatments and control for individual assessment time, 96 h; (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 28\u003c/sub\u003e = 2.38, \u003cem\u003eP\u003c/em\u003e = 0.0246), 144 h; (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 28\u003c/sub\u003e = 6.54, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001), 192 h; (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 28\u003c/sub\u003e = 7.15, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001), and 240 h of observation; (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 28\u003c/sub\u003e = 5.39, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Here, we observed a decline in the rate of food carrying across all treatments and control with an increase in observation period (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e4, 56\u003c/sub\u003e = 172.82, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). In addition, from these results, we established a correlation between the number of surviving ants and the weight of food carried. Here, the significantly low food-carrying rate that was recorded among the axenic Tetra-treated ants and the SLB-control ants was predicted to be partly due to the high mortality rate recorded among these two groups in comparison to other treatment and control groups (data not shown). Bearing this outcome in mind, a second trial was conducted in which dead ants from individual treatments and control were removed and replaced with new ones from the stock at each assessment date. The outcome revealed a similar trend to the first trial, where, aside the Pen-treated ants, the lowest average weight of food carried was recorded across axenic ants, although not statistically different from the average weight recorded in the control and other treatment groups (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e14, 28\u003c/sub\u003e = 1.61, \u003cem\u003eP\u003c/em\u003e = 0.0802; Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Similar to the first trial, a decline in the rate of food carrying across all treatments and control with an increase in observation period was observed (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e4, 56\u003c/sub\u003e = 43.2, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). Generally, within 48 h of observation, the mean weight of sausage carried among the axenic ants\u0026rsquo; ranges from 0.37-0.50g, 0.44-0.63g for the gnotobiotic ants, while 0.53-0.60g and 0.32-0.40g were recorded for the symbiotic and control ants, respectively. Following 240 h of observation, average weight of sausage carried ranges from 0.21-0.25g, 0.22-0.38g, 0.25-0.30g, and 0.26-0.45g for the control, axenic, symbiotic, and gnotobiotic ants, respectively.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe epidermis of insects serves as a protective layer against invading pathogens and can be naturally colonized by bacteria (Douglas, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Pathogenic and protective microbes live on the surface of insect cuticles and can mediate direct or indirect changes in host physiology and other biochemical processes (Davis et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Keiser et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; McFall-Ngai et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The current study tested the hypothesis that the external microbial composition of RIFA influences survival rate and certain social behaviors, including worker aggressiveness, nestmate recognition, and foraging activities. Artificial modulation of the external microbiomes of ants via topical application of antibiotics or microbial monocultures was successful. Data from the validation test revealed significantly greater CFU counts in the symbiotic and gnotobiotic ants than in the axenic and untreated control ants.\u003c/p\u003e\u003cp\u003eThe successful removal of cuticle bacterial symbionts mediated a significant reduction in the survival rate of workers. Higher mortality rates were recorded across the treatments in the axenic group. Specifically, the highest mortality rate was recorded in ants treated with tetracycline. Meanwhile, the symbiotic ants revealed a corresponding induced survival rate that gradually decreased with an increase in the number of days after topical application of bacterial monocultures. Consequently, the results of the survival bioassay showed that successful artificial modification of the cuticle bacterial symbionts of RIFA directly affects the survival rate. This outcome is related to a previous study that suggested that the exosymbiotic microbiome of insects can influence their health status and life history (Ezenwa et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In addition, dissimilarities in survival rates between control and microbial treated red harvester ants (\u003cem\u003ePogonomyrmex barbatus\u003c/em\u003e) were reported by Dosmann et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). When an organism comes in contact with bacteria, either pathogenic or benign, its health status, behavior, and other social functions are affected (Ben-Yosef et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Freitak et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Freitak et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Sharon et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The diversity of microbial communities within each member of a social group is related to individual experiences such as diet, as well as the interactions with the microbiota of nestmates. In addition, the effects on the host can extend over a very long period of time (Wallace et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe evolution of nestmate recognition has been of great interest to the scientific community. Social insects strongly rely on odours to differentiate between within- and between-colony members. In the current study, cuticle microbes influenced the ability of ants to recognize their nestmates. We observed the highest rate of rejection among the microbial-treated ants, whereas for the antibiotics-treated ants, the rate of rejection was relatively low, albeit not statistically different in comparison to the control ants. The results revealed that ants with augmented cuticular microbiota were more likely to be rejected by their nestmates. Although, it appears that the treated ants are less likely to be rejected by their nestmates due to the absence or artificial removal of a familiar odour. Rather, ants are more likely to be rejected because of the presence of a foreign odour or artificial introduction of novel cuticle microbes. This finding is consistent with that of Dosmann et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) in red harvester ants. In a few other related studies, it has been suggested that the rejection of microbial-treated insects could be related to a social immunity response, whereby nestmates perceive insects with augmented cuticular microbiomes as sick nestmates that need to be isolated (Cremer et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Dosmann et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Similar findings have been reported in other economic insects, including leaf-cutting ants (Richard et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), termites (Matsuura, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and \u003cem\u003eDrosophila melanogaster\u003c/em\u003e (Liz\u0026eacute; et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Our findings, in addition to the aforementioned studies, retrace the important roles of external microbes of insects in nestmate recognition. Aside the microbial composition on insect cuticles, other factors have been suggested could be involved. The most commonly mentioned are cuticular chemical hydrocarbons (CHCs), which have been described as important nestmate discriminators in several social insects. CHCs are the predominant compounds responsible for intraspecific recognition within a colony; however, other volatile factors are thought to contribute to the signature odours of individual colonies (Sturgis and Gordon, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; van Zweden and d\u0026rsquo;Ettorre, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Vander Meer and Morel, 1998).\u003c/p\u003e\u003cp\u003eTo understand the role of external microbes in RIFA foraging activities, workers\u0026rsquo; aggressiveness upon contact with prey, the ability to search for food, and the rate of food carrying were examined. The values obtained for the aggressive index varied across control and treatments, where \u003cem\u003eM. odoratus\u003c/em\u003e Z442 strain, tested on the gnotobiotic ants, caused the fastest kill as well as the highest number of interactions, resulting in the highest aggressive index. With respect to the rate of food searching, the symbiotic ants generally needed less time to locate the food in comparison to the other treatment and control groups. However, within 30 min of sausage placement, the number of ants that successfully located the food was similar across treatments and control. The initial period of observation also revealed similarities in the average weight of sausage taken across treatments and control. With an increase in the observation time, a significant reduction was recorded among the antibiotic-treated ants, specifically in ants treated with tetracycline. Across this group, the decreasing number of surviving ants might be partly responsible for the decline in their foraging activities, as the percentage of mortality recorded was generally higher across the antibiotic-treated ants throughout the experiment. Tellingly, a second trial, in which dead ants were replaced, confirmed this assumption. Here, a reduction in foraging activity across antibiotic-treated ants, which was not statistically different from other treatments and control, was recorded. Several factors directly influence ant foraging activities. For instance, the volatiles produced by microorganisms on insect cuticle surfaces may have a direct effect on many aspects of insect social behavior, including foraging activities (Davis et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In other insects, cuticular microbiota are also known to play major roles. For instance, Parks et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported a 10-fold decrease in the foraging aggressiveness of spiders toward a prey artificially placed in their web following topical application of bacterial monocultures of \u003cem\u003eDermacoccus nishinomiyaensis\u003c/em\u003e and \u003cem\u003eStaphylococcus saprophyticus\u003c/em\u003e. The ability of RIFA to successfully invade new areas is determined by a variety of factors, namely, their aggressive behavior, competitive ability, and fighting capability of both single individuals and multiple colonies (Chen et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lai et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Such characteristics can result in the successful defeat of another group, even when the invaders are numerically outnumbered, as seen in social animal battles (Traniello and Beshers, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). According to Obin and Meer (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), species with higher levels of aggression are more likely to gain competitive advantage in interspecific encounters.\u003c/p\u003e\u003cp\u003eIn conclusion, the examined cuticular bacterial species suggested existing interactions between them and their ant host. Furthermore, the topical application of bacterial homogenates to ants significantly improved their survival and foraging aggressiveness. These outcomes indicate the importance of microbial exosymbionts in the ecology and behavior of social insects and suggest that further research is necessary. Although this study has shed light on the beneficial effects of cuticular bacterial symbionts of RIFA, the specific mechanisms underlying these beneficial effects are yet to be fully explored. It is crucial for future research to focus on this area and uncover the mechanisms of action of these symbionts in enhancing host survival and altering ant social behavior.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest Statement\u003c/h2\u003e\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study received financial support through the Research Fund for International Young Scientists (32150410344), a research grant received from National Natural Science Foundation of China (NSFC).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBSB and YX conceived and designed research. BSB, JAS, and LN conducted the experiments. AI and BSB analyzed the data. BSB and JAS prepared the first manuscript draft and was revised by YX and CJ. The funding for the study was obtained by BSB and YX. All authors read and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe are grateful to all of our colleagues at the Red Imported Fire Ants Research Centre of South China Agricultural University, Guangzhou, China, for their assistance in samples collection, culture media preparation, and procurement of experimental items used for the study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData supporting the results can be found in NCBI\u0026rsquo;s Genbank database following this link - http://www.ncbi.nlm.nih.gov/- accessed on February 16, 2025.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBamisile, B.S., Nie, L., Siddiqui, J.A., Ramos Aguila, L.C., Akutse, K.S., Jia, C., and Xu, Y. (2023). Assessment of mound soils bacterial community of the red imported fire ant, Solenopsis invicta across Guangdong province of China. Sustainability\u003cem\u003e 15\u003c/em\u003e, 1350.\u003c/li\u003e\n\u003cli\u003eBen‐Yosef, M., Jurkevitch, E., and Yuval, B. (2008). Effect of bacteria on nutritional status and reproductive success of the Mediterranean fruit fly Ceratitis capitata. Physiological entomology\u003cem\u003e 33\u003c/em\u003e, 145-154.\u003c/li\u003e\n\u003cli\u003eChen, J., Rashid, T., and Feng, G. (2014). A comparative study between Solenopsis invicta and Solenopsis richteri on tolerance to heat and desiccation stresses. PLoS One\u003cem\u003e 9\u003c/em\u003e, e96842.\u003c/li\u003e\n\u003cli\u003eCheng, D., Chen, S., Huang, Y., Pierce, N.E., Riegler, M., Yang, F., Zeng, L., Lu, Y., Liang, G., and Xu, Y. (2019). Symbiotic microbiota may reflect host adaptation by resident to invasive ant species. PLoS pathogens\u003cem\u003e 15\u003c/em\u003e, e1007942.\u003c/li\u003e\n\u003cli\u003eCremer, S., Armitage, S.A., and Schmid-Hempel, P. (2007). Social immunity. Current biology\u003cem\u003e 17\u003c/em\u003e, R693-R702.\u003c/li\u003e\n\u003cli\u003eDavis, T.S., Crippen, T.L., Hofstetter, R.W., and Tomberlin, J.K. (2013). Microbial volatile emissions as insect semiochemicals. Journal of chemical ecology\u003cem\u003e 39\u003c/em\u003e, 840-859.\u003c/li\u003e\n\u003cli\u003eDosmann, A., Bahet, N., and Gordon, D.M. (2016). Experimental modulation of external microbiome affects nestmate recognition in harvester ants (Pogonomyrmex barbatus). PeerJ\u003cem\u003e 4\u003c/em\u003e, e1566.\u003c/li\u003e\n\u003cli\u003eDouglas, A.E. (2015). Multiorganismal insects: diversity and function of resident microorganisms. Annual review of entomology\u003cem\u003e 60\u003c/em\u003e, 17-34.\u003c/li\u003e\n\u003cli\u003eEzenwa, V.O., Gerardo, N.M., Inouye, D.W., Medina, M., and Xavier, J.B. (2012). Animal behavior and the microbiome. Science\u003cem\u003e 338\u003c/em\u003e, 198-199.\u003c/li\u003e\n\u003cli\u003eFreitak, D., Heckel, D.G., and Vogel, H. (2009). Bacterial feeding induces changes in immune-related gene expression and has trans-generational impacts in the cabbage looper (Trichoplusia ni). Frontiers in zoology\u003cem\u003e 6\u003c/em\u003e, 1-11.\u003c/li\u003e\n\u003cli\u003eFreitak, D., Wheat, C.W., Heckel, D.G., and Vogel, H. (2007). Immune system responses and fitness costs associated with consumption of bacteria in larvae of Trichoplusia ni. Bmc Biology\u003cem\u003e 5\u003c/em\u003e, 1-13.\u003c/li\u003e\n\u003cli\u003eGordon, D., Chu, J., Lillie, A., Tissot, M., and Pinter, N. (2005). Variation in the transition from inside to outside work in the red harvester ant Pogonomyrmex barbatus. Insectes Sociaux\u003cem\u003e 52\u003c/em\u003e, 212-217.\u003c/li\u003e\n\u003cli\u003eGrenham, S., Clarke, G., Cryan, J.F., and Dinan, T.G. (2011). Brain\u0026ndash;gut\u0026ndash;microbe communication in health and disease. Frontiers in physiology\u003cem\u003e 2\u003c/em\u003e, 16175.\u003c/li\u003e\n\u003cli\u003eHamby, K.A., and Becher, P.G. (2016). Current knowledge of interactions between Drosophila suzukii and microbes, and their potential utility for pest management. Journal of Pest Science\u003cem\u003e 89\u003c/em\u003e, 621-630.\u003c/li\u003e\n\u003cli\u003eHolway, D.A., Lach, L., Suarez, A.V., Tsutsui, N.D., and Case, T.J. (2002). The causes and consequences of ant invasions. Annual review of ecology and systematics\u003cem\u003e 33\u003c/em\u003e, 181-233.\u003c/li\u003e\n\u003cli\u003eHu, L., Balusu, R., Zhang, W.-Q., Ajayi, O., Lu, Y.-Y., Zeng, R.-S., Fadamiro, H., and Chen, L. (2018). Intra-and inter-specific variation in alarm pheromone produced by Solenopsis fire ants. Bulletin of entomological research\u003cem\u003e 108\u003c/em\u003e, 667-673.\u003c/li\u003e\n\u003cli\u003eJun, H., Yi-Juan, X., Yong-Yue, L., and Ling, Z. (2012). Effects of Solenopsis invicta invasion on the diversity of spider communities in a corn field. Yingyong Shengtai Xuebao\u003cem\u003e 23\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eKafle, L., Wu, W.J., Kao, S.S., and Shih, C.J. (2011). Efficacy of Beauveria bassiana against the red imported fire ant, Solenopsis invicta (Hymenoptera: Formicidae), in Taiwan. Pest management science\u003cem\u003e 67\u003c/em\u003e, 1434-1438.\u003c/li\u003e\n\u003cli\u003eKeiser, C.N., Shearer, T.A., DeMarco, A.E., Brittingham, H.A., Knutson, K.A., Kuo, C., Zhao, K., and Pruitt, J.N. (2016). Cuticular bacteria appear detrimental to social spiders in mixed but not monoculture exposure. Current Zoology\u003cem\u003e 62\u003c/em\u003e, 377-384.\u003c/li\u003e\n\u003cli\u003eLai, L.-C., Hua, K.-H., and Wu, W.-J. (2015). Intraspecific and interspecific aggressive interactions between two species of fire ants, Solenopsis geminata and S. invicta (Hymenoptera: Formicidae), in Taiwan. Journal of Asia-Pacific Entomology\u003cem\u003e 18\u003c/em\u003e, 93-98.\u003c/li\u003e\n\u003cli\u003eLei, W., XU, Y.-j., Ling, Z., and LU, Y.-y. (2019). Impact of the red imported fire ant Solenopsis invicta Buren on biodiversity in South China: A review. Journal of Integrative Agriculture\u003cem\u003e 18\u003c/em\u003e, 788-796.\u003c/li\u003e\n\u003cli\u003eLi, J., Guo, Q., Lin, M., Jiang, L., Ye, J., Chen, D., Li, Z., Dai, J., and Han, S. (2016). Evaluation of a new entomopathogenic strain of Beauveria bassiana and a new field delivery method against Solenopsis invicta. Plos One\u003cem\u003e 11\u003c/em\u003e, e0158325.\u003c/li\u003e\n\u003cli\u003eLiz\u0026eacute;, A., McKay, R., and Lewis, Z. (2014). Kin recognition in Drosophila: the importance of ecology and gut microbiota. The ISME journal\u003cem\u003e 8\u003c/em\u003e, 469-477.\u003c/li\u003e\n\u003cli\u003eLu, Y. (2014). Long‐distance spreading speed and trend prediction of red imported fire ant, Solenopsis invicta Buren, in mainland China. Guangdong Agricultural Science\u003cem\u003e 41\u003c/em\u003e, 70.\u003c/li\u003e\n\u003cli\u003eŁukasik, P., van Asch, M., Guo, H., Ferrari, J., and Charles J. Godfray, H. (2013). Unrelated facultative endosymbionts protect aphids against a fungal pathogen. Ecology letters\u003cem\u003e 16\u003c/em\u003e, 214-218.\u003c/li\u003e\n\u003cli\u003eMatsuura, K. (2001). Nestmate recognition mediated by intestinal bacteria in a termite, Reticulitermes speratus. Oikos\u003cem\u003e 92\u003c/em\u003e, 20-26.\u003c/li\u003e\n\u003cli\u003eMcFall-Ngai, M., Hadfield, M.G., Bosch, T.C., Carey, H.V., Domazet-Lo\u0026scaron;o, T., Douglas, A.E., Dubilier, N., Eberl, G., Fukami, T., and Gilbert, S.F. (2013). Animals in a bacterial world, a new imperative for the life sciences. Proceedings of the National Academy of Sciences\u003cem\u003e 110\u003c/em\u003e, 3229-3236.\u003c/li\u003e\n\u003cli\u003eNie, L., Ni, M., Ning, D., Ran, H., Hassan, B., and Xu, Y. (2019). Comparing mechanisms of competition among introduced and resident ants in China: from behavior to trophic position (Hymenoptera: Formicidae). Myrmecological News\u003cem\u003e 29\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eObin, M.S., and Meer, R.K.V. (1989). Between-and within-species recognition among imported fire ants and their hybrids (Hymenoptera: Formicidae): Application to hybrid zone dynamics. Annals of the Entomological Society of America\u003cem\u003e 82\u003c/em\u003e, 649-652.\u003c/li\u003e\n\u003cli\u003eParks, O.B., Kothamasu, K.S., Ziemba, M.J., Benner, M., Cristinziano, M., Kantz, S., Leger, D., Li, J., Patel, D., and Rabuse, W. (2018). Exposure to cuticular bacteria can alter host behavior in a funnel-weaving spider. Current zoology\u003cem\u003e 64\u003c/em\u003e, 721-726.\u003c/li\u003e\n\u003cli\u003eRen, C., Webster, P., Finkel, S.E., and Tower, J. (2007). Increased internal and external bacterial load during Drosophila aging without life-span trade-off. Cell metabolism\u003cem\u003e 6\u003c/em\u003e, 144-152.\u003c/li\u003e\n\u003cli\u003eRice, E.S., and Silverman, J. (2013). Submissive behaviour and habituation facilitate entry into habitat occupied by an invasive ant. Animal Behaviour\u003cem\u003e 86\u003c/em\u003e, 497-506.\u003c/li\u003e\n\u003cli\u003eRichard, F.-J., Poulsen, M., Hefetz, A., Errard, C., Nash, D.R., and Boomsma, J.J. (2007). The origin of the chemical profiles of fungal symbionts and their significance for nestmate recognition in Acromyrmex leaf-cutting ants. Behavioral Ecology and Sociobiology\u003cem\u003e 61\u003c/em\u003e, 1637-1649.\u003c/li\u003e\n\u003cli\u003eSharon, G., Segal, D., Ringo, J.M., Hefetz, A., Zilber-Rosenberg, I., and Rosenberg, E. (2010). Commensal bacteria play a role in mating preference of Drosophila melanogaster. Proceedings of the National Academy of Sciences\u003cem\u003e 107\u003c/em\u003e, 20051-20056.\u003c/li\u003e\n\u003cli\u003eSturgis, S.J., and Gordon, D.M. (2012). Nestmate recognition in ants (Hymenoptera: Formicidae): a review. Myrmecological news\u003cem\u003e 16\u003c/em\u003e, 101-110.\u003c/li\u003e\n\u003cli\u003eTraniello, J.F., and Beshers, S.N. (1991). Maximization of foraging efficiency and resource defense by group retrieval in the ant Formica schaufussi. Behavioral Ecology and Sociobiology\u003cem\u003e 29\u003c/em\u003e, 283-289.\u003c/li\u003e\n\u003cli\u003evan Zweden, J.S., and d\u0026rsquo;Ettorre, P. (2010). Nestmate recognition in social insects and the role of hydrocarbons. Insect hydrocarbons: biology, biochemistry and chemical ecology\u003cem\u003e 11\u003c/em\u003e, 222-243.\u003c/li\u003e\n\u003cli\u003eVander Meer, R., and Morel, L. (1998). Nestmate recognition in ants. Pheromone communication in social insects. Vander Meer RK, Breed MD, Winston ML \u0026amp; Espelie KE, 79-103.\u003c/li\u003e\n\u003cli\u003eWallace, J.R., Gordon, M.C., Hartsell, L., Mosi, L., Benbow, M.E., Merritt, R.W., and Small, P.L. (2010). Interaction of Mycobacterium ulcerans with mosquito species: implications for transmission and trophic relationships. Applied and environmental microbiology\u003cem\u003e 76\u003c/em\u003e, 6215-6222.\u003c/li\u003e\n\u003cli\u003eWilder, S.M., Holway, D.A., Suarez, A.V., LeBrun, E.G., and Eubanks, M.D. (2011). Intercontinental differences in resource use reveal the importance of mutualisms in fire ant invasions. Proceedings of the National Academy of Sciences\u003cem\u003e 108\u003c/em\u003e, 20639-20644.\u003c/li\u003e\n\u003cli\u003eYang, C.C.S., Shoemaker, D.D., Wu, J.C., Lin, Y.K., Lin, C.C., Wu, W.J., and Shih, C.J. (2009). Successful establishment of the invasive fire ant Solenopsis invicta in Taiwan: insights into interactions of alternate social forms. Diversity and Distributions\u003cem\u003e 15\u003c/em\u003e, 709-719.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-tropical-insect-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtis","sideBox":"Learn more about [International Journal of Tropical Insect Science](http://link.springer.com/journal/42690)","snPcode":"42690","submissionUrl":"https://www.editorialmanager.com/jtis/default2.aspx","title":"International Journal of Tropical Insect Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Formicidae, social insects, physiology, mortality, cuticle bacteria, invasive species","lastPublishedDoi":"10.21203/rs.3.rs-6990545/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6990545/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA wide range of interactions between insects and their associated microbial communities have been documented, with profound implications for host characteristics such as development, biology, and behavior. While much emphasis has been placed on bacterial endosymbionts, the mutual interactions between insects and their external symbionts are often overlooked. In this study, we investigated the potential of the external microbiome to mediate survival and behavioral changes in the workers of red imported fire ants (RIFA). Using culture-based methods, the bacterial species present on the cuticle of the ants were isolated and identified. Experimental manipulation of ant exosymbionts was achieved through the treatment of workers with bacterial monocultures, antibiotics, or a combination of both. Artificial modification of ant cuticle bacterial symbionts revealed significant changes in survival rates and behavioral patterns. The removal of ants\u0026rsquo; cuticle exosymbionts induced about 89% cumulative mortality within 10 days of treatment, significantly higher than that observed in other treatments and the control group. Similarly, artificial manipulation of ant cuticle bacterial symbionts impaired the ants\u0026rsquo; ability to be recognized by their nestmates, as worker ants with an altered cuticle experienced a higher rejection rate compared to those with an intact external microbiome. In addition, foraging activities were affected, including the workers\u0026rsquo; ability to kill prey, search for food, and the weight of food carried over a given duration. These results revealed that cuticular bacteria influence both survival and certain social behaviors in RIFA. Understanding the diversity and potential use of these exosymbiotic bacteria would provide insights into promising biological strategies for RIFA management.\u003c/p\u003e","manuscriptTitle":"Experimental modulation of ants’ external microbiome revealed symbiont-mediated survival and behavioral modifications in workers of Solenopsis invicta","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-08 05:02:46","doi":"10.21203/rs.3.rs-6990545/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-28T15:01:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-24T01:22:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-21T11:26:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158583440033222187781503986008405991459","date":"2025-08-11T17:09:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240076586578188426093489420119271873918","date":"2025-08-11T06:56:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-10T17:14:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"181524294850921643171169988027202239402","date":"2025-08-06T09:49:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13654411283194567193796035382984622134","date":"2025-08-05T16:54:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-05T16:11:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-01T06:25:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-01T06:24:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Tropical Insect Science","date":"2025-06-27T09:59:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-tropical-insect-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtis","sideBox":"Learn more about [International Journal of Tropical Insect Science](http://link.springer.com/journal/42690)","snPcode":"42690","submissionUrl":"https://www.editorialmanager.com/jtis/default2.aspx","title":"International Journal of Tropical Insect Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bd1a766c-b4b5-4e5c-8ba1-0690a0cf459b","owner":[],"postedDate":"August 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:07:36+00:00","versionOfRecord":{"articleIdentity":"rs-6990545","link":"https://doi.org/10.1007/s42690-026-01768-9","journal":{"identity":"international-journal-of-tropical-insect-science","isVorOnly":false,"title":"International Journal of Tropical Insect Science"},"publishedOn":"2026-02-09 15:59:11","publishedOnDateReadable":"February 9th, 2026"},"versionCreatedAt":"2025-08-08 05:02:46","video":"","vorDoi":"10.1007/s42690-026-01768-9","vorDoiUrl":"https://doi.org/10.1007/s42690-026-01768-9","workflowStages":[]},"version":"v1","identity":"rs-6990545","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6990545","identity":"rs-6990545","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}