Entomopathogens and Parasitoids Allied in Biocontrol: A Systematic Review

Biological pest control is an environmentally friendly alternative to synthetic pesticides, using organisms such as viruses, bacteria, fungi, and parasitoids. However, efficacy is variable and combining different biocontrol agents could improve success rates. We conducted a systematic review of studies combining a parasitoid with an entomopathogenic microorganism, the first of its kind. We searched in Web of Science and extracted data from 49 publications matching the pre-defined inclusion criteria. Combinations of 36 hymenopteran parasitoids with 17 entomopathogenic microorganisms used to control 31 target pests were found. Trichogramma pretiosum and Encarsia formosa were the most frequently studied parasitoids, while Beauveria bassiana, Metarhizium anisopliae, Lecanicillium muscarium, Bacillus thuringiensis var. kurstaki, the Spodoptera exigua multiple nucleopolyhedrovirus, and the Spodoptera frugiperda multiple nucleopolyhedrovirus were the main microbial agents assessed. Out of 49 parasitoid–microorganism combinations assessed in the laboratory experiments, thirty-eight were reported as compatible and six as incompatible. Timing and dosage of biopesticides played a crucial role, with later application and appropriate dosage minimizing adverse effects on parasitoid development. More research is needed to assess compatibility and efficacy under real-world conditions. Our review provides valuable insights for researchers and practitioners to optimize the combined use of micro- and macroorganisms for effective pest control.


Introduction
Biological pest control is an alternative to synthetic pesticides with fewer adverse environmental effects [1]. Among the most used organisms in biological control are microorganisms such as entomopathogenic bacteria, fungi, and viruses. Bacillus thuringiensis sp. (Bt) are the most widely used bacteria to control pests in agriculture, forestry, and public health [2]. They release toxins that cause cell lysis and death after binding with specific receptors in the insect midgut [3]. Entomopathogenic bacteria have a wide host range, mainly lepidopteran, dipteran, and coleopteran, but were reported to have minimal to no negative effects on beneficial organisms [3]. Beauveria bassiana (Balsamo) Vuillemin, Metarhizium sp., Paecilomyces farinosus (Holm ex S.F. Gray) Brown & Smith, and Lecanicillium muscarium Zare & Gams (previously known as Verticillium lecanii (Zimmermann) Viegas) are the most used fungi for insect biocontrol. They attack pests by damaging their integument or gut epithelium, using nutrients in their hemocoel, or releasing toxins [2,3]. They are ubiquitous in the environment, have a broad range of arthropod hosts, and often cause epizootics in insect populations. Compatibility with arthropod predators and parasitoids should be tested to ensure compatibility and maximize efficacy [3]. Approximately a dozen viral Given the increasing interest in biological pest control in the past decade and the growing importance of entomopathogen agents [14,15], a new and systematic review of the literature combining all different entomopathogens is timely. We aimed to synthesize the state-of-the-art when combining a parasitoid with an entomopathogenic microorganism. Combining parasitoids and microorganisms may be positive, negative, or with no effect on pest control compared to their use alone. We hypothesized that the effects could be positive and that more efficient pest control could be achieved through combining agents. We, therefore, assessed: (1) Which are the most studied combinations of parasitoids and microorganisms? (2) Which combinations of microorganisms and parasitoids are compatible? (3) How do microorganisms influence the life table parameters of parasitoids? (4) Which key factors influence the compatibility of microorganisms and parasitoids? (5) Can more efficient pest control be achieved by combining a parasitoid with a microorganism instead of using them individually?

Search Criteria
We used the following search string in Web of Science Core Collection on 2 March 2023: ((fung* OR vir* OR entomopathog* OR "vir*-based insecticid*" OR "fung*-based insecticid*" OR "biological insecticid*" OR "microbial insecticid*" OR "natural insecticid*") AND (biocontrol OR biological control)) AND (*parasit* AND (biocontrol OR biological control)) AND (combin* OR interaction OR substitut* OR synergist* OR antagonist*) AND ("integrated pest control" OR "biological pest control" OR "pest biocontrol" OR "pest populations" OR pest OR "pest management" OR IPM). We did not use any date limitation but confined our search to journal articles in English.

Data Inclusion and Exclusion Criteria
We assessed articles by analysing abstracts following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA, [16]) ( Figure S1). We obtained 547 initial hits, of which 121 were excluded as they were review articles. Based on other predefined exclusion criteria, 377 further articles were excluded. Inclusion of a publication was based on three criteria: (1) the study was an experiment concerning the biological control of a pest; (2) the experiment combined at least a parasitoid and an entomopathogenic fungus, bacterium, or virus; and (3) the study design included at least a no-treatment control. Studies combining entomopathogenic nematodes with parasitoids were

Search Criteria
We used the following search string in Web of Science Core Collection on 2 March 2023: ((fung* OR vir* OR entomopathog* OR "vir*-based insecticid*" OR "fung*-based insecticid*" OR "biological insecticid*" OR "microbial insecticid*" OR "natural insecticid*") AND (biocontrol OR biological control)) AND (*parasit* AND (biocontrol OR biological control)) AND (combin* OR interaction OR substitut* OR synergist* OR antagonist*) AND ("integrated pest control" OR "biological pest control" OR "pest biocontrol" OR "pest populations" OR pest OR "pest management" OR IPM). We did not use any date limitation but confined our search to journal articles in English.

Data Inclusion and Exclusion Criteria
We assessed articles by analysing abstracts following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA, [16]) ( Figure S1). We obtained 547 initial hits, of which 121 were excluded as they were review articles. Based on other predefined exclusion criteria, 377 further articles were excluded. Inclusion of a publication was based on three criteria: (1) the study was an experiment concerning the biological control of a pest; (2) the experiment combined at least a parasitoid and an entomopathogenic fungus, bacterium, or virus; and (3) the study design included at least a no-treatment control. Studies combining entomopathogenic nematodes with parasitoids were excluded. Laboratory, semi-field, field, and greenhouse experiments were considered, but modelling and simulations were excluded.

Data Extraction
We used Citavi software (version 6.11.0.0) to import the included studies. We extracted data on (1) pest, (2) control agents, (3) crop, (4) location, (5) study design, (6) evolution of pest and biocontrol agent populations, (7) crop damage, (8) crop yield, and (9) compatibility of biocontrol agents from the 49 selected articles . Each combination of biocontrol agents was considered an experiment, leading to 100 distinct experiments, as some publications studied multiple combinations simultaneously. Different strains of the same microorganism combined with one parasitoid were also counted as distinct experiments. Data from experiments assessing multiple dosages, timing of application, and types of exposition of the parasitoid to the microbial biocontrol agent were extracted as several observations. Each different treatment was considered as a single observation leading to the extraction of 484 individual observations.

Data Synthesis and Analysis
Most studies reported the effect of entomopathogenic microorganisms on the life table parameters of parasitoids. In these studies, parasitoids were the dependent variable. The impact of a treatment with entomopathogenic microorganisms was usually compared with a "no-treatment control" (parasitoids only). Data were synthesized by "vote counting" of the numbers of positive, neutral, and negative effects caused by the entomopathogen on each studied parameter of the parasitoid's fitness. We used "positive" when the microorganism treatment significantly improved the development of the parasitoid in comparison to the no-treatment control. "Negative" was used when the microorganism treatment significantly hindered the development of the parasitoid compared to the no-treatment control. "No effect" was used when no significant difference was found between the treatment and the parasitoid-only control. In a simplified way, combinations with positive or no effect were defined as compatible. However, in the reviewed papers, compatibility was assessed by an overall analysis of all the studied parameters. Being unable to synthesize all interconnected parameters for all reviewed papers, we reported compatibility based on the authors' conclusions. For example, if they mentioned that two biocontrol agents could be combined at a defined timing and dosage despite observed antagonistic effects under other conditions, we reported those as compatible.
When the effect on pest control was reported, "positive" was used to categorize when the pest reduction achieved by the combined biocontrol agents was higher than the reduction achieved by the strongest agent applied individually. Positive effect can be either "synergistic" when the pest reduction achieved by the combined biocontrol agents is higher than the addition of that achieved by each agent applied individually, "additive" when the pest reduction achieved by the combination is equal to the addition of that achieved by each agent used alone, or "less than additive" when the pest reduction achieved by the combined biocontrol agents is significantly higher than that achieved by each agent used alone, yet lower than additive. "No effect" was used when no significant difference was observed between the pest reduction achieved by the combined agents and that achieved by the strongest agent applied individually. "Negative" was used when the combined biocontrol agents achieved a lower pest reduction than the strongest agent used individually.
Among the publications selected for this review, reports on laboratory combination experiments were predominant. From those publications, we extracted life-history data on sixteen parameters for parasitoids, four parameters for pests, and five parameters for entomopathogenic microorganisms. For further analysis, we focused on the parasitism rate, emergence rate, mortality, sex ratio, and longevity of parasitoids, as these were the most documented parameters.

Scope of the Publications
Forty-nine studies detailing one hundred combination experiments were conducted from 2000 to 2022. Eighty-four were laboratory experiments and thus formed the focus of our results. In addition, nine were field experiments, five were semi-field experiments, and two were greenhouse experiments. All combination experiments included a no-treatment control as it was an inclusion criterion. Four experiments included additional controls with either the parasitoid (3) or microorganism (1) alone, and fourteen included both types of controls. Six experiments included a synthetic insecticide control in addition to the no-treatment control. The reviewed studies dealt with 31 target pests. Approximately half of these were Lepidoptera, of which 52% were Noctuidae. The remaining were Hemiptera, Diptera, and Coleoptera ( Figure 2). The studied biocontrol agents included 36 parasitoids and 17 entomopathogenic microorganisms. All parasitoids were Hymenoptera, and the most represented families were Braconidae (44%) and Trichogrammatidae (18%) (Figure 3). Most combination experiments were conducted with fungi (80%), followed by bacteria (11%) and viruses (9%) (Figure 4).
Forty-nine studies detailing one hundred combination experiments were conducted from 2000 to 2022. Eighty-four were laboratory experiments and thus formed the focus of our results. In addition, nine were field experiments, five were semi-field experiments, and two were greenhouse experiments. All combination experiments included a no-treatment control as it was an inclusion criterion. Four experiments included additional controls with either the parasitoid (3) or microorganism (1) alone, and fourteen included both types of controls. Six experiments included a synthetic insecticide control in addition to the no-treatment control.

Effect of Entomopathogenic
Similarly, the first application of Lecanicillium longisporum (Petch) Zare & Gams should be conducted one day after E. formosa enters the pupal stage to reduce competition between the biocontrol agents [29]. Detrimental effects of B. bassiana and M. anisopliae on the development of E. formosa could be reduced by waiting at least four days after parasitoid release to spray the microbial agents [51]. Post-parasitism application of Metarhizium brunneum Petch also appeared to be best suited for Hyposoter didymator as it limited negative effects on the parasitoid due to direct contact with the fungus [45]. A spatial separation of the microbial treatment and the parasitoid release is also possible to avoid these kinds of effects [53]. Fewer A. colemani with a lower rate of females emerged from aphids treated with L. muscarium within five days of parasitization. In contrast, fungal application six or seven days after aphids had been parasitized did not significantly affect the development, emergence rate, or sex ratio of the parasitoid [46]. Four different time intervals between parasitism by Campoletis sonorensis and application of the SfMNPV were tested. Decreasing parasitoid mortality was observed with increasing time interval. The virus did not affect the survival of C. sonorensis when applied six days after parasitization [28]. In an experiment combining Euplectrus plathypenae and the SeMNPV, the parasitoid was only able to complete its development when the viral infection occurred at least two days after parasitization [46].
In contrast, few recommendations to apply the entomopathogenic microorganism prior to the parasitoid release were found in the reviewed publications. Bacillus thuringiensis var. kurstaki and the Helicoverpa armigera nucleopolyhedrovirus (HearNPV) were recommended to be applied two days before releasing H. hebetor to control Helicoverpa armigera on chickpeas [20]. Similarly, it was recommended to apply B. bassiana before releasing Trichogramma dendrolimi so that pest eggs unaffected by the fungal treatment become parasitized [65].
Here, we show for the first time that most studied combinations of biocontrol agents are compatible under controlled conditions. In the best cases, parasitoids are outside the field of action of entomopathogenic microorganisms and remain unaffected when combined with the latter. If not, the timing of application of the biocontrol agents and the biopesticide dose must be carefully determined. Mathematical models such as the one created by Gonthier et al. [69] for the combined use of Necremnus tutae and Phthorimaea operculella granulovirus against Tuta absoluta can be helpful tools for this purpose. If the dose required to control a specific pest is higher than that tolerated by the parasitoid, the compatibility of the two biocontrol agents is compromised. In terms of pest control, combined biocontrol agents had a positive influence in most cases compared with each agent used alone. However, in their narrative review, Roy and Pell [10] highlighted the importance of conducting field experiments in addition to laboratory bioassays to assess the physiological and ecological susceptibility of natural enemies in a realistic environment. In the field, unpredictable climatic conditions could modify the dynamics of the biocontrol agents observed in the laboratory. Furthermore, less precise application of biopesticides and broader spatial dispersion of pests and parasitoids could significantly influence the compatibility and efficacy of the combinations of biocontrol agents. The presence of other insect species could also influence the level of pest control achieved by parasitoids and entomopathogenic microorganisms that have a wide host range.

Parasitoid Life History and Susceptibility to Entomopathogens
Parasitoid life history (e.g., generation time, population structure) can influence their susceptibility to entomopathogens [70,71]. As many entomopathogens target the larval stage of the pest, egg parasitoids are less likely to be in contact or compete with entomopathogens, making them de facto more compatible. The development strategies of larval parasitoids can strongly influence their susceptibility to entomopathogens. Endoparasitoids, which lay their eggs inside the host insect's body, may be less exposed than ectoparasitoids, which lay their eggs on the surface of the host, sometimes in open environments where the microorganisms can directly reach the parasitoid larvae [72]. On the other hand, endoparasitoids typically have a longer development time than ectoparasitoids, which can also influence their susceptibility to entomopathogens. As entomopathogens have a slower mode of action, taking longer to kill the host insect, parasitoids with shorter life cycles may emerge from the host before the entomopathogen has a chance to kill it, reducing their exposure to the pathogen [72].
In fact, endoparasitoids are generally considered to be more susceptible to entomopathogens than ectoparasitoids [73], as the pathogen has more time to act on the immature parasitoid during its extended development period inside the host insect [74]. Additionally, endoparasitoids are more likely to be exposed to entomopathogens that are ingested by the host insect, as the pathogen can spread throughout the host's body and affect the parasitoid's physiological function [75].

Investigated Combinations and Reported Compatibility of Biocontrol Agents in Field, Semi-Field and Greenhouse Experiments
Nine field, five semi-field, and two greenhouse experiments reported the effects of fifteen combinations of parasitoids and entomopathogenic microorganisms on pest control. Thirteen experiments included fungi, one included a bacterium, and the two remaining ones were conducted with viruses [21,31,33,39,40,46,48,61]. As in the laboratory experiments, B. bassiana was the most studied microorganism. It was tested in combination with E. formosa [65], Chelonus bifoveolatus, Coccygidium luteum and Cotesia sp. [48], Anisoptermalus calandrae and Lariophagus distinguendus [33], and Macroglenes penetrans [61] in one experiment, each. In their greenhouse experiment, Labbé et al. [40] found that B. bassiana used in addition to E. formosa resulted in a higher pest reduction than the parasitoid alone without harming the development of the latter. In contrast, the fungus was reported as incompatible with A. calandrae and L. distinguendus [33]. Indeed, in semi-field experiments, B. bassiana affected both parasitoids negatively, resulting in lower pest control of the fungusparasitoid combinations compared with the parasitoid released alone. No answer about the compatibility of this fungus with the other parasitoids mentioned above was given.
In the field experiments conducted by Ngangambe and Mwatawala [48], M. anisopliae was tested in combination with C. bifoveolatus, C. luteum, and Cotesia sp., also in one experiment each. In this study, biopesticides based on B. bassiana and M. anisopliae were reported as less harmful to natural parasitoids than synthetic insecticides based on flubendamide. Fuentes-Contreras and Niemeyer [31] assessed Pandora neoaphidis and Aphidius rhopalosiphi as compatible in two semi-field experiments. Combining these biocontrol agents resulted in more efficient pest control than each agent used alone. It significantly reduced the growth rate of the pest population. In a greenhouse experiment, the SeMNPV and Microplitis pallidipes were found to be compatible [39]. The parasitoid vectored the virus, and their combined use resulted in a significantly higher pest control. Thus, it was recommended to expose the parasitoid to the virus before releasing it.
H. hebetor was tested in combination with Btk and the HearNPV in one field experiment each [21]. These combinations were reported as compatible. Both parasitoid-microbe combinations significantly reduced pest density and crop damage in comparison with each biocontrol agent alone. Crop yield was significantly increased, but so were the control costs. Yield gain was insufficient to cover the additional treatment costs, meaning that combining the biocontrol agents negatively affected the crop's profitability. The remaining investigated combinations were L. muscarium with A. colemani in a semi-field experiment [46] and the SeMNPV with M. pallidipes in a greenhouse experiment [39]. Both were reported as compatible and significantly reduced pest density when deployed together.
Here, we show that combining entomopathogenic microorganisms with parasitoids in the field appears beneficial for pest control; however, profitability may be reduced due to increased control costs. However, the interaction of biocontrol agents on key aspects, namely crop damage, crop yield, and treatment costs, were assessed in only two out of the hundred reviewed experiments. These agronomic and financial parameters must be considered in future research assessing the compatibility of parasitoids and entomopathogenic microorganisms.

Conclusions
Environmental pollution, loss of biodiversity, pest resistances, and risks to human health are among the controversial effects of synthetic pesticides. Alternative methods for pest control are sought after. Combining biocontrol agents can improve pest control and reduce harmful effects on the environment. In this systematic review, we show for the first time that many combinations of parasitoids and entomopathogenic microorganisms are compatible and can be deployed together. Eighty percent of the biocontrol agent combinations included in the reviewed papers were deemed compatible. Combinations, including parasitoids and fungi, were well represented in the literature. In contrast, few experiments were found combining bacteria with parasitoids, despite the large number of Bt-biopesticides. Further research on combining bacterial or viral biopesticides with parasitoids is required. The most studied microorganisms of each category, i.e., B. bassiana and M. anisopliae for fungi, Btk for bacteria, as well as the SeMNPV and the SfMNPV for viruses, were found compatible with many different parasitoids. However, most of the studies were conducted in the laboratory, and new experiments under field conditions are necessary to include agronomic and financial parameters in the final compatibility assessment. Moreover, most of the reviewed studies focused on the impacts of entomopathogenic microorganisms on parasitoids. Few examined the effects of parasitoids on the development and dissemination of microbial biocontrol agents. Further research is required to analyse how both types of biocontrol agents influence each other in the field. Such an assessment should be conducted on more than one generation of parasitoids to highlight possible long-term effects. Appropriate timing of application and dosage must be defined individually for each combination of biocontrol agents against each specific pest, as these are key success factors. Combining biocontrol agents has the potential for pest control, yet interactions between parasitoids and entomopathogenic microorganisms should be further researched. To develop innovative methods, interdisciplinary work should be fostered. Finally, pest biocontrol methods must be viable. Therefore, the availability and the production costs of biocontrol agents should be assessed and further improved.

Data Availability Statement:
No new data were created or analysed in this study. Data sharing is not applicable to this article.