1 Introduction
The leaf miner Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) is a globally distributed pest that infests various host plants, including vegetables and ornamental flowers (Seal et al., 2002; Masetti et al., 2004). The parasitoid Neochrysocharis formosa (Westwood) (Hymenoptera: Eulophidae), a natural enemy of leaf miners, is native to regions where these pests are prevalent (Arakaki and Kinjo, 1998; Konishi, 1998). N. formosa parasitizes the larvae of L. trifolii, ultimately killing the host. This parasitic behavior enables N. formosa to serve as an effective biological control agent and, in Japan, it is now commercially available for managing Liriomyza species. Another significant pest, Hercinothrips femoralis Reuter (Thysanoptera: Thripidae), commonly known as banded greenhouse thrips, is a polyphagous insect with a cosmopolitan distribution (Houston et al., 1991; Trdan et al., 2007). These three insect species harbor endosymbiotic bacteria such as Wolbachia and Rickettsia in Japan and other regions (Adachi-Hagimori et al., 2008; Tagami et al., 2006b; Kumm and Moritz, 2008).
Wolbachia is a well-studied endosymbiotic bacterium that manipulates host reproduction through various mechanisms, including cytoplasmic incompatibility (Werren, 1997), feminization of genetic males (Bouchon et al., 1998), male killing (Hurst and Jiggins, 2000), and thelytokous parthenogenesis, a form of parthenogenesis in which unfertilized eggs develop into females (Stouthamer et al., 1990; Stouthamer and Kazmer, 1994). In L. trifolii, Wolbachia induces cytoplasmic incompatibility, a reproductive barrier in which crosses between infected males and uninfected females result in embryo mortality, thereby reducing reproductive success (Tagami et al., 2006b; Kumm and Moritz, 2008). However, this mechanism does not affect the sex ratio of the offspring.
In contrast, in H. femoralis, Wolbachia induces thelytokous parthenogenesis, a form of parthenogenesis in which unfertilized eggs develop into females, resulting in strongly female-biased populations (Kumm and Moritz, 2008). Similarly, in N. formosa, the endosymbiont Rickettsia induces thelytokous parthenogenesis, leading to highly female-skewed populations (Hagimori et al., 2006) that contribute to the effectiveness of this species as a biological control agent. Notably, H. femoralis females uninfected with Wolbachia and N. formosa females uninfected with Rickettsia produced only male offspring, and these males do not have a function to reproduce, highlighting the essential role of these endosymbionts in reproduction. In pest species like H. femoralis, disrupting symbiont-mediated reproduction may aid in population suppression.
In contrast, for beneficial parasitoids like N. formosa, which are used as biological control agents against L. trifolii, the loss of symbiont-induced thelytoky parthenogenesis could severely reduce female production and compromise control efficacy. Therefore, it is crucial to assess how agricultural treatments, such as antibiotic applications, might differentially affect symbiont-dependent reproduction in both pests and their natural enemies.
The widespread use of agricultural pesticides, including insect growth regulators, biopesticides targeting Bacillus thuringiensis, and chemical classes such as organophosphates, carbamates, neonicotinoids, organocoppers, and organic sulfur compounds, has been central to pest control strategies (Siddall, 1976; Whalon and Wingerd, 2003; Souto et al., 2021; Sharma et al., 2019; Zhang, 2018; Araújo et al., 2023). Antibiotics are also used in agriculture to manage plant diseases (Stockwell and Duffy, 2012). One such bactericide, MycoShield (Maruwa Biochemical, Tokyo, Japan), contains 17.0% oxytetracycline, a tetracycline derivative used to control bacterial diseases such as fire blight (Erwinia amylovora) and infections caused by Pseudomonas and Xanthomonas species (Copping and Duke, 2007). This antibiotic is also effective against diseases caused by mycoplasma-like organisms (Ishiie et al., 1967) and is applied to stone and pome fruits, as well as turfgrass (Copping and Menn, 2000).
Despite the prevalence of bacterial endosymbionts in many insect species, the effects of antibiotic bactericides on symbionts and their hosts are unclear. Therefore, this study aimed to investigate the effects of MycoShield on L. trifolii, H. femoralis, and N. formosa, with a specific focus on (i) the direct effects of MycoShield on these insects, (ii) the uptake routes of MycoShield in L. trifolii and N. formosa, (iii) the relationship between MycoShield concentration and its effect on infected insects, and (iv) the potential use of MycoShield to eradicate H. femoralis populations.
2 Materials and methods
2.1 Insects
Liriomyza trifolii and N. formosa were collected from kidney bean leaves in Iwata, Shizuoka, Japan, in 1991 and placed in cages (width 30 cm, depth 30 cm, height 40 cm). Each cage contained three kidney bean plants, which served as host plants when L. trifolii adults emerged.
Kidney bean leaves in the field were collected for N. formosa, and N. formosa individuals were selected based on morphological characters when leaf miner parasitoids emerged. N. formosa were reared in cages with L. trifolii larvae as hosts.
Hercinothrips femoralis was collected from a beach lily (Crinum asiaticum) in Iwata, Shizuoka, Japan, in 2005 and reared on kidney bean plants in a cage. When host plants were severely damaged, H. femoralis populations were transferred to new cages containing new kidney bean plants.
All three insect species were reared under consistent environmental conditions (25°C, 16 h light:8 h dark photoperiod, relative humidity 70 ± 5%, with light provided by MLR-351, SANYO, Osaka, Japan). Morphological identification of each species was performed based on criteria described previously (Konishi, 1998). The initial Wolbachia and Rickettsia infection status was verified by PCR prior to the experiments.
2.2 Detection of <italic>Wolbachia</italic> and <italic>Rickettsia</italic> and distinguishing between sex
Diagnostic PCR detection of bacterial endosymbionts was performed using a thermal cycler (TP600, TAKARA, Japan) as previously described (Tagami et al., 2006b; Hagimori et al., 2006; Tagami et al., 2006a). Wolbachia was detected by amplifying the wsp gene using the primers wsp81F (5′-TGGTCCAATAAGTGATGAAGAAAC-3′) and wsp691R (5′-AAAAATTAAACGCTACTCCA-3′) (Braig et al., 1998). Rickettsia infection was assessed by targeting the bacterial 16S rRNA gene with the primers NforRick1 (5′-AGTGAGTGATGAAGGCCCTA-3′) and NforRick2 (5′-GGAATTCCATCATCCTCTACTAC-3′) (Tagami et al., 2006a). The PCR conditions (reaction components and thermal cycling profiles) followed those established in the cited literature.
Host sex was identified based on adult morphological characteristics, particularly the structure of the sexual organs, in all three species.
2.3 Effect of MycoShield on <italic>L. trifolii</italic>, <italic>H. femoralis</italic>, and <italic>N. formosa</italic>
Prior to MycoShield treatment, the infection status of each insect species was confirmed by PCR using 30 representative individuals from the laboratory-reared lines. All L. trifolii and H. femoralis individuals tested were found to be 100% infected with Wolbachia, and all N. formosa females were 100% infected with Rickettsia.
To assess the effect of short-term antibiotic exposure, adult insects were placed in cages containing MycoShield-treated plants for 24 h. MycoShield was prepared at a standard concentration (1,000-fold dilution with water) and sprayed evenly onto three kidney bean plants, each bearing four leaves. After the 24-h exposure period, three pairs of L. trifolii or H. femoralis were introduced into separate cages and allowed to reproduce. Once the adults of the next generation emerged, six individuals were randomly collected from each cage and screened for Wolbachia infection using PCR. Each experiment was repeated five times.
For N. formosa, five cages were prepared, and a droplet of honey mixed with MycoShield (1,000-fold dilution) was provided as food for 24 h. After this exposure period, three kidney bean plants parasitized by L. trifolii larvae were introduced into each cage. The Rickettsia infection ratio was determined by analyzing 30 adult individuals (six individuals per cage) of N. formosa from the next generation using PCR.
2.4 MycoShield uptake routes of <italic>L. trifolii</italic> and <italic>N. formosa</italic>
Understanding the uptake routes of MycoShield by L. trifolii and N. formosa is essential for the application of antibiotic agrochemicals to manage bacteria-infected pest insects and their natural enemies.
2.4.1 MycoShield uptake route of <italic>L. trifolii</italic>
The uptake route of L. trifolii was initially analyzed. As L. trifolii does not feed directly on the surface of kidney bean leaves, we hypothesized that MycoShield is primarily ingested by larvae feeding on the mesophyll rather than by adults. Two separate experiments were conducted to test this hypothesis.
First, MycoShield was prepared at a standard concentration (diluted 1,000-fold with water) and sprayed onto 12 leaves from three kidney bean plants. Three pairs of L. trifolii adults were introduced into cages containing the treated plants. After one and three days, the adults were transferred to a new cage containing three untreated kidney bean plants. Eggs laid on untreated plants were collected, and the infection status of Wolbachia in the offspring was analyzed. The experiments were conducted under controlled conditions (25°C, 16 h light:8 h dark photoperiod).
Next, three pairs of L. trifolii adults were introduced into a cage containing three untreated kidney bean plants to examine the uptake of MycoShield by the larvae. After 24 h, the plants with L. trifolii eggs were collected. These plants were then sprayed with MycoShield at the standard concentration and placed in a new cage under controlled conditions (25°C, 16 h light:8 h dark photoperiod). Larvae were collected at four, six, and eight days after treatment, and their Wolbachia infection status was analyzed.
2.4.2 MycoShield uptake route of <italic>N. formosa</italic>
Neochrysocharis formosa does not feed on kidney bean leaves; therefore, it possibly ingests MycoShield through L. trifolii larvae, which consume MycoShield-treated leaves. Adult N. formosa individuals less than 24 h after eclosion were used for these experiments, and all experiments were done at 25°C and a 16 h light:8 h dark photoperiod.
First, it was confirmed that N. formosa adults did not directly ingest MycoShield from the leaves. MycoShields were sprayed onto three unparasitized plants (12 leaves in total) inside a cage, and six N. formosa adults were released. Then these adults were moved to new cages on days one-, three-, five-, and seven-day days later, which introduced untreated kidney bean parasitized by L. trifolii. The infection and male ratios of adult individuals in the next generation were analyzed (Supplementary Figure 1A).
L. trifolii larvae immediately die when parasitized by N. formosa. Second, N. formosa is expected to ingest L. trifolii larvae if L. trifolii larvae die before being sprayed with MycoShield. MycoShield was sprayed after parasitizing N. formosa, and the infection ratio during the pupal stage of N. formosa was determined (Supplementary Figure 1B).
Third, whether N. formosa could ingest MycoShield through adult host feeding was investigated. MycoShield was sprayed onto kidney bean plants that had been oviposited by L. trifolii five days earlier. Three N. formosa individuals were introduced into the cages. N. formosa adults were transferred to new, non-sprayed plants parasitized by L. trifolii on days one, three, five, and seven. Approximately 24 h later, N. formosa eggs were collected, and their infection ratio was analyzed. The experiment was repeated six times (Supplementary Figure 1C).
Fourth, the combined routes of MycoShield uptake by N. formosa were investigated. Five pairs of L. trifolii adults were introduced into the cages containing kidney bean plants. After five days, the oviposited eggs had developed into larvae, and the plants were sprayed with MycoShield before being moved to a new cage. N. formosa adults were introduced in this new cage, where they could only ingest MycoShield through host feeding, whereas N. formosa larvae consumed L. trifolii larvae that had absorbed MycoShield. Emerging N. formosa adults were collected, and their infection and male ratios were analyzed (Supplementary Figure 1D).
2.5 Effect of MycoShield concentration on <italic>L. trifolii</italic>, <italic>H. femoralis</italic>, and <italic>N. formosa</italic> populations
MycoShield solutions were prepared at different concentrations and diluted with water (100-, 1, 000-, 10, 000-, 100, 000-, 1,000,000-fold) with an untreated control. Two kidney bean plants (eight leaves in total) were used in each experiment. Once the plants had grown sufficiently, the MycoShield solutions were sprayed evenly onto their leaves, and these plants were placed inside a cage.
Three pairs of L. trifolii adults were randomly selected and placed in cages. Six H. femoralis and N. formosa adults were introduced into the cage. For N. formosa, two kidney bean plants that had been oviposited with L. trifolii were used. Six adult N. formosa individuals were introduced into the cage when oviposited L. trifolii eggs developed into larvae. The infection and male ratios in the next generation of adults were analyzed.
2.6 Effect of the MycoShield on <italic>H. femoralis</italic> population dynamics
Twelve kidney bean plants in four pots were prepared, which were sprayed with a standard concentration of MycoShield (diluted 1,000-fold with water) and placed inside a cage. Twenty adult female H. femoralis were introduced into cages. Larvae, adult females, and adult males were counted every 20 days.
After each count, new plants treated with a standard concentration of MycoShield were introduced into the same cage, and all H. femoralis individuals were transferred to new plants. Old plants were removed from the cage, but the soil was not replaced because it contained pupae. The study was conducted over 100 days.
2.7 Statistical analyses
All statistical analyses were performed using R software [version 4.4.2; (R Core Team, 2024)]. Logistic regression analysis was performed using the glm function (family = binomial) in R version 4.4.2 to model the relationship between elapsed days and infection probability. Data normality was assessed using the Shapiro–Wilk test. Nonparametric tests were applied if normality was violated (p < 0.05). All statistical tests were two-tailed, and significance was set at α = 0.05. Data visualization was performed using ggplot2 in R. Fisher’s exact test was used to evaluate the differences in infection rates among the experimental groups. For post hoc analysis, the Dunn–Bonferroni test was used. A logit transformation was performed prior to statistical analysis as the sex ratio data were proportional. The Kruskal–Wallis test was used for group comparisons, followed by the Dunn–Bonferroni test for post hoc analysis. The number of larvae was analyzed using the nonparametric Friedman test to assess temporal differences (p < 0.01). The Dunn–Bonferroni test was conducted as a post hoc analysis when significant differences were detected. A repeated-measures analysis of variance was performed to evaluate temporal changes in the number of H. femoralis adults. Mauchly’s sphericity test was applied, and the Greenhouse–Geisser correction was used if the assumption of sphericity was violated (p < 0.05).
4 Discussion
Wolbachia was almost eliminated from L. trifolii and H. femoralis when MycoShield was sprayed at the standard concentrations on kidney bean leaves. Additionally, Rickettsia was completely removed (Table 1) when N. formosa was fed MycoShield with honey. Although only one agrochemical antibiotic was tested, two species of endosymbionts were successfully removed. A wide variety of agrochemical antibiotics exist globally, and Wolbachia and Rickettsia infect 52% and 24% of arthropod species, respectively (Weinert et al., 2015; Zug and Hammerstein, 2012), with numerous other symbionts also present in many species (Douglas, 1998; Kikuchi et al., 2007; Rupawate et al., 2023). These findings suggest that agrochemical antibiotics can be used as effective tools to manage agricultural pests.
The application of MycoShield to leaf surfaces resulted in a significant reduction in Wolbachia infection rates of L. trifolii and H. femoralis adults, suggesting that Wolbachia was eliminated through the gradual uptake of the antibiotic by adults (Figure 1A and Table 1). Although L. trifolii larvae burrow into the leaves, the application of MycoShield to the leaf surface also reduced the Wolbachia infection rate (Figure 1B). This finding suggests that Wolbachia were eliminated as a result of larval ingestion of MycoShield, which penetrated the leaf tissue.
The route of MycoShield uptake by N. formosa, which parasitizes L. trifolii, was also investigated. N. formosa did not ingest antibiotics upon contact with its leaves. However, when its mouthparts came into contact with L. trifolii exposed to MycoShield or when it fed on the host’s body fluids or internal tissues, antibiotic absorption occurred, leading to a reduced infection rate (Figures 2A, B). These findings suggest that antibiotic uptake by N. formosa is indirect, occurring through exposure to an antibiotic-treated host. Key stages of this process include feeding on the host and larval consumption of infected tissues.
In agricultural settings, MycoShield is typically diluted 1,000-fold with water before application. At this concentration, the Wolbachia infection rate in L. trifolii decreases to 2.8%, the Wolbachia infection rate in H. femoralis decreases to 1.4%, and the Rickettsia infection rate in N. formosa decreases to 1.4% (Table 1). These results indicate that MycoShield application in agricultural settings can effectively eliminate symbiotic bacteria from insects.
When N. formosa was exposed to MycoShield diluted 1,000-fold, Rickettsia was eliminated, resulting in the loss of thelytoky parthenogenesis and the production of male offspring (Figure 2B). This finding suggests that MycoShield may also influence N. formosa populations in agricultural settings, particularly in controlling leaf miners. Some parasitoid wasps are commercially available as biological control agents, playing a crucial role in pest population management via parasitism (van Lenteren, 2012). As female wasps are essential for successful biological control, careful selection of pest management methods is necessary to avoid compromising parasitoid populations.
Although selective elimination of Wolbachia without affecting Rickettsia would be ideal for preserving the biological control capacity of N. formosa, currently no antibiotics are known that can achieve such specificity. This is because Wolbachia and Rickettsia are both members of the Alphaproteobacteria (Dunning Hotopp et al., 2006) and share many fundamental molecular targets, such as ribosomal components and replication machinery (Andersson et al., 1998; Ioannidis et al., 2007; Zug and Hammerstein, 2015). Consequently, the application of broad-spectrum antibiotics like tetracycline inevitably impacts both symbionts (Nguyen et al., 2014). However, previous studies in aphids have demonstrated selective removal of specific endosymbionts through dose-dependent antibiotic treatments—e.g., Serratia via ampicillin and Buchnera via rifampicin (Koga et al., 2007). These findings suggest that with careful optimization of antibiotic type and concentration, selective targeting of symbionts like Wolbachia and Rickettsia may be achievable in other insect systems. Further research to explore such possibilities could significantly improve the integration of antibiotic-based approaches with biological control programs.
To evaluate long-term suppression, we conducted a 100-day experiment in which MycoShield was applied every 20 days at a field-relevant concentration (1,000-fold dilution). In theory, eliminating thelytoky parthenogenesis-inducing Wolbachia should result in all-male offspring, leading to population collapse (Table 1, female ratio: 1.4%). This outcome was observed in two plots where both larvae and adults were absent by day 100 (Figure 3C). However, in most cages, female production persisted and complete suppression was not achieved, likely due to partial clearance of symbionts. Although Table 1 shows a sharp decline in the female ratio among next-generation adults following treatment, Table 2 indicates that female ratios remained at 10–20% in most cages during the 40–100 day period. This discrepancy can be explained by several biological and methodological factors.
First, the life cycle of H. femoralis progresses from egg to adult in approximately 18–20 days at 27°C (Laughlin, 1971). Since insect development generally slows down at lower temperatures, the cycle at our experimental condition (25°C) is expected to be slightly longer. Therefore, the 100% female ratio observed on day 20 likely reflects surviving F0 females rather than newly emerged F1 individuals. Second, although larvae likely ingest antibiotics during feeding, the efficacy of symbiont elimination depends on both the timing and cumulative dose of exposure (Koga et al., 2007; Shan et al., 2016). Since plants were replaced every 20 days, all larval stages likely encountered treated leaves; however, variability in ingestion among instars may have led to insufficient antibiotic uptake in some individuals. Moreover, as H. femoralis pupates on the soil surface, prepupae and pupae may have escaped continued exposure, especially if they entered these protected stages shortly after initial ingestion. This partial exposure scenario may explain why endosymbionts persisted in some individuals.
Interestingly, five cages had only male adults by day 100, suggesting that eradication may occur over a longer timeframe. These male-only populations were effectively reproductively sterile, as no functional females remained to sustain reproduction. This condition reflects a severe reproductive bottleneck, where antibiotic-induced elimination of symbionts disrupts thelytokous parthenogenesis. In such small, male-biased groups, genetic drift further impedes the chance of symbiont persistence or spontaneous recovery, ultimately leading to local extinction. These findings highlight the importance of optimizing treatment timing and frequency to achieve consistent disruption of symbiont-mediated reproduction. However, field conditions may introduce additional variables—such as environmental fluctuation and immigration—that could buffer or counteract this process, warranting further investigation in open settings.
The diverse beneficial roles of symbiotic microorganisms in insect hosts are well-documented. The partial or complete suppression of these microorganisms can result in nutrient deficiencies, weakened immunity, impaired plant defense mechanisms, and increased susceptibility to pathogens, parasites, and predators. Such disruptions may contribute to the partial or complete elimination of pest populations in the field by disrupting essential host functions (Rupawate et al., 2023; Gonella and Alma, 2023). Consequently, targeting symbiotic microorganisms is a promising strategy for integrated pest management.
This study used MycoShield (17.0% oxytetracycline) as the antibiotic treatment; however, numerous antibiotic agrochemicals are commercially available worldwide (McManus et al., 2002). Given the diversity of these compounds, alternative antibiotic agrochemicals may exert broader or more potent antibacterial effects, not only against obligate endosymbionts but also against gut-associated microbiota and mycetocyte-residing microorganisms in infected insects. Additionally, their effects on insect physiology, feeding behavior, and population dynamics warrant further investigation. Investigating the differential effects of various agrochemical antibiotics on insect pests and their associated microbiota is essential for optimizing symbiont-targeted pest control strategies.