Phthorimaea absoluta (Lepidoptera: Gelechiidae) is an invasive insect pest of tomato and other solanaceous plants, which is rapidly expanding its geographic range (Mutamiswa et al., 2017). The pest is reported to have originated in the central highlands of Peru in South America in 1917 (Shiberu and Getu, 2018; Chang and Metz, 2021). In Africa, the P. absoluta invasion was first reported in Morocco, Tunisia, and Algeria in 2008, and later in 2014 in Witu and Mpeketoni fields in Lamu County, Kenya (Tonnang et al., 2015; Kinyanjui et al., 2021). Since then, the pest has spread to all other major tomato-growing regions in Kenya (Kinyanjui et al., 2021). P. absoluta poses a significant threat to the sustainable production of tomatoes, resulting in yield losses ranging from 80 to 100% with substantial price increases for tomatoes in the market (Chepchirchir et al., 2021; Kinyanjui et al., 2021). The high yield and economic losses are linked to P. absoluta’s high reproduction potential, short life cycle, the nocturnal activity of adults, and the larval stage’s concealed feeding behavior inside the leaf mesophyll, making it the most devastating insect pest present in tomato-producing areas in sub-Saharan Africa (Tonnang et al., 2015; Birhan, 2018).

Chemical pesticides are the most commonly used management practices against P. absoluta because they are easily accessible, cheap, and quick-acting (Skinner et al., 2014). However, farmers often mis- and overuse these pesticides as they are applied prophylactically without following the labels and even without detecting the pest (Chidege et al., 2016; Pandey et al., 2023). Heavy application of pesticides has negative consequences on the environment, leads to the development of insecticide resistance, and endangers the production of healthy foods due to the accumulation of chemical residues (Rwomushana et al., 2019). Due to these shortcomings, alternative and environmentally friendly control approaches against P. absoluta are being developed to supplement or entirely replace chemical pesticides (Agbessenou et al., 2020).

The search for alternatives to chemical pesticides has turned to natural microbial pathogens of insects (Skinner et al., 2014). Naturally-occurring entomopathogens have been shown to regulate insect populations in the wild (Ding et al., 2020). Several entomopathogenic fungi (EPF) species, such as M. anisopliae and Beauveria bassiana, have been adopted as biological control agents against insect pests (Qu and Wang, 2018; Akutse et al., 2020a,b). Due to their negligible effect on non-target organisms and easy mass production, EPF are considered the next-generation control agent for insect pests (Jiang et al., 2020). M. anisopliae isolates (ICIPE 665, ICIPE 18, ICIPE 20) (Agbessenou et al., 2021) have recently been identified as potential control agents for the sustainable management of P. absoluta adults (Akutse et al., 2020a,b). However, their efficacy against insect pests is affected by abiotic factors such as temperature, humidity, light, and pH (Dimbi et al., 2009; Silva et al., 2020). The relatively slow mode of action of EPF compared to chemical insecticides has sometimes hindered their adoption and widespread application (Wei et al., 2017). In addition, insect pests have evolved defense mechanisms to counter the infection (Qu and Wang, 2018). Understanding the physiological mechanisms by which EPF exerts control over P. absoluta can lead to more targeted and effective pest control strategies (Wang et al., 2021).

The EPF infection starts with the attachment of spores to the outer layer of the insect’s cuticle. The spores then germinate, penetrate the cuticle, and grow inside the insect’s hemocoel (Wang et al., 2021). Upon infection, the insect senses the invading pathogen and activates cellular and humoral immune responses (Gwokyalya and Altuntaş, 2019). Cellular immune responses are activated immediately after infection, while humoral responses appear hours post-infection (Zhong et al., 2017). Cellular immunity is mediated by hemocytes, which directly recognize invading pathogens or indirectly recognize humoral receptors that bind to and opsonize the invader’s surface. Hemocytes protect the host from invading pathogens through various immune responses, including melanization, nodulation, encapsulation, phagocytosis, and release of antimicrobial peptides (Eleftherianos et al., 2021). However, EPF releases chemical metabolites such as destruxins and beauvericin that paralyze the immunity of the host insects (Jiang et al., 2020; Kryukov et al., 2021). This circular battle results in an evolutionary arms race between insect hosts and pathogens (Qu and Wang, 2018). In addition, the insect gut contains a diverse community of commensal bacteria, the microbiome, which influence the host immune system by modulating immune cell development, maintaining gut barrier integrity, and secreting metabolites that affect immune responses (Liu et al., 2013; Gichuhi et al., 2020; Zeng et al., 2022). These interactions promote tolerance beneficial to microbes while guarding against pathogens, impacting overall immune function and disease susceptibility (Liu et al., 2013; Zeng et al., 2022). The gut microbiome is involved in digestion, detoxification of allelochemicals, and stress responses (Kryukov et al., 2021). It also protects the insect from pathogen infection by inhibiting fungal growth and differentiation through systemic effects such as producing metabolites or signaling molecules that enter the hemocoel, activating immune responses and creating an environment less conducive to fungal growth (Wei et al., 2017; Zhang et al., 2017). Most studies investigating host-pathogen models have focused on the evolutionary and functional aspects, leaving a knowledge gap about the physiological mechanisms underpinning the efficacy of EPF as a control agent against insect pests (Gwokyalya et al., 2022). Thus, a significant lack of detailed information exists regarding the intricate physiological processes that drive the successful control of this pest through EPF. Understanding the physiological mechanisms by which EPF exerts control over P. absoluta can lead to more targeted and effective control strategies (Qu and Wang, 2018). This knowledge could help develop methods that optimize EPF applications, increasing their impact on pest populations while minimizing environmental impact (Yaneth et al., 2020).

To better understand these physiological mechanisms, we assessed the survival of P. absoluta adults upon exposure to M. anisopliae isolates (ICIPE 665, ICIPE 20, ICIPE18) previously identified for sustainable management of P. absoluta adults under different temperature conditions (15, 20, 25, and 30°C). Since the efficacy of EPF is affected by abiotic factors such as temperature (Onsongo et al., 2019), the experiment was conducted under varying temperature regimes to mimic the ecological conditions in which the insect is found in the field. Subsequently, we evaluated the cellular immune responses of surviving P. absoluta moths by focusing on cell population dynamics and morphological characterization of different hemocytes produced when adult tomato leafminers are exposed to the M. anisopliae isolates at varying temperature regimes. EPF triggers varying immune responses in insect pests (Zafar et al., 2020). Counting and identifying different hemocyte populations could offer insights into the types of immune cells activated and their abundance, shedding light on the specificity and intensity of the immune response (Marringa et al., 2014). Finally, we examined the effect of the most potent M. anisopliae isolates (ICIPE 20 and ICIPE 18) on the composition and diversity of bacterial communities within P. absoluta after infection at different temperature regimes.

The survival of P. absoluta following infection with M. anisopliae isolates is likely influenced by hemocyte counts and gut microbial diversity. Hemocyte counts reflect immune responses affecting the ability to combat the infection. On the other hand, gut microbial diversity could impact immune function, potentially influencing survival outcomes by affecting immune activation and overall physiological resilience. The relationship among these factors informs how immune reactions and microbial interactions contribute to survival outcomes in these host-pathogen interactions. Therefore, this study aimed to offer valuable insights into the impact of the M. anisopliae isolates on the biology of P. absoluta adults across various temperature conditions. This research will enhance our comprehension of the specific physiological mechanisms that underlie the effectiveness of EPF as potential control agents against P. absoluta adults, a crucial knowledge for developing targeted and sustainable pest control strategies.

Entomopathogenic fungi control insects by infecting and killing them through complex mechanisms (Migiro et al., 2010; Jiang et al., 2020). When the spores of these fungi come into contact with the host insect, they can attach to the cuticle and germinate, penetrating the insect’s body through the cuticle, mouthparts, or spiracles (Wang et al., 2021). Once inside the insect, the fungi grow and produce enzymes and toxins that break down the insect’s tissues and organs, ultimately leading to death (Skinner et al., 2014). However, the effectiveness of EPF depends on several factors, such as the fungal species, the target host insect, and the environmental conditions (Bugeme et al., 2008; Shamakhi et al., 2019). Our research offers new insights into EPF infections and interactions with the innate immune responses and gut microbiome dynamism of P. absoluta. The cellular immune response and gut immunity protect insects against infections with entomopathogenic filamentous fungi (Yassine et al., 2012; Ramirez et al., 2019).

When P. absoluta moths were exposed to dry M. anisopliae conidia, their survival rate gradually declined as the temperature increased from 15 to 30°C. After infection, all three fungal isolates induced the highest mortality rate at 25°C, which seemed to be the optimal temperature for the maximum impact of these fungal strains’ infection on the pest. Adult P. absoluta challenged with ICIPE 18 had the lowest survival rate, followed closely by those infected with ICIPE 20, while moths challenged with ICIPE 665 showed the highest survival rate. Our findings align with earlier research studies reported by Akutse et al. (2020a,b) and Agbessenou et al. (2021), who demonstrated the substantial potential of M. anisopliae isolates ICIPE 18 and ICIPE 20 against adult P. absoluta. Onsongo et al. (2019) also reported comparable results, where M. anisopliae isolates, ICIPE 18 and ICIPE 69 caused the most significant mortality in adult Tephritid fruit flies (Zeugodacus cucurbitae) at 25°C. Tumuhaise et al. (2015) conducted a study on temperature-dependent effects, which revealed that the most significant mortality rates were observed between 25 and 30°C when combating adult legume pod borer (Maruca vitrata Fabricius) using M. anisopliae isolate ICIPE 69. The rapid germination of the M. anisopliae isolates at 25°C could probably explain the fastest/highest mortality they induced in the host insect pests. This phenomenon confirms that fungal-based insecticides kill insect pests most rapidly at the optimum temperature of their vegetative growth (Tumuhaise et al., 2015). Our findings demonstrate that ICIPE 20 and ICIPE 18 could be deployed in ecological areas with temperatures around 25°C for sustainable management of adult P. absoluta, which has also been reported by Agbessenou et al. (2021). Since the efficacy of EPF against insect pests is temperature-dependent, the virulence pattern observed in this study is crucial in choosing key application areas for these biopesticides.

When EPF penetrate the body of insect pests, they activate both cellular and humoral immune responses that work to protect the insect host from the invading pathogens (Wang J.-L. et al., 2022). In response, the infected insects release more hemocytes to fight the external stimuli (Shamakhi et al., 2019). These hemocytes play a critical role in mediating cellular immune responses, which are important in helping insects fight against invading pathogens (Stoepler et al., 2013). In the process, hemocytes may adhere to the surface of the fungal hyphae, which can reduce their invasion and virulence to defend against the infection.

In this study, we observed a significant reduction in the total hemocyte counts (THCs) in P. absoluta adults exposed to M. anisopliae isolates, with the decline increasing steadily from 15°C to 25°C. The declining pattern in the concentration of THCs could be linked to the ability of the tested M. anisopliae isolates to produce toxins that affect the hemocytes’ viability or function/virulence. In addition, the immune response to the fungal infection may have led to the depletion of hemocytes, as they are recruited and consumed by the host insect to fight the infection. Freitas et al. (2015) also witnessed a comparable reduction in the number of hemocytes 48 h after infecting Rhipicephalus microplus ticks with Beauveria bassiana CG 206 conidia. Additionally, Zhong et al. (2017) reported a decrease in THCs following infection of the cotton bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) with M. anisopliae. In contrast, Mishra et al. (2015) noted an opposite effect: the THCs increased in housefly larvae Musca domestica Linnaeus (Diptera: Muscidae) after exposure to B. bassiana.

Our findings also demonstrated significant post-infection alterations in the constitutive hemocyte types in host insects. For instance, variations in the granulocyte counts after the fungus challenge, may indicate fungus-induced differentiation and secretion of granulocytes into the hemolymph. However, a decline in plasmatocyte density was observed in P. absoluta infected with fungus. The observed pattern of granulocyte increase and plasmatocyte decrease following M. anisopliae infection may be linked to increased granulocyte differentiation and selective plasmatocyte apoptosis. Among the differential hemocyte counts observed in this study, the specific roles of spherulocytes and adipohemocytes in innate immunity are unknown (Altuntaş et al., 2021; Gwokyalya et al., 2022). However, oenocytoids have been stated to play an essential role in mediating melanization by activating the phenol oxidase cascade (Gwokyalya et al., 2022). Therefore, the increase in oenocytoids following fungal infection may be due to the host’s response to stimulate the phenoloxidase melanization of the fungal germ tubes and consequently block the pathogen activity within the host. Although M. anisopliae conidia is easily transmitted to the pest through contact, the duration of infection is relatively long (24–72 h) (Sinha et al., 2016). This explains the variations in hemocyte types observed on day 0 (2 h post-infection) that may be due to other factors such as temperature changes.

The decrease in circulating hemocytes following infection led us to investigate the microbial community in the gut of the infected P. absoluta adults, as the fungal infection could directly impact the gut microbiome. Fungal infections can disrupt gut bacterial diversity by altering the microbial balance, causing inflammation, and affecting the gut environment (Wei et al., 2017). This disruption can lead to imbalances in the microbiota, impacting digestion, immune responses, and overall gut health (Siddiqui et al., 2022). Our results showed an upsurge in bacterial load in infected P. absoluta moths on the tenth-day post-infection. The level of microbiome loads increase appeared to reflect the magnitude of the immune response since P. absoluta adults that had recorded the highest THCs decline (ICIPE 20 and ICIPE 18-infected P. absoluta) also had the highest upsurge in microbiota diversity at 25°C. The effect of EPF on the gut microbiome was previously examined by Wei et al. (2017) in Anopheles stephensi Liston (Diptera: Culicidae) mosquitoes after infection with B. bassiana. The study demonstrated an increase in gut bacterial loads in mosquitoes. In addition, the authors found that increased bacterial loads led to accelerated deaths of infected mosquitoes. Therefore, the rise in gut bacterial load is most likely due to the indirect result of the multifaceted response mounted by P. absoluta against the fungal infection challenges/stresses. A microbiome analysis between the treatment groups showed no significant microbial shifts in the gut bacterial composition in P. absoluta adults incubated at 15 and 20°C. The slow mode of action of M. anisopliae infection at these temperature regimes could cause an unnoticeable change in bacterial abundances in P. absoluta. However, M. anisopliae infection caused significant microbial shifts at 25°C. The variations in the relative abundance of Wolbachia to other bacterial genera, including Hydrogenophilus, Ewingella Bacillus, Klebsiella, Escherichia, Sphigomonas, Pseudomonas, Ewingella, Vibrio, and Enterobacter, highly suggest that this endosymbiont might play an essential role in the course of fungal infection. Wang J.-L. et al. (2022b) observed a similar gut microbial shift pattern following topical fungal infection of brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacini) with M. anisopliae.

Wolbachia was the predominant bacterial genera in infected P. absoluta groups and their control counterparts (control groups) across all the temperature regimes. It is a crucial endosymbiont in many insects (Gichuhi et al., 2020). Wolbachia has been proposed as a potential component that can be explored to develop control approaches against host insects (Rancès et al., 2012). Studies show that Wolbachia induces host reproductive manipulation, including cytoplasmic incompatibility, feminization, male killing, and parthenogenesis (Oliver et al., 2022). In adult P. absoluta infected with ICIPE 18 and incubated at 25°C, we noted an upsurge of Ewingella, Vibrio, and Hydrogenophilus. In addition, at the same temperature regime, we noted an upsurge in Acinetobacter, Bacillus, Klebsiella, Providencia, Pseudomonas, Staphylococcus, and Serratia in ICIPE 20-infected P. absoluta. Ewingella and Vibrio are pathogenic gram-positive bacteria that have been reported to cause high mortality in insects and humans (Meisler et al., 2020). Acinetobacter is a bacterial genus found in insect gut that provides the host with nutrients and insecticidal potential that could be pathogenic against their host (Wang et al., 2022a). Bacillus is an entomopathogenic bacteria used for microbial pest management on various insect pests, including Spodoptera frugiperda (Lepidoptera: Noctuidae) (Ruiu, 2015). Other bacteria showing pathogenic potential against insect pests include Serratia, Providencia, and Pseudomonas (Ruiu, 2015; Deka et al., 2021). As far as we know, no research has been conducted to investigate the pathogenic effects of Hydrogenophilus on P. absoluta.

Our results indicated that at 25°C, P. absoluta adults infected with ICIPE 20 had the highest evenness index, while those challenged with ICIPE 18 exhibited the greatest microbial diversity. These findings suggest that ICIPE 20 caused all bacterial communities within P. absoluta to have a higher expression rate of equal abundance within the microbiome than the insects challenged with ICIPE 18. Analysis of species richness showed that P. absoluta challenged with ICIPE 20 had the most bacterial taxa and, thus, were likely to have a more complex microbiota. The species evenness and richness in fungus-challenged P. absoluta were positively correlated. Ajene et al. (2020) reported that species’ evenness and richness positively correlate within invertebrate communities. The Jaccard Dissimilarity indices showed significantly different clustering of fungus-challenged P. absoluta moths and uninfected insects. This suggests that the bacterial community structure in M. anisopliae-infected P. absoluta differed from that of their control counterparts. Overall, the alpha and beta diversity of the metagenome of adult P. absoluta supports the hypothesis that infection with M. anisopliae disrupts gut bacterial community structure.

We also expected the survival rate, THCs, and gut microbial abundance to be positively correlated when P. absoluta was infected with ICIPE 20 at different temperature regimes (Onchuru and Kaltenpoth, 2019). However, this assumption held for prohemocytes, plasmatocytes, spherulocytes, THCs, plasmatocytes, and survival rate at 15°C. It seems likely that the high numbers of THCs are responsible for the defense of host insects, hence the higher survival. At 20°C, THCs were negatively correlated with survival rate, which might explain the decline in survival of P. absoluta. In addition, the positive correlation of plasmatocytes and gut microbial abundance might indicate increased immune response in the host-insect after M. anisopliae infection. A similar correlation was observed at 25°C, seemingly indicating hemocytes and gut microbiome are the primary defense mechanism used by P. absoluta when infected by M. anisopliae.

In conclusion, our study provides valuable insights into the potential of M. anisopliae isolates, specifically ICIPE 18 and ICIPE 20, as effective biocontrol agents against P. absoluta adults. The findings of this study underscore the significance of immunological changes and gut microbial shifts in influencing the survival and fitness of P. absoluta adults. The mortality induced by ICIPE 18 suggests strong pathogenicity, and ICIPE 20’s impact underscores its potential as a potent agent of insect pest population control. The significant reduction in THCs in response to these isolates points to the disruption of the insect’s immune response. This immune system suppression could render pests more susceptible to infections and environmental stressors. Moreover, the observed gut microbial shifts induced by both isolates at 25°C present a crucial mechanistic insight. Alteration in the gut microbiota can significantly affect insect digestion, nutrient utilization, and overall fitness. By weakening both immune defenses and gut health, ICIPE 18 and ICIPE 20 act synergistically to compromise the survival and vitality of P. absoluta adults. These findings collectively support the potential of ICIPE 18 and ICIPE 20 as biocontrol agents. Their ability to disrupt physiological processes, weaken immune responses, and induce gut microbial shifts suggests a multi-pronged approach to pest management. Future research could delve deeper into the mechanisms underlying these effects and further explore their long-term ecological implications to validate their role as sustainable alternatives to chemical insecticides.

The data presented in the study are deposited in the NCBI GenBank repository https://www.ncbi.nlm.nih.gov/genbank, Bioproject number PRJNA970552.

The manuscript presents research on animals that do not require ethical approval for their study.

FM: Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – original draft, Writing – review & editing. KA: Conceptualization, Data curation, Funding acquisition, Resources, Supervision, Validation, Writing – review & editing. IA: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – review & editing. KO: Resources, Supervision, Writing – review & editing. FK: Conceptualization, Data curation, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing.