# Sugar supplementation affects ant-mealybug mutualism and promotes natural enemies for mealybug control in pineapple systems

Marie Bourel, Eva Faustin, Bernard Raymond Abufera, Capucine Perrey, Camille Stasiolojc, Anaïs Chailleux, Felix Wäckers, Philippe Tixier, Dominique Carval

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Marie Bourel, Eva Faustin, Bernard Raymond Abufera, Capucine Perrey, Camille Stasiolojc, et al. Sugar supplementation affects ant-mealybug mutualism and promotes natural enemies for mealybug control in pineapple systems. Biological Control, 2026, 213, pp.105975. 10.1016/j.biocontrol.2026.105975. hal-05504424

HAL Id: hal-05504424

https://hal.science/hal-05504424v1

Submitted on 11 Feb 2026

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<figure><img src="image_2.png" /></figure>
Sugar supplementation affects ant-mealybug mutualism and promotes
natural enemies for mealybug control in pineapple systems

Marie Bourel<sup>a,b</sup>, Eva Faustin<sup>a,b</sup>, Bernard Raymond Abufera<sup>a,b</sup>, Capucine Perrey<sup>a,b</sup>, Camille Stasiolojc<sup>a,b</sup>, Anaïs Chailleux<sup>c,d</sup>, Felix Wäckers<sup>e</sup>, Philippe Tixier<sup>a,f</sup>, Dominique Carval<sup>a,b,*</sup><sup>g</sup>

HIGHLIGHTS

* Mealybug abundance was positively associated with ant abundance.

* Sugar supplementation reduced ant abundance on pineapple plants by 42%.

* Predator abundance increased by 105% near sugar feeders in supplemented plots.

* Three dominant ant species strongly interacted with mealybugs in sentinel prey assays.

* Sugar supplementation may enhance biological control in tropical pineapple systems.

ARTICLE INFO

Keywords:

Sugar feeders

Mutualism disruption

Tropical agronomy

Spiders

Coccinellid

Computer vision

YOLO

Sentinel prey

A B S T R A C T

Honeydew-producing hemipterans establish mutualistic interactions with ants, which collect honeydew while providing protection against natural enemies and other services. Although this association benefits hemipterans, it often exacerbates crop damage by increasing pest abundance. Providing alternative sugar resources has been proposed as a strategy to disrupt these associations, but its effectiveness remains poorly tested in tropical agrosystems. Here, we investigated whether sugar feeders could reduce ant-mealybug mutualism in pineapple (Ananas comosus) fields on La Réunion, where the mealybug *Dysmicoccus brevipes*, a vector of Pineapple Mealybug Wilt-associated viruses (PMWaVs), poses a major threat to production. We deployed sugar feeders in replicated field plots and monitored ant, mealybug and predator abundances over eleven months, complemented by sentinel prey assays to quantify ant-mealybug interaction rates and mealybug removal. Mealybug abundance was positively associated with ant presence, while sugar supplementation reduced ant abundance by 42% on pine- apple plants compared to controls. Predator abundance was 105% higher in supplemented zones, likely due to both direct nutritional benefits and lower ant interference, but declined with distance from feeders. Three ant species, *Solenopsis geminata*, *Pheidole megacephala* and *Brachymyrmex cordemoyi* accounted for most interactions with mealybugs, with species-specific temporal dynamics but no response to sugar supplementation. Sugar supplementation represents a promising management lever, but its effect on mealybug remains context- dependent. Integrating sugar provisioning with complementary agroecological practices, such as biological control and habitat diversification, may maximize its impact and contribute to sustainable management of pineapple mealybug wilt disease.

<footnote><sup>a</sup> GECO, Univ Montpellier, CIRAD, Montpellier, France</footnote>

<footnote><sup>b</sup> CIRAD, UPR GECO, F-97455 Saint-Pierre, La Réunion, France</footnote>

<footnote><sup>c</sup> HortSys, Univ Montpellier, CIRAD, Montpellier, France</footnote>

<footnote><sup>d</sup> CIRAD, UPR Hortsys, F-97455 Saint-Pierre, La Réunion, France</footnote>

<footnote><sup>e</sup> Biobest, Westerlo, Belgium</footnote>

<footnote><sup>f</sup> CIRAD, UPR GECO, Vientiane, the Lao People's Democratic Republic</footnote>

<footnote>* Corresponding author at: GECO, Univ Montpellier, CIRAD, Montpellier, France.</footnote>

<footnote>E-mail address: dominique.carval@cirad.fr (D. Carval).</footnote>
# 1. Introduction

Ant-hemipteran mutualism is a widespread ecological interaction in which ants provide honeydew-producing hemipterans with services, including protection against natural enemies, in exchange for carbohydrate-rich secretions (Campos et al., 2006; Cheng et al., 2015; Feng et al., 2015; Oliver et al., 2008; Tanga et al., 2015; Way, 1954). While these interactions often provide hemipterans with protection and can lead to higher population levels, they can also have negative consequences for plants by increasing hemipteran densities and associated feeding damage (Cudjoe et al., 1993; Daane et al., 2007; Itioka & Inoue, 1996; Mgocheki & Addison, 2009; Zhou et al., 2013).

Early observations suggested that providing alternative carbohydrate resources to ants could disrupt these associations. Way (1954) showed that when honey was supplied to *Oecophylla longinoda* (Latreille, 1802) (Hymenoptera: Formicidae), workers neglected their mutualistic partner *Saissetia zanzibarensis* Williams, 1953 (Hemiptera: Coccidae) and even preyed upon them, a behavior reversed when honey was removed. Building on this idea, Becerra & Venable (1989) proposed the "ant distraction hypothesis" in which Extra-Floral Nectaries (EFNs) may serve to divert ants from tending honeydew-producing hemipterans, thereby reducing the intensity of hemipteran feeding damage. According to this hypothesis, the primary benefit of EFNs to plants is not only the recruitment of ants against herbivores, but also the reduction of ant services provided to hemipterans.

Since then, a number of field studies have demonstrated that providing sugar reduces ant attendance on hemipterans, sometimes within a few days, and sometimes after prolonged supplementation (Correa et al., 2023; Foronda et al., 2025; Nagy et al., 2013, 2015; Pérez-Rodríguez et al., 2021; Wäckers et al., 2017). This reduction in tending can also increase parasitism and predation pressure on hemipterans (Nagy et al., 2013, 2015; Pérez-Rodríguez et al., 2021; Wäckers et al., 2017). Additionally, several studies have shown that sugar supplementation can directly benefit natural enemies, resulting in reduced hemipteran densities (Leman et al., 2023; Tena et al., 2015). However, the consequences for hemipteran populations appear context-dependent: while some studies reported significant declines in hemipteran densities, others found no clear effect depending on the season, location, or hemipteran species (Correa et al., 2023; Foronda et al., 2025; Nagy et al., 2013, 2015).

Pineapple (*Ananas comosus* (L.) Merr.) agrosystems provide a relevant system to test these hypotheses. The pineapple mealybug *Dysmigococcus brevipes* (Cockerell, 1893) (Hemiptera: Pseudococcidae) forms a mutualistic relationship with ants, facilitating its population growth (Dey et al., 2018; González-Hernández et al., 1999; Rohrbach et al., 1988). This mutualism has major consequences, as *D. brevipes* is the primary vector of Pineapple Mealybug Wilt-associated viruses (PMWaVs), the causal agents of pineapple wilt disease, which severely impacts yields in many tropical regions including La Réunion (Dey et al., 2018; Massé et al., 2021; Nurbel et al., 2021; Sether et al., 1998). In La Réunion, pineapple mealybug management has long relied almost exclusively on chemical control, primarily through the use of spirotetramat, the only insecticide authorized against this pest. At the European level, the approval of the active substance spirotetramat expired on 30 April 2024, as no renewal application was submitted (European Commission, 2022). Consequently, all plant protection products containing spirotetramat were withdrawn in France as of April 2024, with use of existing stocks permitted until October 2025 (ANSES, 2024). To date, no formal integrated pest management (IPM) strategy based on biological control has been implemented on the island, and no predator or parasitoid species has been officially evaluated under local conditions. Although several studies are ongoing, no published data are yet available. Despite its importance, the potential to manipulate ant-mealybug interactions in pineapple through sugar supplementation remains unexplored.

Through an 11-month field survey, combining plant sampling and

sentinel prey experiment, we assessed whether the provision of alternative sugar resources can disrupt the ant-mealybug mutualism in pineapple fields in La Réunion. Specifically, we hypothesized that sugar supplementation (i) reduces ant abundance on pineapples, (ii) reduces mealybug abundance on pineapples, (iii) increases predator abundance on pineapples and (iv) alters the frequency and outcome of ant-mealybug interactions.

# 2. Methods

## 2.1. Field characteristics and experimental design

This study was carried out between April 2024 and February 2025 in southern La Réunion. The southern region of La Réunion Island is characterized by a humid tropical climate with marked seasonal and spatial variability. The austral summer is the warmest period (October to May), while the austral winter (June to September) is cooler and relatively drier, hence the study covered both seasons.

The experiment was conducted on two pineapple farms, each containing two experimental plots. A farm with nine-month-old plots was located at 300 m above sea level and used plastic mulching combined with sprinkler irrigation, whereas a farm with eleven-month-old plots was situated at 700 m above sea level, also with plastic mulching but without sprinkler irrigation. All four plots were managed under conventional agricultural practices during the study period. Under this management, pineapple plants received a preventive treatment with spirotetramat, and weed control was managed using glyphosate. Within each plot, three adjacent zones were established: a treatment zone (12 m long × 30 m wide), a control zone (12 m long x 30 m wide) and a buffer zone (15 m long × 30 m wide) separating them. This experimental design resulted in a total of 4 replicates per modality (2 per farm).

## 2.2. Initial field populations

Before feeder deployment, an initial sampling session was conducted to assess ant, mealybug and predator abundances. During this session, we randomly collected 18 pineapple plants from both the treatment and control zones of each plot (= 144 plants in total for the session). The plants were then placed in hermetic bags and transported to the laboratory, where they were dissected to collect and count ants, mealybugs and other arthropods. Ants were identified under a binocular microscope (ZEISS Stemi 508) using identification keys (Bolton, 2007; Fischer & Fisher, 2013; Guerrero, 2018; Hita Garcia & Fisher, 2015; LaPolla et al., 2011; LaPolla & Fisher, 2014; Mackay et al., 1990; Ortiz-Sepulveda et al., 2019). Other arthropods were identified at the order or family level.

## 2.3. Ant, mealybug and predator abundances on pineapples

In the treatment zones, we used prototype sugar feeders currently under development by the Biobest company. Ten feeders were installed along a central transect crossing each treatment zone, spaced 1 m apart. They consisted of a translucent 250-mL plastic bag containing sugar solution, with a wick that allowed the liquid to rise by capillarity and become accessible through a 28 mm-diameter grid, whose mesh was adjusted to the size of small arthropods like ants. The sugar solution was prepared with brown cane sugar solution at 50%. Each month, we checked the sugar feeders and to refill them. Each feeder was enclosed in a metal wire cage to prevent access by rats and dogs. No feeders were deployed in the control and buffer zones.

After feeder deployment, sampling sessions were conducted every two months (except for one plot that was harvested before the 10th month) to monitor ant, mealybug and predator abundances. Pineapple plants were collected and processed as described in section 2.2. The distance between sampled plants and feeders was recorded to assess spatial effects on ant and predator abundance. A total of 798 pineapple
plants were sampled, for a total of 5 sessions over the study period.

## 2.4. Sentinel prey experiment

As mealybug consumption by ants could not be directly observed, we designed a sentinel prey experiment to quantify ant-mealybug interactions and mealybug disappearance. For this purpose, two cameras per treatment and control area were installed once per month in the pineapple interrows on either side of the central transect. Each camera was located 1 m from the line of sugar feeders and 4 m or 8 m from the edge of the zone. Images were captured every 15 s for 3 h during two sessions per treatment and control zone: one in the morning during daylight conditions (8:30–11:30) and one in the evening, beginning at dusk and extending into nocturnal conditions (17:30–20:30). This experimental design resulted in a total of 8 replicates per modality (4 per farm).

For each sentinel prey replicate, ten adult mealybugs of *D. brevipes* from our rearings were acclimated on a piece of pineapple leaf at least 48 h prior to camera deployment, allowing them to insert their stylets and establish feeding behavior. The camera was fixed on a tripod (Manfrotto Befree Advanced) and was covered with an umbrella to protect from rain and sun. The leaf was then fixed with double-sided tape onto a white ceramic plate (22 x 18 cm) to provide a standardized image background, at a distance of 10 cm from the camera lens (Panasonic DMC-FZ 300). In each picture, the captured area of the tile measured 182.52 cm² (15.6 cm x 11.7 cm). Remaining mealybugs on the leaf pieces were also counted.

We used the same version of the Corigan pipeline (Tresson et al., 2019) as in Bourel et al., 2025. We retrained the convolutional neural network for 40,000 iterations on the same image dataset as in Bourel et al., 2025 but completed it with *D. brevipes* labelled images. The images were then processed through the trained pipeline to automatically identify and count individuals of species present and to count the number of interactions between them and mealybug prey.

## 2.5. Statistical analyses

Data analysis was performed on R version 4.2.2 (R Core Team 2022) and all statistical tests were performed at an alpha confidence level of 0.05.

### 2.5.1. Initial field populations analyses

To assess potential initial differences between treatments, we modeled ant, mealybug and predator abundances as a function of treatment (sugar feeders vs. control) before sugar supplementation. For all models, the random effect structure was evaluated using Likelihood Ratio Tests (LRTs) to determine whether including random intercepts for the farm and the plot identity improved model fit and adequately accounted for the hierarchical sampling design. All models were fitted using a negative binomial distribution through the *glmmTMB* package (Brooks et al., 2017), or the *MASS* package (Venables & Ripley, 2013) if no random effect was selected. The significance of the treatment effect was assessed through a LRT using the *drop1* function with a chi-squared test from the stats package. Finally, models were validated using the *DHARMa* package (Hartig, 2017).

### 2.5.2. Ant, mealybug and predator abundances on pineapple analyses

We used Structural Equation Modeling (SEM) to evaluate the direct and indirect relationships among experimental treatments, distance to the sugar feeders/control and the number of months since the beginning of the experiment (respectively called the distance and the months hereafter), ant abundance, mealybug abundance and predator abundance on pineapples.

First, we modeled ant abundance as a function of treatment (sugar feeders vs. control), distance and months. Second, we modeled mealybug abundance (Mealybugs) as a function of ant abundance and

months. Third, we modeled predator abundance as a function of treatment, distance and months. For all models, the random effect structure was examined through LRTs to determine whether including random intercepts for the farm and the plot identity improved model fit and adequately captured the hierarchical sampling design. All models were fitted using a negative binomial distribution through the *glmmTMB* package (Brooks et al., 2017) or the *MASS* package (Venables & Ripley, 2013) if no random effect was selected. These individual models were then combined into a SEM using the *piecewiseSEM* package (Lefcheck, 2016). The overall fit of the piecewise SEM was evaluated using tests of directed separation (d-separation), which assess whether the hypothesized model structure adequately accounts for the observed conditional independencies, and by calculating Fisher's C statistic. A non-significant Fisher's C statistic indicates that the data do not reject the conditional independencies implied by the model, supporting the overall fit of the model. The individual models were then validated using the *DHARMa* package (Hartig, 2017).

### 2.5.3. Sentinel prey analyses

We used Spearman rank correlation tests between the abundance-activity of each ant species and the number of interactions of each ant species with mealybugs. Interactions included physical contact, handling and transport of mealybugs by ants. We then used SEM to evaluate the direct and indirect relationships among experimental treatments (sugar feeders vs. control), time (months), day-night period, number of interactions between sentinel mealybugs and each selected ant species, and mealybug disappearance rate. Based on the matrix of interactions and the results of the Spearman rank correlation tests, we selected the species that may play a role in predation of sentinel mealybugs. First, we modeled the number of interactions between sentinel mealybugs and each selected ant species as a function of treatment, day-night, and months. Second, we modeled mealybug disappearance rate as a function of the number of interactions between sentinel mealybugs and each selected ant species and months. For each model, the random effect structure was examined through LRTs to determine whether including random intercepts for farm and plot improved model fit and adequately captured the hierarchical sampling design. Each model of the number of interactions between sentinel mealybugs and each selected ant species was fitted using a negative binomial distribution through the *glmmTMB* package (Brooks et al., 2017) or the *MASS* package (Venables & Ripley, 2013) for the models without random effects. The mealybug disappearance rate model was fitted using a binomial distribution. These individual models were then combined into a SEM using the *piecewiseSEM* package (Lefcheck, 2016). The overall fit of the piecewise SEM was evaluated using tests of directed separation (d-separation) and by calculating Fisher's C statistic. The individual models were also validated using the *DHARMa* package (Hartig, 2017).

# 3. Results

## 3.1. Arthropods on pineapple plants

In total, 9 ant species/genera were identified on the plants (Table 1): *Brachymyrmex cordemoyi* Forel, 1895, *Hypoponera* spp. Santschi, 1938, *Nylanderia bourbonica* (Forel, 1886), *Pheidole megacephala* (Fabricius, 1793), *Solenopsis geminata* (Fabricius, 1804), *Technomyrmex* spp. Mayr, 1872, *Paratrechina longicornis* (Latreille, 1802), *Tetramorium bicarinatum* (Nylander, 1846) and *Monomorium floricola* (Jerdon, 1851). In addition, 7 arthropod groups were recorded on pineapple plants, including Araneae, Coccinellidae, Coleoptera (except Coccinellidae), Myriapoda, Collembola, Orthoptera and Dermaptera. Among these taxa, only spiders and ladybeetles were considered as potential predators of pineapple mealybugs, based on their known predatory behavior and documented ability to prey upon hemipterans (Escalona et al., 2017; Hajer & Hrubá, 2007). No parasitoids of mealybugs were observed and no signs of
<caption>**Table 1**</caption>

Ant species collected on plants throughout the study (before and after sugar supplementation).

<table><thead><tr><th>Ant species</th><th>Total abundance</th><th>Frequency on plant (%)</th><th>Mean per plant ± SE</th></tr></thead><tbody><tr><td>Brachymyrmex cordemoyi</td><td>916</td><td>14.39</td><td>1.16 ± 0.22</td></tr><tr><td>Hypoponera spp.</td><td>24</td><td>1.89</td><td>0.03 ± 0.01</td></tr><tr><td>Nylanderia bourbonica</td><td>23</td><td>0.76</td><td>0.03 ± 0.02</td></tr><tr><td>Paratrechina longicornis</td><td>1</td><td>0.13</td><td>0.00 ± 0.00</td></tr><tr><td>Pheidole megacephala</td><td>5475</td><td>23.11</td><td>6.57 ± 2.88</td></tr><tr><td>Solenopsis geminata</td><td>431</td><td>2.78</td><td>0.54 ± 0.28</td></tr><tr><td>Technomyrmex spp.</td><td>123</td><td>3.91</td><td>0.16 ± 0.05</td></tr><tr><td>Tetramorium bicarinatum</td><td>2</td><td>0.25</td><td>0.00 ± 0.00</td></tr><tr><td>Monomorium floricola</td><td>1</td><td>0.13</td><td>0.00 ± 0.00</td></tr></tbody></table>

parasitism (e.g., mummified mealybugs or emergence holes) were detected throughout the study.

## 3.2. Initial field population

At the beginning of the experiment, a total of 1 085 mealybugs were recorded across sampled pineapple plants (mean ± SE: 7.53 ± 3.52 individuals per plant), ant abundance reached 373 individuals (mean ± SE: 2.59 ± 0.89 individuals per plant), while predator abundance totaled 24 individuals (mean ± SE: 0.17 ± 0.04 individuals per plant).

The results of the random-effects structure selection are provided in the *Supplementary Materials* (Section 1). We found no significant initial differences between treatment (LRT = 2.35, df = 1, p = 0.125 for the ant model and LRT = 0.64, df = 1, p = 0.425 for the mealybug model). Model validation using *DHARMa* indicated no deviations: residuals were uniform (KS test: p = 0.205 for the ant model, p = 0.861 for the mealybug model and p = 0.729 for the predator model) and no overdispersion was detected (dispersion test: p = 0.537 for the ant model, p = 0.806 for the mealybug model and p = 0.959 for the predator model).

## 3.3. Ant, mealybug and predator abundances on pineapples

After sugar supplementation, we measured ant, mealybug and predator abundances on 648 pineapples (324 per treatment). A total of 11 460 mealybugs (mean ± SE: 17.7 ± 2.55 individuals per plant), 6 623 ants (mean ± SE: 10.2 ± 3.70 individuals per plant) and 257 predators (mean ± SE: 0.40 ± 0.05 individuals per plant) were recorded.

The results of the random-effects structure selection are provided in the *Supplementary Materials* (Section 2a). Our piecewise SEM showed a good overall fit to the data (Fisher's C = 5.83, df = 4, p = 0.212). The addition of sugar feeders significantly decreased by 42% (± 14.4%) the

<caption>**Table 2**</caption>

**Summary of path coefficients from the piecewise structural equation model on ant and mealybug abundances on pineapple.** Values in boldface indicate a significant effect at an alpha level of 0.05. Abbreviation: Std. Error: Standard Error.

<table><thead><tr><th>Response</th><th>Predictor</th><th>Estimate</th><th>Std. Error</th><th>t / z value</th><th>p-value</th></tr></thead><tbody><tr><td rowspan="3">Ants</td><td>Distance</td><td>0.0223</td><td>0.0262</td><td>0.85</td><td>0.396</td></tr><tr><td>Months</td><td>0.2479</td><td>0.0355</td><td>6.98</td><td><strong>&lt;0.001</strong></td></tr><tr><td>Sugar feeders</td><td>-0.5439</td><td>0.2474</td><td>-2.20</td><td><strong>0.027</strong></td></tr><tr><td rowspan="3">Mealybugs</td><td>Ants</td><td>0.0107</td><td>0.0051</td><td>2.11</td><td><strong>0.035</strong></td></tr><tr><td>Months</td><td>0.0299</td><td>0.0433</td><td>0.69</td><td>0.490</td></tr><tr><td>Predators</td><td>0.2316</td><td>0.1729</td><td>1.34</td><td>0.180</td></tr><tr><td rowspan="3">Predators</td><td>Ants</td><td>0.0005</td><td>0.0008</td><td>0.63</td><td>0.526</td></tr><tr><td>Distance</td><td>-0.0615</td><td>0.0222</td><td>-2.77</td><td><strong>0.005</strong></td></tr><tr><td>Months</td><td>0.1412</td><td>0.0339</td><td>4.16</td><td><strong>&lt;0.001</strong></td></tr><tr><td></td><td>Sugar feeders</td><td>0.7193</td><td>0.1890</td><td>3.80</td><td><strong>&lt;0.001</strong></td></tr></tbody></table>

ant abundance compared to the control (Table 2). Ant abundance was also positively associated with the number of months since the beginning of the experiment (Fig. 1), whereas the distance had no significant effect (Table 2). Mealybug abundance was positively related to ant abundance (Fig. 1), but not significantly related to the abundance of predators, nor to the number of months (Table 2). The addition of sugar feeders significantly increased by 105% (± 38.8%) the predator abundance compared to the control (Table 2). The predator abundance was also positively associated with the number of months since the beginning of the experiment, whereas it was negatively related to the distance to the sugar feeders (Table 2). The ant abundance had no effect on the predator abundance (Table 2). However, there was no significant direct effect of treatment or distance on mealybug abundance (p = 0.163 and p = 0.332 in d-separation tests, respectively).

Model validation using *DHARMa* indicated no deviations for the mealybug model: residuals were uniform (KS test: p = 0.511) and no overdispersion was detected (dispersion test: p = 0.343). Concerning the predator model, we detected no deviation from uniformity in the residuals (KS test: p = 0.805) and we found evidence of overdispersion (dispersion test, p = 0.020). Additionally, we detected a slight deviation from uniformity in the residuals (KS test: p = 0.034) and evidence of overdispersion (dispersion test: p = 0.030) for the ant model. This pattern was likely driven by the presence of a *P. megacephala* nest on one pineapple, which had an exceptionally high ant abundance (2,210 individuals). We re-ran the analysis excluding this outlier, which yielded qualitatively similar results (Supplementary Materials, Section 2b). After removal, model validation no longer indicated deviations: residuals were uniform (KS test: p = 0.104 for the ant model, p = 0.680 for the mealybug model and p = 0.064 for the predator model) and no overdispersion was detected (dispersion test: p = 0.067 for the ant model, p = 0.593 for the mealybug model and p = 0.297 for the predator model) (Supplementary Materials, Section 2b).

## 3.4. Sentinel prey experiment

We obtained satisfactory metrics for the detection of arthropods on the sentinel prey experiments (Supplementary Materials, Section 3a, Supplementary Table 2). Confusion between species identification was low, with, for instance, 2.84% of misidentification when the actual species is *P. megacephala* but the predicted was *S. geminata* (Fig. 2). However, despite this low error percentage, given the relatively high abundances of these 2 species, we checked for possible confusion between the two species in the images where co-occurrence of these species was reported by the pipeline. This led to a removal of 1.60% of the detections from the prey sentinel dataset.

Overall, image-based data were obtained for 328 sentinel prey replicates, comprising 727,765 images. A total of 8 ant species were

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Mean abundance of ants and mealybugs throughout months. Error bars represent standard errors of the means.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Fig. 2. Confusion matrix of the image analysis image.</figcaption></figure>

identified on the images: *B. cordemoyi*, *N. bourbonica*, *P. longicornis*, *T. melanocephalum* (Fabricius, 1793), *Technomyrmex* spp., *P. megacephala*, *S. geminata*, and *T. bicarinatum* (Table 3). Interactions with mealybugs occurred mainly with 3 ant species: *S. geminata*, *P. megacephala* and *B. cordemoyi* (Fig. 3). *Brachymyrmex cordemoyi*, *P. megacephala* and *S. geminata* were present on 84.8%, 46.6% and 15.2% of sentinel prey experiments (Table 3). The abundance-activity was positively correlated with the number of interactions with mealy-bugs for *S. geminata* (ρ = 0.218, p < 0.001), *P. megacephala* (ρ = 0.447, p < 0.001) and *B. cordemoyi* (ρ = 0.136, p = 0.019). This relationship was

<table><caption>**Table 3**

Ant species identified on sentinel prey images.</caption><thead><tr><th>Ant species</th><th>Number of occurrences</th><th>Frequency on images (%)</th></tr></thead><tbody><tr><td>Brachymyrmex cordemoyi</td><td>278</td><td>84.8</td></tr><tr><td>Nylanderia bourbonica</td><td>34</td><td>10.4</td></tr><tr><td>Paratrechina longicornis</td><td>112</td><td>34.1</td></tr><tr><td>Pheidole megacephala</td><td>153</td><td>46.6</td></tr><tr><td>Solenopsis geminata</td><td>50</td><td>15.2</td></tr><tr><td>Tapinoma melanocephalum</td><td>65</td><td>19.8</td></tr><tr><td>Technomyrmex spp.</td><td>42</td><td>12.8</td></tr><tr><td>Tetramorium bicarinatum</td><td>49</td><td>14.9</td></tr></tbody></table>

not significant for *P. longicornis* (ρ = -0.011, p = 0.854) and *T. melanocephalum* (ρ = -0.095, p < 0.102), while we detected no interactions with mealybugs for *N. bourbonica*, *Technomyrmex* spp. and *T. bicarinatum*.

The results of the random-effects structure selection are provided in the Supplementary Materials (Section 3b). Our piecewise structural equation model showed a good overall fit to the data (Fisher's C = 10.34, df = 10, p = 0.411). The number of interactions between *S. geminata* and mealybugs was positively related to the number of months since the beginning of the experiment, while the number of interactions between *P. megacephala* and mealybugs was negatively related to the number of months (Table 4). From night to day, the number of interactions with mealybugs decreased by 84% (± 12.9%) for *S. geminata* and by 96% (± 2.0%) for *P. megacephala* (Table 4, Fig. 4). The number of interactions between *B. cordemoyi* and mealybugs was not significantly related to the number of months since the beginning of the experiment nor to the day-night periods (Table 4, Fig. 4). The sugar feeders had no significant effect on the number of interactions between mealybugs and any of the three ant species (Table 4, Fig. 5). The mealybug disappearance rate was positively related to the number of months since the beginning of the experiment (Table 4, Fig. 6) and to the number of interactions with *S. geminata*, *P. megacephala* and *B. cordemoyi* (Table 4, Fig. 7). However, there was no significant direct effect of
<figure><img src="image_5.png" /><figcaption>Fig. 3. Matrix of interactions. Numbers indicate the sum of interactions that have been detected between two species.</figcaption></figure>

<caption>Table 4</caption>

Summary of path coefficients from the piecewise structural equation model on interactions of *S. geminata*, *P. megacephala* and *B. cordemoyi* with mealybugs. Values in boldface indicate a significant effect at an alpha level of 0.05. Abbreviation: Std. Error: Standard Error.

<table><thead><tr><th>Response</th><th>Predictor</th><th>Estimate</th><th>Std. Error</th><th>t / z value</th><th>p-value</th></tr></thead><tbody><tr><td rowspan="3">S. geminata – mealybug interactions</td><td>Number of months</td><td>0.7684</td><td>0.1702</td><td>4.51</td><td>&lt;0.001</td></tr><tr><td>Sugar feeders</td><td>-0.4719</td><td>0.7984</td><td>-0.59</td><td>0.554</td></tr><tr><td>Night</td><td>1.8311</td><td>0.8026</td><td>2.28</td><td><strong>0.022</strong></td></tr><tr><td rowspan="3">P. megacephala – mealybug interactions</td><td>Number of months</td><td>-0.2072</td><td>0.1030</td><td>-2.01</td><td><strong>0.044</strong></td></tr><tr><td>Sugar feeders</td><td>0.5460</td><td>0.5009</td><td>1.09</td><td>0.276</td></tr><tr><td>Night</td><td>3.2675</td><td>0.5194</td><td>6.29</td><td>&lt;0.001</td></tr><tr><td rowspan="3">B. cordemoyi – mealybug interactions</td><td>Number of months</td><td>-0.0270</td><td>0.1191</td><td>-0.23</td><td>0.821</td></tr><tr><td>Sugar feeders</td><td>-0.4630</td><td>0.6718</td><td>-0.69</td><td>0.491</td></tr><tr><td>Night</td><td>0.7531</td><td>0.6722</td><td>1.12</td><td>0.263</td></tr><tr><td rowspan="4">Mealybug disappearance rate</td><td>Number of months</td><td>0.1038</td><td>0.0142</td><td>7.30</td><td>&lt;0.001</td></tr><tr><td>S. geminata – mealybug interactions</td><td>0.0101</td><td>0.0027</td><td>3.71</td><td>&lt;0.001</td></tr><tr><td>P. megacephala – mealybug interactions</td><td>0.0243</td><td>0.0047</td><td>5.19</td><td>&lt;0.001</td></tr><tr><td>B. cordemoyi – mealybug interactions</td><td>0.0753</td><td>0.0331</td><td>2.28</td><td><strong>0.023</strong></td></tr></tbody></table>

treatment or day-night periods on mealybug disappearance rate (p = 0.656 and p = 0.077 in d-separation tests, respectively, Fig. 8).

Model validation using DHARMA indicated no deviations and no overdispersion for the models of interactions of mealybugs with *S. geminata* (KS test: p = 0.13; dispersion test: p = 0.46), with *P. megacephala* (KS test: p = 0.12; dispersion test: p = 0.58) or with

*B. cordemoyi* (KS test: p = 0.85; dispersion test: p = 0.91). Similarly, model validation using DHARMA indicated no deviations for the mealybug disappearance rate model: residuals were uniform (KS test: p = 0.71) and no overdispersion was detected (dispersion test: p = 0.97).

## 4. Discussion

We conducted an eleven-month field study combining long-term monitoring of ant, mealybug and predator abundances on pineapple plants with sentinel prey experiments designed to assess ant-mealybug interactions. Overall, sugar supplementation led to a marked reduction in ant abundance on plants, which was accompanied by lower mealybug densities and higher predator occurrence in supplemented zones. In parallel, the sentinel prey experiments revealed that only a few dominant ant species, *Pheidole megacephala*, *Solenopsis geminata* and *Brachymyrmex cordemoyi*, interacted with mealybug prey, and that the number of these interactions was positively related to mealybug disappearance rates.

### 4.1. Ant, mealybug and predator abundances on pineapples

We found that sugar supplementation significantly reduced ant abundance by 42% on pineapple plants. Additionally, the structural equation model revealed that mealybug abundance was positively related to ant abundance. These results provide evidence that sugar supplementation can reduce ant presence on host plants, and indirectly negatively influence mealybug populations. Thus, in accordance with the literature, our findings support that sugar supplementation can reduce mealybug infestation in pineapple crops (Correa et al., 2023; Foronda et al., 2025; Nagy et al., 2013, 2015; Pérez-Rodríguez et al., 2021; Wäckers et al., 2017).

In addition, predators, i.e. spiders and ladybeetles, were more abundant on pineapples in sugar-supplemented zones, with an increase of 105% compared to control zones. Two non-exclusive explanations may account for this pattern. First, sugar supplementation can have a
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Mean number of interactions between mealybugs and the 3 ant species during sentinel experiments according to the day and the night. Error bars represent standard errors of the means. Asterisks indicate a significant effect: \*: p ≤ 0.05, \*: p ≤ 0.01, \*: p ≤ 0.001. "n.s." indicates a non-significant effect at an alpha level of 0.05.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Mean number of interactions between mealybugs and the 3 ant species during sentinel experiments according to the treatment. Error bars represent standard errors of the means. "n.s." indicates a non-significant effect at an alpha level of 0.05.</figcaption></figure>

direct effect on predators. Many predatory arthropods rely on carbohydrate-rich resources to complement their carnivorous diet (Sunderland, 2009). Access to sugars can enhance predator survival, fecundity and foraging activity (Carvell et al., 2015; Stowe et al., 2021; Taylor & Bradley, 2009; Taylor & Pfannenstiel, 2009). Accordingly, several studies have reported that sugar supplementation favors natural enemy communities of hemipterans, increasing their associated services as biocontrol agents (Leman et al., 2023; Tena et al., 2015). Second, the reduction in ant abundance at plants may indirectly favor predator presence, as ants are known to deter or exclude predators and parasitoids (Campos et al., 2006; Cheng et al., 2015; Itioka & Inoue, 1996). In our SEM, however, the link between ant and predator abundance was not statistically significant. This may reflect the fact that the effect of ants on predators depends more on their spatial presence on plants than

on their absolute abundance. However, Nève de Mevergnies et al. (2022) found that the total abundance of spiders was not altered by the presence of the dominant ant *Oecophylla longinoda* (Latreille, 1802) in mango orchards. We considered in our study ladybeetles and spiders as members of the predator functional group. Possibly, these two types of predators may be affected differently by the presence and/or abundance of ants.

Indeed, ladybeetles are more specialized predators, feeding mostly on hemipterans such as mealybugs (Escalona et al., 2017), while spiders are more generalist predators of arthropods (Andelić Dmitrović et al., 2023). Alternatively, the relationship may have been too weak to be detected, given the relatively low numbers of predators sampled in our study. Predator abundance also declined with increasing distance from sugar feeders, whereas ant abundance was unaffected by distance. This
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Mean of the disappearance rate of mealybugs as a function of the number of months since the beginning of sentinel experiments. Error bars represent standard errors of the means.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Mean of the number of interactions between ants of species *S. geminata*, *P. megacephala* and *B. cordemoyi* with mealybugs as a function of the number of months since the beginning of sentinel experiments. Error bars represent standard errors of the means.</figcaption></figure>

discrepancy likely reflects differences in foraging strategies: polydomyous ant species such as *Pheidole megacephala* and *Solenopsis geminata* can forage over large areas and exploit distant resources, while predators may be more locally concentrated around feeders.

Although mealybug abundance was positively related to ant abundance, we did not detect a significant direct effect of sugar supplementation on mealybug infestation levels. Sugar feeders were primarily designed to alter ant behavior, rather than to act directly on mealybugs. In addition, predator abundance remained relatively low throughout the experiment and no parasitoids were detected. Under these conditions, reduced ant attendance alone may be insufficient to translate into a measurable decrease in mealybug densities, which likely also depends on the effectiveness of natural enemy regulation. Consequently, additional measures, such as enhancing natural enemy populations, could

further increase the effectiveness of sugar supplementation.

## 4.2. Sentinel prey experiment

The sentinel prey experiment identified three ant species interacting with mealybug sentinel prey: *B. cordemoyi*, *P. megacephala* and *S. geminata*. The number of interactions was positively related to mealybug disappearance rates, indicating that these species contributed to mealybug removal from plants. Indeed, in some image sequences, ants were observed transporting mealybugs (Supplementary Materials, Fig. S1), which could represent predation or plant/nest relocation. However, without additional behavioral observations (e.g., confirmation of consumption), we cannot firmly conclude that this behavior reflects predation. Instead, we consider it as a plausible hypothesis
<figure><img src="image_10.png" /><figcaption>**Fig. 8.** Mean of the disappearance rate of mealybugs during sentinel experiments according to the day-night periods and to the treatment. Error bars represent standard error of the means. "n.s" indicates a non-significant effect at an alpha level of 0.05.</figcaption></figure>

requiring further investigation.

Interestingly, sugar supplementation had no detectable effect on the number of interactions between ants and sentinel mealybugs, nor on mealybug disappearance rates, as observed in Pérez-Rodríguez et al. (2021) findings. This result may indicate that feeders did not alter ant's behaviors toward mealybugs, contrasting with studies suggesting that sugar supplementation can lead to an increase in ant aggressiveness (Grover et al., 2007; Offenberg, 2001). Plant chemical mediation may also have masked the effect of sugar provisioning through feeders. Indeed, ants respond to plant chemicals to the benefit or the detriment of the plants (Nelson et al. 2019). The cutted leaves used in our sentinel prey experiments may have emitted senescence-related volatiles that are attractive to ants, such as terpenes (Portillo-Estrada et al., 2020) or herbivore-induced plant volatiles in response to the mealybugs fixed through their stylet (Nelson et al. 2019). Finally, the quantity of the supplemental sugar might have been insufficient to alter the ant aggressiveness toward the mealybugs in this experiment. Although, in this sentinel prey experiment, we used living mealybugs fixed on an ananas leaf to mimic real situations, the use of entire plants should be considered for further research.

Elsewhere, we found that *P. megacephala* and *S. geminata* were more active at night, with significantly higher interaction rates with mealy-bugs compared to the day. This nocturnal peak in activity, combined with seasonal differences in their responses, suggests that the outcome of ant-mealybug interactions is dynamic and influenced by environmental factors (Bronstein, 1994; Van Baalen & Jansen, 2006). For instance, *S. geminata* activity increased during the warmer summer months, while *P. megacephala* showed the opposite trend. These contrasting seasonal niches may shape their relative importance in regulating mealybug populations over time (Foronda et al., 2025). Indeed, ants vary seasonally in their reliance on carbohydrate resources: in temperate regions colonies are known to increase sugar uptake when sugar is scarce, and to intensify foraging during summer (Josens & Roces, 2000; Sudd & Sudd, 1985).

Although our experimental design provided effective results, several adjustments could further improve its impact on mealybug populations. First, we deployed only a single line of feeders per plot: increasing the number or density of feeders could provide more accessible sugar, potentially further enhancing the attractivity for ants, as well as the abundance and activity of predators and other natural enemies. Second,

feeders were placed along paths between pineapple rows, whereas ant nests are usually located at the base of plants. Adjusting feeder placement to colony location may therefore improve their impact (Correa et al., 2023; Pérez-Rodríguez et al., 2021). Finally, the scarcity of predators and parasitoids in pineapple monocultures may have limited the indirect consequences of reduced ant presence. This low density of natural enemies is likely linked to the structural simplicity of pineapple agrosystems, which provide few refuges apart from plant themselves. Strong sun exposure, the use of plastic mulching, and the absence of alternative resources further constrain the establishment of predator and parasitoid populations. Management strategies that increase habitat diversity could enhance the conservational biological control by facilitating the establishment and persistence of natural enemies, thereby amplifying the benefits of reduced ant protection for mealybugs.

## 5. Conclusions

To conclude, our findings support the potential of sugar supplementation as a tool to reduce mealybug infestations in pineapple systems. By reducing ant abundance on plants and promoting predators, feeders can contribute to more favorable conditions for biological control (Nagy et al., 2013, 2015; Wäckers et al., 2017). Besides, increasing feeder density may improve their effectiveness. Importantly, sugar supplementation alone may not be sufficient to suppress mealybug populations. Integrating feeders with habitat management practices that promote natural enemies, such as providing floral resources or refuges, could amplify the conservational biological control (Huang et al., 2011; Pérez-Rodríguez & Messelink, 2023; Wade et al., 2008). In this context, recent studies have shown that combining sugar provisioning with predator or parasitoid releases yield stronger reductions in hemipteran pests than either method alone (Burgio et al., 2025; Parrilli et al., 2021). Such integrated strategies are likely to be more robust and sustainable in the long term.

## Funding sources

This work was carried out as part of the CIRAD DPP SADur agro-nomical research program. This program is co-funded by the European Union, the French State, and La Réunion Region. Europe is committed to La Réunion through the EAFRD (grant REU77071-1-000011), for which
La Réunion Department is the managing authority, and through the ERDF (grant 2024—1248-583 005756), for which La Réunion Region is the managing authority.

## CRediT authorship contribution statement

**Marie Bourel:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization. **Eva Faustin:** Writing – review & editing, Methodology, Investigation, Formal analysis, Conceptualization. **Bernard Raymond Abufa:** Investigation. **Capucine Perrey:** Investigation. **Camille Stasiolojc:** Investigation. **Anàs Chailleux:** Writing – review & editing. **Felix Wäckers:** Writing – review & editing. **Philippe Tixier:** Writing – review & editing, Supervision, Methodology, Conceptualization. **Dominique Carval:** Writing – review & editing, Writing – original draft, Supervision, Software, Project administration, Methodology, Investigation, Formal analysis, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

The authors thank the producer who agreed to participate in this study.

Data availability statement.

The data and R code used in this study are publicly available on Figshare: https://figshare.com/s/4937213ea398b9262e13 (10.6084/m.9 figshare.30404875).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biocontrol.2026.105975.

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