Insect Frass as an Agricultural Resource Against Abiotic and Biotic Crop Stresses: Mechanisms of Action and Possible Negative Effects

Abstract

The relentless growth of the global population, coupled with increasing biotic and abiotic stresses on crops, poses a major challenge: enhancing agricultural productivity while mitigating these stresses and reducing chemical inputs. Insect farming has led to the large-scale production of insect frass, a nutrient-rich by-product with biofertilizer and biostimulant potential. This review examines the effects of frass on plant stress responses, including its mechanisms of action and possible negative effects. Regarding abiotic stress, frass from certain insects improves plant resilience to drought, waterlogging and salinity, while facilitating heavy metal sorption and complexation in contaminated soils. For biotic stress, frass contains antifungal, antibacterial, and nematicide compounds, as well as entomopathogenic fungi, all of which can reduce pest survival. Additionally, frass activates plant defense mechanisms, such as the increased expression of the defense-related genes involved in stress signaling and immune activation. However, some studies report negative effects, including pathogen dispersion, pest attraction, and the inhibition of beneficial microorganisms commonly used as biopesticides. Despite these risks, frass is a promising alternative for sustainable agriculture, reducing chemical dependency while improving plant resilience. Nevertheless, further research is needed to mitigate its potential risks and optimize its agricultural application.

1. Introduction

With roughly 16.5 gigatons of carbon dioxide equivalent (GtCO₂e) annually out of a total of 54 GtCO₂e, food systems represent one-third of global anthropogenic greenhouse (GHG) gas emissions, making them a significant environmental impact driver [1]. Furthermore, according to the United Nations, the world’s population, currently at 8.2 billion, is projected to grow steadily over the next six decades, and peak at approximately 10.3 billion by the mid-2080s [2]. Agriculture represents 9.91% of GDP [3], yet many countries prioritize it as a “national security” concern, given the essential role these products play in their survival.

Agricultural production is currently facing one of humanity’s biggest challenges: how to meet the food needs of a growing global population while minimizing reliance on chemical inputs. By 2050, global food production must increase by 70%, which calls for a transition to more sustainable farming methods [4]. Achieving this goal will require a significant boost in agricultural output despite limited resources and increasingly degraded soils. The challenge is further exacerbated by the escalating impacts of climate change, such as more frequent droughts, extreme weather events, and the spread of new pests and diseases, all of which place considerable stress on crops and food systems [5].

The extensive use of agrochemicals, including fertilizers and pesticides, has greatly increased agricultural productivity, supporting the growth of the global population. However, excessive use has resulted in significant environmental and health issues [6]. These problems include soil and water contamination, a decline in biodiversity, and the accumulation of harmful residues in food chains, posing a threat to ecosystems and human health [7]. To address these concerns, initiatives like the European Green Deal, led by the European Union, aim to transition toward more sustainable agricultural practices. Through the Farm to Fork Strategy, the plan targets a 50% reduction in pesticide use and a 20% decrease in chemical fertilizers by 2030 [8].

With the expansion of insect farming, the growing availability of insect excrement presents new opportunities for agricultural applications. It is estimated that frass production in Europe already exceeds several thousand tons per year and could reach 1.5 million tons annually by 2025 [9]. This review explores its potential as a novel agricultural input, focusing on how it can help alleviate both abiotic and biotic stresses. Additionally, the discussion analyzes the viability of frass as a sustainable substitute for synthetic agrochemicals and its contribution to promoting eco-friendly farming practices, along with a thorough evaluation of its future prospects. The possible negative effects resulting from its application, such as the spread of plant pathogens, the inhibition of local plant defense mechanisms, or even an increase in pest populations, will also be discussed.

2. Abiotic Stresses of Agricultural Importance

Crop stress poses a significant threat to food security worldwide by lowering agricultural output and restricting yield potential [10]. Abiotic stresses, exacerbated by climate change, have a major effect on food production and need to be further investigated. Environmental factors, such as water stress, high soil salinity, and pollution have also been shown to hinder plant growth and productivity by imposing physiological and biochemical constraints [10]. These pressures threaten plant resilience, can result in yearly agricultural losses of over USD 170 billion [11], and yield 60% below ideal conditions [12].

Drought is among the most damaging abiotic stresses affecting agricultural systems, accounting for a significant portion of agricultural losses and severely impacting essential crops, such as wheat, maize, rice, and soybeans, with estimated annual losses of approximately USD 44 billion [5]. However, other studies suggest that these losses may reach as high as USD 80 billion annually [11]. Approximately 11% of total croplands and 14% of pastures are affected by recurrent droughts, while more than 60% of irrigated croplands experience high water stress [5].

Heat waves affect plant growth differently depending on the species; however, they are undeniably a highly disruptive factor in crop development worldwide [13], with their frequency projected to increase fourfold by 2040 [14]. Climate-related events have tripled in severity in Europe, reducing cereal yields by 9% and non-cereal crops by 3.8% [15]. Southern Europe is particularly vulnerable, as wheat and corn production are expected to decline sharply due to water shortages and rising temperatures driven by climate change [16]. Heat waves in Africa and Asia have caused yield losses of 15–35% [14].

Soil degradation is a growing concern in agriculture, with salinity affecting approximately 10% of arable land, including both irrigated and rainfed areas, resulting in an estimated global economic cost of USD 30 billion per year [11,17]. Some authors claim that salinity affects 1100 Mha of soil, which represents a total of 7% of the world’s surface [18].

Additionally, heavy metal contamination further accelerates soil degradation, impacting over 20 million hectares of land with pollutants [19]. In Europe, an estimated 2.5 million locations may be contaminated with organic pollutants and heavy metals, while in the United States, between 235,000 and 355,000 sites are considered in need of remediation. In China, by contrast, approximately 16% of the territory is thought to harbor heavy metals and other pollutants above soil quality levels [20].

Mitigating the consequences of abiotic stress on plants and improving their resistance to environmental changes largely depends on biological and chemical approaches. Among the chemical methods, the application of exogenous abscisic acid (ABA), since it controls stomatal closure and hence lowers water loss under drought and salt stress, and also regulates toxic metal transport, minimizes damage from heavy metal stress [21,22]. A novel strategy involves the use of nanotechnology, such as zinc oxide nanoparticles, which have shown promise in enhancing antioxidant defenses and reducing oxidative stress under extreme conditions [23]. Also, certain plant polymers, such as lignin, are less sensitive to environmental relative humidity [24], which enables plants to better withstand the effects of drought [25].

From a biological perspective, leveraging plant–microbe interactions offers a promising and sustainable strategy for stress mitigation. Beneficial microorganisms, such as endophytic fungi, arbuscular mycorrhizal fungi (AMF), plant growth-promoting rhizobacteria (PGPR), or cyanobacteria, enhance plant resilience by improving nutrient and water uptake, stimulating stress-related hormone production, and regulating reactive oxygen species (ROS) to limit cellular damage [26,27,28]. These microorganisms also contribute to soil structure enhancement by producing biofilms and exopolysaccharides, which improve water retention and nutrient flow around plant roots [29].

3. Agricultural Biotic Stresses: Pests and Pathogens

Living organisms including viruses, bacteria, fungi, protists, nematodes, insects and parasitic plants, cause plant damage through their biological interactions. These pathogens and pests can affect any stage of a plant’s life cycle including seedling emergence, plant maturation, or grain and fruit development [30]. Biotic stress agents extract nutrients directly from their host, thereby reducing plant vigor and, in serious instances, causing host death. As a result of co-evolution with crops, pests and pathogens (P&P) are integral components of agricultural systems. Cultivated plants tend to exhibit genetic uniformity, which facilitates the rapid proliferation of highly adapted P&P species, as they share similar phenological and physiological characteristics [31].

Currently, it is estimated that global yield losses due to pests and diseases average at 21.5% for wheat, 30% for rice, 22.5% for maize, 17.2% for potato, and 21.4% for soybean. Overall, they account for 20–40% of total crop yield losses worldwide [31]. According to other researchers, arthropod pests alone are responsible for the destruction of 18–20% of the world’s annual crop production, amounting to losses valued at over USD 470 billion [32]. P&P are widely recognized as major threats to stable and resilient food systems [33].

Agricultural P&P species pose a significant threat to global food security, causing substantial economic and yield losses [34]. Fungal pathogens from the genus Fusarium exemplify this challenge, with species such as Fusarium graminearum and Fusarium oxysporum ranked among the ten most significant plant pathogenic fungi [35]. Notably, F. graminearum is widely regarded as the primary agent of Fusarium head blight (FHB), with estimates from natural epidemics, fungicide trials, and controlled inoculation studies indicating yield losses in small grain cereals of up to 74% [36].

Similarly, the fall armyworm (Spodoptera frugiperda J.E. Smith) is a major pest in the maize-growing regions of tropical and subtropical Africa, where it is responsible for yield losses of up to 6.25% [31]. In Sub-Saharan Africa, it is estimated to cause annual maize, rice, sorghum, and sugarcane losses of USD 2.5–6.3 billion, with some estimates going up to USD 13 billion [37].

In response to this scenario, numerous chemical and biological strategies are being developed to improve plant responses to biotic stresses. Silver and copper NPs have demonstrated potent antimicrobial activity, serving as effective nanopesticides against a wide range of phytopathogens, including Alternaria alternata, F. oxysporum, and Sclerotinia sclerotiorum [38]. Silicon is an emerging strategy against pests and diseases, as it accumulates in plant tissues [39], forming protective layers beneath the cuticle and reinforcing cell walls to hinder pathogen invasion [40]. Additionally, silicon enhances plant defense mechanisms by stimulating key enzymes (e.g., peroxidase or chitinase) and activating hormonal pathways (salicylic acid, jasmonate, and ethylene). It also limits pest feeding and reproduction by altering plant tissue composition, making it less digestible for herbivores [41]. As observed in abiotic stress management, biological approaches are gaining interest for their potential in pest and disease control. The use of endophytic microorganisms, AMF and PGPR has shown promising results, enhancing resistance against pests and diseases, as documented in various studies [30,42]. Endophytic fungi, in particular, produce a wide range of bioactive metabolites with antifungal, antimicrobial, and insecticidal properties, reinforcing their potential for plant protection and sustainable agricultural practices [43].

4. The Insect Farming Industry Around the World

Particularly in hotter regions, the consumption of insects has been a component of human diets for millennia [44]. Today, approximately two billion people regularly consume insects in 113 countries, primarily in tropical regions of Africa, Asia, and America [45]. However, natural insect harvesting is declining due to anthropogenic pressures and environmental challenges, including agricultural expansion, desertification, and urbanization [46]. In response to these challenges, large-scale insect farming has become a sustainable option for traditional animal protein, offering an eco-friendly solution to satisfy the rising demand food and feed [47].

Making up 31% of edible insect species, beetles (Coleoptera) are the most widely consumed insect worldwide [48]. Next come caterpillars (Lepidoptera) at 18%, bees, wasps, and ants (Hymenoptera) at 14%, grasshoppers, crickets, and katydids (Orthoptera) at 13%, true bugs (Hemiptera) at 10%, termites (Isoptera) at 3%, dragonflies and damselflies (Odonata) at 3%, flies (Diptera) at 2%, and miscellaneous at 5% [49]. According to [50], among the over 2100 recognized edible insect species, the most commercially farmed include the house cricket (Acheta domesticus L.), the black soldier fly (Hermetia illucens L.), the yellow mealworm (Tenebrio molitor L.), and the grain mold beetle (Alphitobius diaperinus P.).

With more than 27,000 registered farms, Thailand is one of the top producers of house crickets in Asia [51]. These farms, which mostly operate on a small scale, collectively produce between 3000 and 7000 tons of crickets annually [52]. China has raised insect output for both animal feed and human consumption by several orders of magnitude, raising species including T. molitor, silkworm (Bombyx mori L.), black soldier fly, and housefly (Musca domestica L.). In African countries, insect farming is also expanding rapidly, with more than 2300 active insect farms across the continent [53]. The estimated annual production of dried black soldier fly larvae, one of the most farmed insect species in Africa, is around 19,732 tons, with the potential to scale up to 60 million tons of conventional animal feed [51,53]. In Latin America, the annual production of edible insects is estimated to range between 4500 and 6000 tons, representing approximately 6.7% to 10% of the global total, which is around 60,000 to 67,000 tons per year [54]. Despite ongoing challenges in establishing a stable market, the edible insect sector has grown rapidly, particularly in Europe and the United States, with even greater expansion in Western countries in general [55]. Key barriers to further growth include the need for production automation, reduced substrate costs and patent development to drive innovation [56]. The International Platform for Food and Feed (IPIFF) reports that its members currently produce around 6000 tons of insects annually, with projections suggesting an increase to 5 million tons by 2030 [57].

While insect farming primarily focuses on protein production, it inevitably generates by-products that can significantly enhance industry profitability [50]. Their application in various sectors can enhance their value, either by creating additional revenue streams or reducing disposal costs. Key by-products, such as fats (extracted during defatting processes), chitin, and excreta—commonly referred to as frass—can be repurposed for industrial applications, including surfactant and biodiesel production, plant protection agents, and fertilizers, respectively [47]. Among the by-products, frass stands out due to the large volumes in which it is generated. In Europe, current frass production already exceeds several thousand tons annually and is expected to reach 1.5 million tons by 2025. In North America, estimates of annual frass production and future trends are still emerging. However, it is known that for every ton of edible insect biomass produced, approximately 2 to 4 tons of frass are generated, highlighting its substantial contribution as a by-product within the industry [9].

To illustrate frass production volumes, T. molitor generates approximately 200–300 g of frass for every 100 g of biomass produced [58]. In contrast, the exact frass yield of H. illucens, which is widely used in waste treatment, remains uncertain, with the primary focus being placed on protein production. Its frass output varies significantly depending on the substrate characteristics, as shown in [59]. Additionally, a study by [60] analyzing the frass of nine insect species reared on waste substrates found that frass production varies by species, with H. illucens showing the most promising results in terms of frass production rate.

5. Insect Frass as an Agricultural Resource

Mostly used as a fertilizer due to its nutrient content, insect frass also acts as a phytofortifier, improving plant growth and health beyond its nutritional contribution [61,62,63,64]. Furthermore, its use as a natural amendment increases soil carbon content, which is especially helpful in Mediterranean areas with little organic content [65]. Given these characteristics, frass can be incorporated into carbon farming plans to help slow climate change and promote sustainable agriculture [66]. In addition, some studies have investigated the use of H. illucens frass in aquaculture diets as a partial substitute and have found encouraging results with species, such as hybrid tilapia (Oreochromis niloticus x O. mozambique) or white shrimp (Litopenaeus vannamei) [67,68].

What makes frass such an interesting biofertilizer is its rich nutritional profile, which is easily assimilated to by plants [62,69] and packed with essential nutrients, the presence of beneficial microorganisms that can support plant health and soil vitality [70], and its ability to release nitrogen gradually [71]. Frass has the potential to be used as a soil improver and plant fertilizer by supplying soil not only with nitrogen, phosphorus, and potassium [72], but also secondary (calcium, magnesium, and sulfur) and micro-nutrients (manganese, copper, iron, zinc, boron, and sodium) [60,73]. However, each type of frass has unique characteristics in terms of nutritional composition and microbiota, with diet significantly influencing its nutrient content [60,74], suggesting that its agricultural application should be tailored to the needs of the soil and crops. A comparative study conducted by [75] analyzed the frass of H. illucens, T. molitor, and Gryllus assimilis (JFC), highlighting differences in composition and microbiota. H. illucens frass exhibited the highest ammonium and plant-available phosphorus content, along with high microbial activity, while T. molitor frass had higher nitrate levels and lower biological activity. In contrast, G. assimilis frass contained the highest total nitrogen content and a distinct microbiota. These differences suggest that the agricultural application of each type of frass should be tailored to the specific needs of the soil and crops.

Despite frass’ potential, challenges remain regarding its large-scale application as a fertilizer. Ref. [76] found that 98% of mineral nitrogen in H. illucens frass is ammonium, which may pose risks of phytotoxicity at high doses and potentially reduce mycorrhizal colonization. Ref. [77] observed that a 1.5% application rate of T. molitor and A. diaperinus frass improved plant growth and nitrogen mineralization, increasing NH₄⁺ and NO₃⁻ availability. However, a 3% application inhibited germination, likely due to ammonium toxicity or high salinity.

When comparing insect frass to more widely recognized fertilizers, such as manure or vermicompost, the scientific literature remains limited. Studies indicate that T. molitor frass has shown effects comparable to those of chicken manure, with no significant differences in edible biomass yield. However, plants treated with frass exhibited a higher flower production [78]. Furthermore, research by [79] found that frass application led to increased chlorophyll content in treated plants compared to those receiving vermicompost.

To regulate its production and commercialization, the European Union has recently approved the use of insect frass as a fertilizer, a decision expected to boost its adoption in crop production and expand its market [80]. Valued at approximately 96 million dollars in 2023, the market is expected to exceed 135 million dollars by 2030, with an approximate compound annual growth rate (CAGR) of 6% from 2024 to 2030 [81].

6. Analysis Conducted

A systematic literature review was performed together with a quantitative analysis of publications based on year, journal, and countries. The compilation of all publications was carried out with the keywords “(insect frass OR insect excreta OR insect feces) AND (abiotic stress OR salinity OR drought OR flooding OR heavy metal OR pollutant OR biotic stress OR pest OR pathogen)”. The bibliographic database Web of ScienceTM (Web of Science Core Collection—WoS) (https://www.webofscience.com) and the Elsevier^® Scopus library services metabase (https://www.scopus.com) were selected due to their higher level of scientific rigor compared to other free and more open-access databases, such as Google Scholar [82].

In WoS, after searching for keywords in “All Fields”, without time restriction, and by selecting the document type “articles”, 496 results were retrieved (search performed on 28 January 2025). Of these 496 articles, 433 were excluded as they were not relevant to the subject; therefore, 63 articles were included in the review. Similarly, in Scopus, after applying the same search criteria, 169 results were obtained, of which 167 were deemed unrelated to the research topic, leaving only 2 articles for inclusion. It is important to note that some articles were present in both databases. Of the 63 articles selected from WoS and the 2 from Scopus, 1 article overlapped, meaning that 62 unique articles were sourced from WoS and 1 from Scopus. Therefore, the total number of final articles in the review on the use of insect frass to combat agricultural biotic and abiotic stresses, as well as its possible negative effects, was 64 articles.

As indicated, in both Scopus and in WoS the search provided many more articles than those included in this review because they were not related to the subject. In order to decide which articles were included and which were discarded, the title, abstract, keywords, and methodology of all the works were thoroughly read in all cases. Through this procedure, the articles unrelated to the subject of the review could be discarded.

The first publication on this topic dates back to 1982 [83], with only four articles being published between 1982 and 1988. However, no further publications were recorded until 1999, followed by a steady average of two articles per year until 2011. From 2015 onward, interest in this subject increased annually, with three articles published in 2015 and eight articles in 2024, the latter marking the highest number of publications to date (Figure 1a).

Regarding the geographical distribution of the reviewed articles, the United States contributed the largest number, with 18 publications. China and France followed, with eight and six articles, respectively. Germany, Kenya, and Mexico (five articles each) ranked fourth, followed by Spain and the UK (four articles), Canada, Italy, South Africa, Sweden, and The Netherlands (with three articles), and Estonia, Norway, Pakistan, Poland, Senegal, and South Korea (with two articles). Additionally, single publications were identified in 11 other countries across Asia (India, Iran, Japan, and Taiwan), Africa (Benin), the Americas (Brazil and Costa Rica), and Europe (Austria, Greece, Hungary, and Switzerland) (Figure 1b).

These authors published their articles in 47 different journals, including the Journal of Pest Science and Pest Management Science (with four articles), Agronomy, Annals of Applied Biology, and Journal of Chemical Ecology (with three articles), and Applied Soil Ecology, Biological Control, Bulletin of Entomological Research, Environmental Entomology, and Phytopathology (with two articles). The remaining journals only published one article (

Table 1. Journals in which the reviewed papers were published.

Journal	Number of Papers	Paper References
Journal of Pest Science	4	[84,85,86,87]
Pest Management Science	4	[88,89,90,91]
Agronomy	3	[92,93]
Annals of Applied Biology	3	[83,94,95]
Journal of Chemical Ecology	3	[96,97,98]
Applied Soil Ecology	2	[61,77]
Biological Control	2	[99,100]
Bulletin of Entomological Research	2	[101,102]
Environmental Entomology	2	[103,104]
Phytopathology	2	[105,106]
African Entomology	1	[107]
Applied Biological Chemistry	1	[108]
Applied Sciences	1	[109]
Behavioral Ecology	1	[110]
Biocatalysis and Agricultural Biotechnology	1	[111]
BioControl	1	[112]
BMC Evolutionary Biology	1	[113]
Chemoecology	1	[114]
Current Science	1	[115]
Ecological Entomology	1	[116]
Ecotoxicology	1	[117]
Elife	1	[118]
Entomologia Experimentalis et Applicata	1	[119]
Environmental Pollution	1	[120]
Environmental Science and Pollution Research	1	[20]
Forest Pathology	1	[121]
Frontiers in Microbiology	1	[122]
Frontiers in Plant Science	1	[123]
Fungal Ecology	1	[124]
Insects	1	[125]
Journal of Applied Entomology	1	[126]
Journal of Economic Entomology	1	[127]
Journal of Entomological Science	1	[128]
Journal of Insects as Food and Feed	1	[129]
Journal of Insect Science	1	[130]
Journal of Plant Protection Research	1	[131]
Journal of Stored Products Research	1	[132]
Microbial Ecology	1	[133]
Microorganisms	1	[134]
Nematology	1	[135]
Neotropical Entomology	1	[136]
Oecologia	1	[137]
Physiological Entomology	1	[138]
Phytoparasitica	1	[139]
Plant Physiology	1	[97]
PLoS ONE	1	[140]
The Plant Pathology Journal	1	[141]

).

Regarding the number of citations of these articles, the article published in the journal Oecologia in 2008 [137] stood out, with 148 citations (Scopus). In second place, we found an article published in 2019 in the journal Applied Soil Ecology [61], with 109 (Scopus) and 98 (WoS) citations, and another one published in 2000 in the journal Ecological Entomology [116], with 98 citations in WoS. Only one article less than 5 years old ranked among the top 10 most cited articles, published in the journal Applied Soil Ecology in 2021 [77], with 37 (WoS) citations (

Table 2. Number of citations of the 10 most cited articles.

Reference	Journal	WoS Citations	Scopus Citations
[137]	Oecologia	-	148
[61]	Applied Soil Ecology	98	109
[116]	Ecological Entomology	98	-
[120]	Environmental Pollution	69	-
[97]	Plant Physiology	56	-
[142]	Journal of Chemical Ecology	49	-
[96]	Journal of Chemical Ecology	44	-
[103]	Environmental Entomology	43	-
[77]	Applied Soil Ecology	37	-
[98]	Journal of Chemical Ecology	32	-

).

To the best of our knowledge, this is the first systematic review that comprehensively analyzes the effects of insect frass on both abiotic and biotic agricultural stresses, as well as its potential drawbacks. This review provides a critical comparison of the existing studies and a practical perspective on the agricultural applications of insect frass, setting the foundation for future research and its potential implementation in sustainable farming.

7. Insect Frass Against Abiotic Plant Stresses

Climate change is increasing the abiotic stresses affecting crops, which causes significant agricultural losses (as discussed in Section 2). So far, several studies have been carried out on the use of insect frass to reduce the negative effects of abiotic stresses on plants, which are summarized in

Table A1. Works on improving plants under abiotic stress through the use of insect excrement, indicating the described effects and the mechanisms of action involved.

Insects		Crop/Plant	Form of Application and Dosage	Abiotic Stress	Effects	Mechanism of Action	References
Order	Species
Coleoptera	Alphitobius diaperinus	-	Frass: in soil at 2.5% or 5% (w/w)	Heavy metal	Heavy metal soil removal	Sorption and complexation of heavy metals	[77]
	Tenebrio molitor	Bean	Frass: in soil at 2% (v/v)	Salinity Drought Flooding	Greater plant biomass formation under abiotic stress	Presence of bacteria and fungi that induce plant tolerance under abiotic stress Absorbent action of saline ions and/or water of the chitin/chitosan present in the frass	[61]
		-	Biochar from frass (using pyrolysis at 600 °C for 90–120 min)	Heavy metal	Heavy metal soil removal	Sorption of heavy metals	[120]
		-	Frass: in soil at 2.5% or 5% (w/w)	Heavy metal	Heavy metal soil removal	Sorption and complexation of heavy metals	[77]
		-	Frass: in soil at 2% or 4%	Heavy metal	Heavy metal soil removal	Complexation of heavy metals	[93]
		-	Frass	Heavy metal	Heavy metal soil removal	Sorption of heavy metals	[108]
Diptera	Hermetia illucens	Rice	Frass: in soil at 2–8% (w/w)	Heavy metal	Heavy metal soil removal	Sorption and complexation of heavy metals	[20]
		-	Frass: in soil at 2.5% or 5% (w/w)	Heavy metal	Heavy metal soil removal	Sorption and complexation of heavy metals	[77]
		Lettuce	Frass: in field at 1.5 t ha−1	Heavy metal	Less accumulation of Pb in the plant	Increase in microbial enzymatic activity in the soil	[92]
		Barley	Frass: in soil at 10–12.5 g/L	Drought	Increased plant tolerance to drought	Promotion of plant photosynthetic activity	[131]
		Lettuce	Frass: 12.5 g per plant	Drought	Increased plant tolerance to drought	Promotion of plant photosynthetic activity	[109]

.

In response to plant stress due to drought, several studies have been carried out on the use of insect frass from the mealworm (T. molitor) and the black soldier fly (H. illucens) as a strategy to reduce its negative effects. The frass from T. molitor is characterized by a chitinous layer derived from the pleiotropic intestinal membrane of the insect. Chitin in this frass has been reported to enhance drought tolerance in bean plants due to its ability to retain water [69].

Furthermore, by using unsterilized frass from the same insect species, researchers determined that certain microorganisms present in the frass induced drought tolerance responses in bean plants, complementing the effects previously reported for sterile frass [61]. In the case of insect frass from H. illucens, its potential to increase plant tolerance to drought in barley and lettuce has been described, although the underlying mechanism remains unclear. In both crops, it was described how the application of insect frass to the soil enhanced photosynthetic capacity of plants under drought stress, as well as decreased accumulation of polyphenolic compounds, a sign of a lower presence of stress in these plants [109,131].

Regarding flood stress, only one study has been carried out that uses insect frass to try to reduce it. Using both sterile and non-sterile T. molitor frass, it was determined that certain microorganisms present in non-sterile frass promoted flood tolerance in bean plants. This suggests that the observed tolerance was microbially mediated, as no benefits were reported with sterile frass application [61].

Under salinity stress, plant tolerance has been successfully increased through the application of insect frass, although only one study has addressed this topic to date. The application of frass from T. molitor to the growing medium increased the tolerance of bean plants to saline stress. This effect was attributed to both the presence of microorganisms capable of inducing plant tolerance and the ability of chitin in the frass to bind salt ions, as demonstrated using sterile and non-sterile frass treatments [61].

Finally, several studies have been carried out on the use of insect frass to combat the presence of heavy metals in the soil, using different mechanisms of action. The insect frass of T. molitor has been shown to absorb heavy metals such as cadmium (Cd) in its own structure when applied to the soil [108]. Additionally, when this frass is processed into biochar via pyrolysis at 600 °C, its structure enables the sorption of lead (Pb), copper (Cu), zinc (Zn), and chromium (Cr) present in the soil [120]. Another mechanism of action described for mealworm frass in soils contaminated by heavy metals is complexation, which works for Zn, but not for Cd or Pb [93]. Therefore, both mechanisms of action (sorption and complexation) can act simultaneously when frass from T. molitor is applied to the soil. This dual effect has been described for the removal of Zn, Cu, Cd, and nickel (Ni), using both T. molitor and lesser mealworm (A. diaperinus) frass [77]. Furthermore, in the case of frass from H. illucens, sorption and complexation mechanisms have been identified as key processes in removing Cd and Pb from the soil, thereby reducing their uptake and accumulation in rice plant tissues [20]. However, another study conducted with lettuce plants in Pb-contaminated soils treated with H. illucens frass suggested that the lower Pb accumulation in plant tissues was linked to increased microbial enzymatic activity in the soil following frass application. Enhanced enzymatic activity included increases in acid phosphatase, alkaline phosphatase, arylsulfatase, N-acetyl-β-D-glucosaminidase, dehydrogenase, and total hydrolase activity [92].

8. Insect Frass Against Agricultural Pests and Pathogens

Biotic stresses (pathogens and pests) cause significant crop losses, so new sustainable strategies are needed to obtain a reduction in the negative effects of these organisms on plants (problem developed in Section 3). One of these alternatives could be the use of insect frass, such as it has been investigated in the studies compiled in

Table A2. Works on improving plants under biotic stresses through the use of insect frass, indicating the described effects and mechanisms of action involved.

Insects		Crop/Plant	Form of Application and Dosage	Abiotic Stress	Effects	Mechanism of Action	References
Order	Species
Coleoptera	Allomyrina dichotoma	Rice Tomato Wheat Barley Pepper	As microorganisms isolated from frass	Pathogens: Rhizoctonia solani (fungus), Phytophthora capsici (oomycete), Colletotrichum coccodes (fungus), and Fusarium oxysporum (fungus)	Inhibition of fungal and oomycete growth (in vitro)	Production of antifungal and oomyceticidal compounds via Bacillus amyloliquefaciens (bacteria)	[141]
	Calosoma sayi	-	Frass	Insect pest: Spodoptera frugiperda (Lepidoptera)	Insect pest death	Entomopathogens vehicle (nuclear polyhedrosis virus and Vairimorpha sp.)	[128]
	Diaprepes abbreviates	Citrus crops	As frass extracts	Insect pest: Diaprepes abbreviatus (Coleoptera)	Insect pest death	Entomopathogens attraction (Steinernema diaprepesi and Heterorhabditis indica)	[114]
	Hylobius abietis	Scots pine (Pinus sylvestris)	As fungal volatiles (extracted from frass)	Insect pest: Hylobius abietis (Coleoptera)	Reduction in infestation	Fungal volatiles cancel out the volatiles from the host plant	[124]
		Scots pine (Pinus sylvestris)	As bacteria volatiles (extracted from frass)	Insect pest: H. abietis (Coleoptera)	Reduction in infestation	Bacteria volatiles act as antifeedant compounds	[133]
	Hylotrupes bajulus	-	Frass	Insect pest: Hylotrupes bajulus (Coleoptera)	Not identified	Parasitoid attraction (Sclerodermus cereicollis and S. domesticus)	[132]
	Hypothenemus hampei	-	As frass extracts	Insect pest: Hypothenemus hampei (Coleoptera)	Not identified	Parasitoid attraction (Cephalonomia stephanoderis and Prorops nasuta)	[102,112]
	Osphranteria coerulescens	Apricot	Frass	Insect pest: Osphranteria coerulescens (Coleoptera)	Insect pest death	Entomopathogens vehicle (Heterorhabditis bacteriophora and Steinernema carpocapsae)	[135]
	Phyllophaga vandinei	-	Frass	Insect pest: Phyllophaga vandinei (Coleoptera)	Insect pest death	Entomopathogen vehicle (invertebrate iridescent virus 6)	[130]
	Prostephanus truncatus	-	As frass extracts	Insect pest: Prostephanus truncatus (Coleoptera)	Not identified	Predator attraction (Teretrius nigrescens)	[138]
	Protaetia brevitarsis	-	As bacteria isolated from frass	Pathogens: Sclerotinia sclerotiorum, S. rolfsii and F. oxysporum (fungi)	Inhibition of fungal growth (in vitro)	Production of antifungal compounds via Bacillus subtilis (bacteria)	[134]
		-	As bacteria isolated from frass	Pathogens: Fusarium oxysporum, Pyricularia grisea, Rhizoctonia solani, and Stagonosporopsis cucurbitacearum (fungi)	Inhibition of fungal growth (in vitro)	Production of antifungal compounds via Streptomyces albidoflavus and Nocardiopsis flavescens (bacteria)	[88]
	Rhyzopertha dominica	Wheat (grains)	Frass	Insect pest: Rhyzopertha dominica (Coleoptera)	Not identified	Parasitoid attraction (Theocolax elegans)	[125]
	Tenebrio molitor	Arabidopsis thaliana	Frass: in soil at 2% (v/v)	Pathogen: Botrytis cinerea (fungus)	Less fungal lesions	Induction of systemic defensive responses	[111]
	Trichoferus holosericeus	-	Frass	Insect pest: Trichoferus holosericeus (Coleoptera)	Not identified	Parasitoid attraction (Sclerodermus cereicollis and S. domesticus)	[132]
Collembola *	Heteromurus nitidus	Annual meadow grass (Poa annua) (in microcosm chambers) White clover (Trifolium repens) (in microcosm chambers)	Frass	Insect pest: Myzus persicae (aphids)	Reduction in aphid reproduction	Not identified	[137]
	Onychiurus scotarius	Annual meadow grass (P. annua) (in microcosm chambers) White clover (T. repens) (in microcosm chambers)	Frass	Insect pest: M. persicae (aphids)	Reduction in aphid reproduction	Not identified	[137]
Dermaptera	Forficula auricularia	-	Frass	Pathogens: Staphylococcus aureus (bacteria) and Aspergillus niger (fubgus)	Inhibition of bacterial multiplication and fungal growth	Presence of antimicrobial compounds	[113]
Diptera	Ceratitis capitata	Coffee (in field)	As frass extracts	Insect pest: Ceratitis capitata (Diptera)	Reduction in infestation	Pheromone-deterrent effect	[101]
	Hermetia illucens	Kale Swiss chard	Frass: in field at 10.3 t ha−1	Insect pest: Aphis spp. (Hemiptera), Plutella xylostella (Lepidoptera), Bemisia tabaci (Hemiptera), and Liriomyza spp. (Diptera)	Reduces pest infestation	Induction of systemic defensive responses	[143]
		Brussels sprouts	Frass: in soil at 5 g/kg	Insect pest: Delia radicum (Diptera)	Insect pest death	Unidentified	[87]
		-	As frass extracts	Pathogens: Alternaria solani, Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sclerotiorum (fungi), and Phytophthora capsici (oomycete)	Inhibition of fungal and oomycete growth	Presence of antifungal and oomyceticidal compounds from microorganisms	[129]
		Mustard (Brassica rapa)	Frass: in soil at 2 g/kg	Insect pest: D. radicum (Diptera) and P. xylostella (Lepidoptera)	Insect pest death	Unidentified	[119]
		Spinach	As frass extracts: 100 mL per plant	Pathogen: Meloidogyne incognita (nematode)	Suppressed nematode egg hatchability J2s paralysis and death Suppression of gall development	Presence of nematicidal compounds	[123]
		Brussels sprouts	Frass: in soil at 5 g/Kg	Insect pest: D. radicum (Diptera)	Insect pest death	Unidentified	[126]
Lepidoptera	Anticarsia gemmatalis	Soya	Frass	Insect pest: Anticarsia gemmatalis (Lepidoptera)	Insect pest death	Entomopathogen vehicle (Anticarsia gemmatalis multiple nucleopolyhedrovirus)	[100]
	Busseola fusca	Maize	Frass	Plant pathogen: Fusarium verticillioides (fungus)	Not identified	Biological control agent vehicle: Acremonium zeae (fungus)	[107]
	Cydalima perspectalis	-	As frass extracts (volatile compounds)	Insect pest: Cydalima perspectalis (Lepidoptera)	Not identified	Oviposition-repellent effect	[84]
	Cydia pomonella	Apple (in vitro)	Frass	Insect pest: Cydia pomonella (Lepidoptera)	Not identified	Parasitoid attraction (Hyssopus pallidus)	[96]
	Helicoverpa zea	Tomato	As protein extract from frass	Insect pest: S. frugiperda (Lepidoptera)	Reduction in plant biomass consumption by the insect pest	Induction of systemic defensive responses	[98]
	Hyblaea puera	-	Frass	Insect pest: Hyblaea puera (Lepidoptera)	Insect pest death	Entomopathogen vehicle (Hyblaea puera nucleopolyhedrovirus)	[115]
	Lymantria dispar	Poplar (Populus nigra) (in vitro)	Frass	Insect pest: Lymntria dispar (Lepidoptera)	Insect pest death	Parasitoid attraction (Glyptapanteles flavicoxis)	[116]
		-	Frass	Insect pest: L. dispar (Lepidoptera)	Insect pest death	Entomopathogens vehicle (Nosema lymantriae and Vairimorpha disparis)	[99]
	Ostrinia nubilalis	Maize	As protein extract from frass	Insect pest: Ostrinia nubilalis (Lepidoptera)	Reduction in plant biomass consumption by the insect pest	Induction of systemic defensive responses	[98]
	Phthorimaea operculella	-	As frass extracts (volatile compounds)	Insect pest: Phthorimaea operculella (Lepidoptera)	Not identified	Oviposition-repellent Effect	[139]
	Spodoptera frugiperda	-	Frass	Insect pest: Spodoptera frugiperda (Lepidoptera)	Not identified	Parasitoid attraction (Cotesia marginiventris)	[117]
		Maize	As protein extract from frass	Pathogen: Cochlioblus heterostrophus (fungus)	Reduction in disease severity	Induction of systemic and local defensive responses	[97]
		Maize Rice	As protein extract from frass	Insect pest: S. frugiperda (Lepidoptera)	Reduction in plant biomass consumption by the insect pest	Induction of systemic defensive responses	[98]
	Trichoplusia ni	Cabbage	As protein extract from frass	Insect pest: Trichoplusia ni (Lepidoptera)	Reduction in plant biomass consumption by the insect pest	Induction of systemic defensive responses	[98]
	Tuta absoluta	-	Frass	Insect pest: Tuta absoluta (Lepidoptera)	Not identified	Predator attraction (Nesidiocoris tenuis)	[89]
		-	Frass	Insect pest: T. absoluta (Lepidoptera)	Not identified	Parasitoid attraction (Dolichogenidea gelechiidivoris)	[86]

* It is not an order of insects, but a class within the hexapods. However, there are occasions when they have been referred to as an order of insects.

.

8.1. Against Plant Pathogens

To help crops fight diseases caused by pathogens, different insect frass-mediated mechanisms of action have been described. Both frass itself and extracts derived from it have been described as products with antimicrobial properties, due to the presence of antibacterial, antifungal, and nematicidal compounds. The frass obtained from the common earwig (Forficula auricularia) is capable of inhibiting the in vitro growth of the plant pathogenic bacterium Staphylococcus aureus and the pathogenic fungus Aspergillus niger, due to the possible presence of antimicrobial compounds that have not yet been identified [113]. On the other hand, extracts obtained from the frass of H. illucens also inhibited the in vitro growth of different phytopathogenic fungi and oomycetes, such as Alternaria solani, Botrytis cinerea, F. oxysporum, Rhizoctonia solani, S. sclerotiorum, and Phytophthora capsici [129]. Furthermore, these frass extracts also exhibited nematicidal activity when applied to spinach plants, causing the suppression of nematode egg hatchability (by 42%), the paralysis and death of J2s (100% and 95%, respectively), and the suppression of gall development (by 85%) in the root-knot nematode (Meloidogyne incognita) [123].

This antimicrobial activity reported in insect frass may result from the synthesis of antimicrobial compounds via the microorganisms present in this product, as has been previously described in several works with beetle and lepidopteran frass. Different bacteria with antifungal properties have been isolated from the insect frass obtained from the beetle Protaetia brevitarsis through in vitro confrontation assays. For example, Bacillus subtilis was capable of inhibiting the growth of the pathogenic fungi S. sclerotiorum, S. rolfsii, and F. oxysporum [134]; or Streptomyces albidoflavus and Nocardiopsis flavescens, which acted against F. oxysporum, Pyricularia grisea, R. solani, and Stagonosporopsis cucurbitacearum [122]. These antimicrobial effects have also been confirmed in in planta assays. From the frass of the Japanese rhinoceros beetle (Allomyrina dichotoma), the KB3 strain of the bacterium Bacillus amyloliquefaciens was obtained, which produces the antifungal and oomyceticidal lipopeptides iturin A and surfactin. The cell-free filtrates obtained from this bacterium inhibited the in vitro growth of the pathogenic fungi R. solani, F. oxysporum, and Colletotrichum coccodes, as well as the oomycete P. capsici; their biocidal effects were confirmed in planta after infecting rice, tomato, wheat, barley, and pepper plants [141]. Furthermore, fungi with a potential antimicrobial capacity have been isolated from insect frass. The fungus Acremonium zeae was isolated from the frass of the maize stalk borer (Busseola fusca), a fungus capable of producing the antifungal compounds pyrrocidines A and B, which have been described as effective in the control of the maize pathogen Fusarium verticillioides. However, the real antifungal activity of this isolate from insect frass has not yet been described [107].

Another frass-mediated mechanism of action for plant pathogen control is the induction of plant defenses. To date, only two studies have addressed this topic; however, it is an interesting area of research with many future possibilities. Using frass from T. molitor, different strategies for activating plant defenses in the model plant Arabidopsis thaliana were studied. Firstly, callose deposition in the roots was analyzed as a marker of local plant defense activation after exposure to mealworm frass. Results indicated that this frass did not activate local plant defenses. Conversely, systemic plant defense induction was studied by applying this frass to the soil and using the fungus B. cinerea as a foliar pathogen. It was found that the plant activated systemic defense responses upon recognizing T. molitor frass in the roots, a response mediated by the increased expression of the genes ZAT10, ERF5, WRKY33, and CML37 [111]. Another study described how protein extracts from the frass of the pest insect S. frugiperda induced systemic defense responses in maize plants against the fungal pathogen Cochliobolus heterostrophus. A pathogenesis-related gene was implicated in this response. Additionally, it was determined that the specific proteins recognized by the plant, which induced defensive responses, were chitinases derived from the maize plant itself, excreted in the frass of the pest insect after feeding on maize biomass [97,142]. However, these same chitinases also suppressed a defensive response against the same insect pest that produced the insect frass [142]. Therefore, the activation of plant defenses against pathogens via insect frass also requires the study of defensive responses to insect pests.

8.2. Against Insect Pests

The most developed field of study on the use of insect frass against biotic stresses is aimed at reducing the negative effects of insect pests on crops. Several works have reported the effectiveness of insect frass in reducing the damage caused by insect pests on crops, but without identifying the mechanism of action involved. By using insect frass from two species of springtail (Heteromurus nitidus and Onychiurus scotarius), in contact with the roots of annual meadow grass (Poa annua) and white clover (Trifolium repens), it was possible to reduce the reproduction of the aphid Myzus persicae in the aerial part of the plant by 45%, only in the grass [137]. Other subsequent work has focused on the species widely exploited in insect farming, H. illucens and T. molitor. The application of frass from the black soldier fly to the soil caused a reduction in the survival and pupal biomass of the cabbage fly (Delia radicum) in Brussels sprouts [87,126] and mustard [119] crops. Similar results have been reported with the use of mealworm frass mixed with soil for mustard cultivation, both in relation to the cabbage fly and the diamondback moth (Plutella xylostella) [119]. Although none of these studies identify the mechanism of action involved in these results, the presence of entomopathogens in the frass, such as the bacterium Bacillus thuringiensis, is suggested as a possibility [126].

In this sense, several works have been carried out describing how insect frass can be used as a vehicle for entomopathogenic microorganisms that effectively contribute to the biological control of agricultural insect pests. In the case of viruses, several insect pests of plants transmit viruses that are capable of killing them through their own frass. This insect frass could be used to control these same pests. For example, the frass from the caculo beetle (Phyllophaga vandinei) contains the invertebrate iridescent virus 6, capable of killing this beetle [130], or the frass from the Hyblaea puera moth horizontally transmits its nucleopolyhedrovirus [115]. Furthermore, viruses present in insect frass from one species can be effective biocontrol agents against other insect pests. This is the case of frass from the lepidopteran predator beetle Calosoma sayi, which, by feeding on the insect pest fall armyworm larvae (S. frugiperda), represents an important source of nuclear polyhedrosis virus inoculum that kills the lepidopteran [128]. In addition to in vitro and in vivo studies with insect pests, this use of insect frass as a vehicle for entomopathogenic viruses has been confirmed in planta. Velvetbean moth (Anticarsia gemmatalis) larvae are able to actively transmit their multiple nucleopolyhedrovirus to soybean plants via frass, causing the death of the insect pest [100].

In addition to viruses, insect frass has been described as an effective vehicle for entomopathogens of the protozoan and nematode groups. Several microsporidian protozoa have been actively transmitted through insect frass, such as Endoreticulatus schubergi, Nosema lymantriae, and Vairimorpha disparis, which are present in the frass of the gypsy moth (Lymantria dispar) and capable of killing the insect pest [99]. Furthermore, microsporidian protozoa can be actively transmitted through the frass of insects that prey on pest insects. This happens with the protozoan Vairimorpha sp., which is capable of killing the lepidopteran S. frugiperda and is actively transmitted through the frass of the lepidopteran-predating beetle C. sayi [128]. In the case of nematodes, they can also be transmitted through the frass of insect pests as biological control agents against themselves. This happens with the rosaceae longhorned beetle (Osphranteria coerulescens), a pest insect of woody crops whose frass actively transmits the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae, both of which can kill the beetle on apricot tree branches [135].

In addition to being used as a vehicle for entomopathogens, insect frass has been widely studied as a source of volatiles with great potential for controlling insect pests in plants. These volatiles include pheromones from pest insects present in their frass, which act as powerful attractants for entomopathogens, such as nematodes. This mechanism has been described in the frass from the citrus root weevil (Diaprepes abbreviatus), which emits a specific pest insect pheromone that actively attracts the entomopathogenic nematodes Steinernema diaprepesi and Heterorhabditis indica, even when applied in citrus orchards [114]. Therefore, the frass of the insect pest itself can be used for its control by actively attracting entomopathogenic nematodes. Similarly, other works have described how frass from various insect pests actively attracts their predators. For instance, for the frass from the lepidopteran pest tomato pinworm (Tuta absoluta), different volatile compounds have been described that actively attract the pest’s predator. These compounds are the monoterpenes α-pinene, α-phellandrene, 3-carene, β-phellandrene, and β-ocimene, which attract the polyphagous heteropteran Nesidiocoris tenuis [89]. Similar effects were reported with frass extracts produced by the insect pest larger grain borer (Prostephanus truncatus), which contained volatile compounds that actively attracted its predator, the specialist beetle Teretrius nigrescens [138].

These insect frass volatiles can also act as attractants of parasitoids of insect pests, a field of study that has been widely addressed in the 21st century. In vivo studies have reported the attractiveness of coleopteran pest frass and its extracts to parasitoids that control the pest [102,112,132]. Furthermore, coleopteran frass has been used for the active control of insect pests. This is the case of the frass produced by the lesser grain borer (Rhyzopertha dominica), which has been described as an effective attractant for the generalist parasitoid Theocolax elegans when applied to stored wheat grains [125]. In the case of lepidopteran insect pests, this mechanism of action has been described for various species in vitro, such as S. frugiperda [117] or T. absoluta [86]. Specifically, in the case of T. absoluta, the volatile compounds responsible for attracting the parasitoid Dolichogenidea gelechiidivoris were identified as α-pinene, β-myrcene, α-phellandrene, α-terpinene, β-ocimene, methyl salicylate, and (E)-β-caryophyllene [86]. These attracting effects on parasitoids have also been confirmed by applying frass directly to plant material, bringing the use of this frass closer to actual commercial use to reduce the losses caused by these pests. In apple fruits, the application of frass from the codling moth (Cydia pomonella) effectively attracted its parasitoid, the wasp Hyssopus pallidus [96]. Similar results were reported for the wasp Glyptapanteles flavicoxis on poplar leaves (Populus nigra) through the application of frass from L. dispar [116].

Some of these volatile compounds with attractive properties for entomopathogens, predators, or parasitoids may be produced by microorganisms present in the frass. However, the volatiles obtained via these microorganisms have not been described with these attracting mechanisms so far. These microorganisms have been studied in the frass from the large pine weevil (Hylobius abietis), which produces volatiles that reduce the infestation of Scots pines (Pinus sylvestris) by the insect pest. The bacteria isolated from this frass are capable of producing the volatile compound 2-phenylethanol, which acts as an antifeedant compound against H. abietis [133]. In addition, the filamentous fungi Ophiostoma canum and O. pluriannulatum, and the yeast Debaryomyces hansenii, isolated from this frass, are capable of producing the volatile compounds 6-protoilludene and methyl salicylate, whose function is to cancel out the volatiles produced by the host plant, preventing their detection by the insect pest [124]. Therefore, it is possible that frass microorganisms are involved in the oviposition-repellent and pheromone-deterrent activity reported for frass from other insect pests, such as the box tree moth (Cydalima perspectalis) [84], the potato tuber moth (Phthorimaea operculella) [139], and the Mediterranean fruit fly (Ceratitis capitata) [101].

In addition to all the mechanisms of action described that involve the direct action of frass and its components without mediating with the plant, several works have been conducted where insect frass is capable of activating systemic defensive responses against insect pests. The field application of frass from H. illucens has been described as an effective activator of systemic defenses in kale and Swiss chard, reducing infestation by different hemipterans (Aphis spp. and Bemisia tabaci), lepidopterans (P. xylostella), and dipterans (Liriomyza spp.) [143]. It is possible that the molecule recognized by plant roots and responsible for the induction of systemic defenses is a protein, as has been demonstrated in several works with protein extracts obtained from frass from insect pests. By applying these extracts to tomato, maize, rice, and cabbage plants, it was possible to reduce plant biomass consumption by the insect pests, namely the tomato fruit worm (Helicoverpa zea), European corn borer (Ostrinia nubilalis), the fall armyworm (S. frugiperda), and the cabbage looper (Trichoplusia ni) [98]. The activation of systemic defensive responses in plants using insect frass against insect pests requires further study in order to elucidate how it happens and its possible real-world application in agricultural systems.

9. Negative Stress-Related Effects of the Use of Insect Frass in Agriculture

Despite the effects of insect frass in reducing the negative effect of biotic and abiotic stresses described above, some studies have reported negative effects related to these stresses in plants where frass is applied. All of these studies are compiled in

Table A3. Works reporting negative effects with regard to abiotic or biotic plant stresses due to the use of insect frass in agriculture.

Insects		Crop/Plant	Form of Application and Dosage	Abiotic Stress	Effects	Mechanism of Action	References
Order	Species
Blattodea	Blattella germanica	-	Bacteria isolated from frass	Insect pests: unspecified	Inhibition of fungal bioinsecticides action	Production of antifungal compounds	[88]
Coleoptera	Acalymma vittatum	Cucumber	Frass	Pathogen: Erwinia tracheiphila (bacteria)	Pathogen dissemination	Plant pathogen vehicle: Erwinia tracheiphila (bacteria)	[103]
	Cosmopolites sordidus	Banana	Frass	Pathogen: F. oxysporum f.sp. cubense (fungus)	Pathogen dissemination	Plant pathogen vehicle: F. oxysporum f.sp. cubense (fungus)	[90]
	Leptinotarsa decemlineata	Potato	Frass: 20 mg diluted in 20 μL of sterile water per plant	Insect pest: Leptinotarsa decemlineata (Coleoptera)	Inhibition of local plant defense responses	Bacteria in the frass act as inhibitors of plant defenses	[91]
	Odontotaenius disjunctus	-	Bacteria isolated from frass	Insect pests: unspecified	Inhibition of fungal bioinsecticides action	Production of antifungal compounds	[118]
	Tenebrio molitor	Wheat	Frass	Pathogen: Fusarium proliferatum (fungus)	Pathogen dissemination	Plant pathogen vehicle: Fusarium proliferatum (fungus)	[140]
		Mustard (Brassica rapa)	Frass: in soil at 2 g/kg	Insect pest: D. radicum (Diptera) and P. xylostella (Lepidoptera)	Increased insect pest survival	Unidentified	[119]
	Xyleborinus Saxesenii X. affinis X. ferrugineus X. perforans	ʻŌhiʻa lehua (Metrosideros polymorpha)	Frass	Pathogen: Ceratocystis lukuohia and Ceratocystis huliohia (fungi)	Pathogen dissemination	Plant pathogen vehicle: Ceratocystis lukuohia and Ceratocystis huliohia (fungi)	[121]
Dermaptera	Forficula auricularia	-	Frass	Insect pest: Forficula auricularia (Dermaptera)	Increase in pest insect population	Coprophagy	[110]
Diptera	Bradysia impatiens	-	Frass	Pathogen: Thielaviopsis basicola (fungus)	Pathogen dissemination	Plant pathogen vehicle: Thielaviopsis basicola (fungus)	[95]
	Drosophila suzukii	Raspberry	Frass	Pathogens: Cladosporium cladosporioides and C. pseudocladosporioides (fungi)	Pathogen dissemination	Plant pathogen vehicle: Cladosporium cladosporioides and C. pseudocladosporioides (fungi)	[104]
		Raspberry	Frass	Pathogens: Cladosporium spp. and Botrytis spp. (fungi)	Pathogen dissemination	Plant pathogen vehicle: Cladosporium spp. and Botrytis spp. (fungi)	[139]
	Psychoda spp.	-	Frass	Pathogen: Thielaviopsis basicola (fungus)	Pathogen dissemination	Plant pathogen vehicle: Thielaviopsis basicola (fungus)	[95]
	Scatella stagnalis	-	Frass	Pathogen: T. basicola (fungus)	Pathogen dissemination	Plant pathogen vehicle: T. basicola (fungus)	[105]
		-	Frass	Pathogen: T. basicola (fungus)	Pathogen dissemination	Plant pathogen vehicle: T. basicola (fungus)
Lepidoptera	Busseola fusca	Maize	Frass	Pathogen: Aspergillus spp. and Fusarium spp. (fungi)	Pathogen dissemination	Plant pathogen vehicle: Aspergillus spp. and Fusarium spp. (fungi)	[107]
	Spodoptera frugiperda	Maize	As protein extract from frass	Insect pest: Spodoptera frugiperda (Lepidoptera)	Increased consumption of plant biomass by the insect pest	Inhibition of systemic and local defensive responses	[97,98]
	S. littoralis	Cotton	As bacteria volatiles (extracted from frass)	Insect pest: Spodoptera littoralis (Lepidoptera)	Increased plant infection	Bacterial volatiles act as attractants for insect pest larvae	[85]
Orthoptera	Locusta migratoria	Maize	Frass	Pathogen: Colletotrichum graminicola (fungus)	Pathogen dissemination	Plant pathogen vehicle: Colletotrichum graminicola (fungus)	[83]
	Melanoplus bivittatus	Alfalfa	Frass (10 fecal pellets per plant)	Pathogen: Verticillium albo-atrum (fungus)	Pathogen dissemination	Plant pathogen vehicle: Verticillium albo-atrum (fungus)	[106]
	M. sanguinipes	Alfalfa	Frass (10 fecal pellets per plant)	Pathogen: V. albo-atrum (fungus)	Pathogen dissemination	Plant pathogen vehicle: V. albo-atrum (fungus)	[106,127]
	Schistocerca gregaria	Maize	Frass	Pathogen: C. graminicola (fungus)	Pathogen dissemination	Plant pathogen vehicle: C. graminicola (fungus)	[83]

.

The most widely described negative effect related to the use of insect frass in agriculture is the spread of plant pathogens. In this sense, insect frass has been described as a vector for the transmission of phytopathogenic bacteria, such as Erwinia tracheiphila [103] and phytopathogenic fungi, such as Colletotrichum graminicola [83], Verticillium albo-atrum [106,127], F. oxysporum f.sp. cubense [90], Fusarium proliferatum [140], Ceratocystis lukuohia, C. huliohia [121], and Cladosporium spp. or Botrytis spp. Therefore, a plant health safety analysis of insect frass is required before it is placed on the market, in order to avoid the possible presence of plant pathogens.

Furthermore, strategies for treating insect frass must be studied and established to try to prevent the spread of plant pathogens. As with other animal excrement, the composting process is an effective strategy for eliminating plant pathogens before their application to crops [144]. In the specific case of the European Union, the possible transmission of plant pathogens in the agricultural application of insect frass is less probable, due to Regulation 2021/1925 of the European Commission, which includes this insect product in Annex I of Regulation No 142/2011. Specifically, in order to market insect frass within the European Union, a prior heat treatment of at least 70 °C for 1 h [80] is mandatory.

With regard to insect pests that affect crops, several mechanisms of action described for insect frass may favor its negative effects. Insect frass can be directly used as a food resource by plant-eating insects in a process of coprophagy, as is the case with frass from F. auricularia, which enables the pest to survive in the absence of a plant host [110]. Insect frass can also act as an attractant of insect pests through the emission of volatile compounds. In the case of the insect pest African cotton leafworm (Spodoptera littoralis), this attracting effect of its own frass is a consequence of the emission of the volatile compound guaiacol by the bacteria Serratia marcescens, Enterobacter cloacae, E. ludwigii, and Klebsiella sp. [85].

With regard to the induction of defensive plant responses, insect frass has also been described as an effective inhibitor. This is the result of an evolutionary response of insect pests to facilitate their feeding on plant biomass. For example, the insect frass produced by the Colorado potato beetle (Leptinotarsa decemlineata) contains bacteria of the genera Acinetobacter and Citrobacter, which, upon contact with potato plant tissue, are capable of inhibiting jasmonic acid-mediated plant defense responses, such as the local accumulation of steroidal glycoalkaloids [91].

The last negative mechanism of action of insect frass for crops is the inhibition of microorganisms used as bioinsecticides. This mechanism is a survival strategy of the insect against different entomopathogens. For example, against the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana, the patent leather beetle (Odontotaenius disjunctus) and the German cockroach (Blattella germanica) present in their frass bacteria that produce powerful antifungal metabolites [88,118]. Therefore, it is important to understand the microbial diversity associated with the insect frass used in agriculture, not only to avoid spreading plant pathogens, but also to avoid negative effects on other biological strategies that may be used in crops.

To conclude, and as a summary, an infographic (Figure 2) has been created showing all the positive and negative effects of insect frass in agricultural use, in relation to abiotic and biotic plant stresses.

10. Conclusions and Future Prospects

The ever-increasing world population is intensifying the demands on food production; however, this increase has to take place in parallel with a reduction in chemical inputs and in a scenario where biotic and abiotic stresses are constantly on the increase. This challenge is quite enormous and needs to be met by focusing efforts on new strategies. The industrial-scale rearing of insects has led to a significant increase in the generation of by-products that require valorization, particularly those with added value, such as insect frass. Due to its rich nutritional profile and diverse microbial content, frass holds great potential as a biofertilizer. Moreover, numerous studies have demonstrated its ability to enhance plant responses to both biotic and abiotic stresses. As highlighted in this review, the current scientific literature supports the beneficial effects of insect frass in mitigating various stress factors, from improving plant tolerance and salinity to exhibiting antifungal activity against plant diseases. However, some studies have also reported potential negative effects. Therefore, continued research is essential to better understand and mitigate these drawbacks, ensuring frass’ safe and effective application in agriculture.

For future research directions, it would be valuable to further investigate the mechanisms activated in plants upon the application of insect frass, as well as the metabolic pathways involved. Additionally, it is crucial to consider the practical application of frass in real-world agriculture and increase field trials to assess its functionality under natural conditions. Long-term studies on soil health are also necessary to determine the cumulative effects of frass application over time, particularly its influence on nutrient cycling, microbial communities, and soil structure. Furthermore, regulatory assessments should be conducted to establish standardized guidelines for its use, ensuring both efficacy and safety while addressing potential concerns regarding contaminants or variability in frass composition. A deeper exploration of the microbial diversity associated with frass and its interactions with the existing soil microbiome is also needed to mitigate potential negative effects on crops and soil health.

In summary, insect frass is a promising agricultural input with potential applications as a growth promoter and in mitigating both abiotic and biotic stresses, contributing to a more sustainable approach that reduces the reliance on chemical inputs in the field. This is particularly relevant given the increasing regulatory restrictions, which advocate for a gradual reduction in chemical usage. However, its application requires a thorough evaluation to prevent potential negative effects, ensure its safety and validate its effectiveness under real agricultural conditions.