**Chemical Compositions, Mosquito Larvicidal and Antimicrobial Activities of Essential Oils from Five Species of** *Cinnamomum* **Growing Wild in North Central Vietnam**

**Do N. Dai 1,2,\*, Nguyen T. Chung 1, Le T. Huong 3, Nguyen H. Hung 4, Dao T.M. Chau 5, Nguyen T. Yen 3 and William N. Setzer 6,7,\***

Academic Editor: Giovanni Benelli

Received: 20 January 2020; Accepted: 17 February 2020; Published: 12 March 2020

**Abstract:** Members of the genus *Cinnamomum* (Lauraceae) have aromatic volatiles in their leaves and bark and some species are commercially important herbs and spices. In this work, the essential oils from five species of *Cinnamomum* (*C. damhaensis*, *C. longipetiolatum*, *C. ovatum*, *C. polyadelphum* and *C. tonkinense*) growing wild in north central Vietnam were obtained by hydrodistillation, analyzed by gas chromatography and screened for antimicrobial and mosquito larvicidal activity. The leaf essential oil of *C. tonkinense*, rich in β-phellandrene (23.1%) and linalool (32.2%), showed excellent antimicrobial activity (MIC of 32 μg/mL against *Enterococcus faecalis* and *Candida albicans*) and larvicidal activity (24 h LC50 of 17.4 μg/mL on *Aedes aegypti* and 14.1 μg/mL against *Culex quinquefasciatus*). *Cinnamomum polyadelphum* leaf essential oil also showed notable antimicrobial activity against Gram-positive bacteria and mosquito larvicidal activity, attributable to relatively high concentrations of neral (11.7%) and geranial (16.6%). Thus, members of the genus *Cinnamomum* from Vietnam have shown promise as antimicrobial agents and as potential vector control agents for mosquitoes.

**Keywords:** Lauraceae; *Aedes aegypti*; *Aedes albopictus*; *Culex quinquefasciatus*; antibacterial; antifungal

### **1. Introduction**

The Lauraceae is a large family of tropical and subtropical trees and shrubs [1]. In this family, the genus *Cinnamomum* is comprised of around 250 species with concentrations in east and southeast Asia [1]. Vietnam is home to 45 species of *Cinnamomum* [2,3], many of which are used in traditional medicine, for essential oils, as well as for timber [4,5].

We are in the midst of a post-antibiotic era. Numerous pathogenic microorganisms have developed resistance to commonly used antibiotic agents [6,7]. For example, *Klebsiella pneumoniae* [8], *Pseudomonas aeruginosa* [9] and *Staphylococcus aureus* [10], three organisms that are major causes of nosocomial

infections, have developed extremely drug resistant (XDR) strains. Likewise, antibiotic resistance is increasing in fungi such as *Candida* ssp. and *Aspergillus* spp. [11]. Essential oils have shown promise as complementary or adjuvant therapies for combating antimicrobial resistance [12–19].

Mosquitoes have been and continue to be the deadliest animals on earth. *Aedes aegypti* (L.) (Diptera: Culicidae) and *Ae. albopictus* (Skuse) are vectors for the arboviral diseases dengue, Zika, chikungunya and yellow fever and *Ae. aegypti* is also a vector for the emerging Rift Valley fever virus [20]. *Culex quinquefasciatus* (Say) is a vector of West Nile virus, Saint Louis encephalitis virus and lymphatic filariasis [21]. *Culex quinquefasciatus* may also serve as a vector in emerging viral diseases such as Zika virus [22], Sindbis virus [23] and Usutu virus [24]. Unfortunately, insecticidal resistance of these mosquito species is increasing leading to failure of vector control programs in many locations [25]. Furthermore, populations of *Ae. aegypti* [26], *Ae. albopictus* [27] and *Cx. quinquefasciatus* [28] are showing widespread resistance to commonly used larvicidal agents. It has been suggested that essential oils may serve as alternative and more ecologically benign mosquito larvicidal agents [29–31].

Because of the biological activities and traditional uses of members of the *Cinnamomum* genus, we hypothesize that *Cinnamomum* species from Vietnam may also exhibit potentially useful biological activities. As part of our ongoing investigations into the essential oils of Vietnamese *Cinnamomum* [32–34], we have obtained, analyzed and carried out antimicrobial and larvicidal screening of *Cinnamomum ovatum* C.K. Allen (syn. *Cinnamomum rigidissimum* H.T. Chang), *Cinnamomum tonkinense* (Lecomte) A. Chev. (syn. *Cinnamomum albiflorum* var. *tonkinense* Lecomte), *Cinnamomum damhaensis* Kosterm., *Cinnamomum longipetiolatum* H.W. Li and *Cinnamomum polyadelphum* (Lour.) Kosterm. (syn. *Laurus polyadelpha* Lour., *Cinnamomum litseafolium* Lecomte, *Cinnamomum litseafolium* var. *denticupulatum* Liou, *Cinnamomum saigonicum* Farw, *Camphorina saigonica* Farw).

### **2. Results**

### *2.1. Essential Oil Collection and Analysis*

Plant materials were collected from mature *Cinnamomum* trees from different locations in north central Vietnam. The collection details and essential oil yields of the *Cinnamomum* species are summarized in Table 1. The essential oils were analyzed by gas-chromatography–mass spectrometry (GC-MS) and gas chromatography–flame ionization detector (GC–FID). The chemical compositions of the *Cinnamomum* species are presented in Table 2.

**Table 1.** Collection details for *Cinnamomum* species from north central Vietnam.

**Table 2.** Chemical compositions (%) of *Cinnamomum* essential oils from north central Vietnam.

**Table 2.** *Cont.*

a RI = Retention Index determined on an HP-5ms column. b RI from the databases. c *C. ov.* = *Cinnamomum ovatum*.

d *C. to.* = *Cinnamomum tonkinense*. e *C. da.* = *Cinnamomum damhaensis.* f *C. lo.* = *Cinnamomum longipetiolatum.* g *C. po.* = *Cinnamomum polyadelphum.*

### *2.2. Antimicrobial Screening*

The *Cinnamomum* essential oils were screened for antimicrobial activity against Gram-positive (*Enterococcus faecalis*, *Staphylococcus aureus*, *Bacillus cereus*) and Gram-negative (*Escherichia coli*, *Pseudomonas aeruginosa*, *Salmonella enterica*) bacteria and a yeast (*Candida albicans*). Minimum inhibitory concentrations and IC50 values were determined using the microbroth dilution assay (Table 3).

**Table 3.** Antimicrobial activities of *Cinnamomum* essential oils from north central Vietnam.

a Micrograms of essential oil per milliliter of test solution.

### *2.3. Larvicidal Screening*

The *Cinnamomum* essential oils were screened for mosquito larvicidal activity against *Aedes aegypti*, *Aedes albopictus* and *Culex quinquefasciatus*. The 24 h and 48 h LC50 and LC90 values are summarized in Tables 4 and 5.

**Table 4.** Twenty-four-hour mosquito larvicidal activities (μg/mL) of *Cinnamomum* essential oils from north central Vietnam.

**Table 5.** Forty-eight-hour mosquito larvicidal activities (μg/mL) of *Cinnamomum* essential oils from north central Vietnam.

### **3. Discussion**

### *3.1. Cinnamomum ovatum*

The leaf and stem bark essential oils of *C. ovatum* demonstrated broad antimicrobial activity against the organisms tested with MIC values ranging from 16 to 128 μg/mL (Table 3). The major components of the leaf and stem essential oils were eugenol (70.5% and 71.2%, respectively), eugenyl acetate (9.5% and 9.3%, respectively) and linalool (5.9% and 8.3%, respectively) (Table 2). The high concentration of eugenol in these two essential oils is likely responsible for the observed antimicrobial effects. Eugenol has shown broad spectrum antibacterial [35,36] and antifungal [37–39] activities. Likewise, the mosquito larvicidal activity of *C. ovatum* leaf essential oil is likely due to eugenol; that compound has shown larvicidal activity against *Ae. aegypti* [40], *Ae. albopictus* [41] and *Cx. quinquefasciatus* [42]. *Cinnamomum cambodianum* leaf [34] and stem bark [33] essential oils from Vietnam have also shown high concentrations of linalool (27.0% and 33.1%, respectively).

### *3.2. Cinnamomum tonkinense*

*Cinnamomum tonkinense* leaf essential oil showed excellent antimicrobial activity against *E. faecalis* and *C. albicans* with MIC of 32 μg/mL and good activity against *B. cereus* and *S. aureus* (Table 3). The essential oil is rich in monoterpenes, α-pinene (4.0%), sabinene (3.4%), α-phellandrene (4.8%), β-phellandrene (23.1%), 1,8-cineole (9.8%), linalool (32.2%) (Table 2). Both α-pinene and linalool have shown antibacterial activity against *E. faecalis* [35] and *S. aureus* [43]; α-pinene and 1,8-cineole have shown antifungal activity against *C. albicans* [43]. Sabinene, on the other hand, has shown little [44] or no [45] antimicrobial activity. Likewise, α-phellandrene has shown no activity against *C. albicans* [46]. The leaf essential oils of *C. cordatum* and *C. scortechini* from Pahang, Malaysia, both rich in β-phellandrene (9.0% and 17.3%, respectively) and linalool (17.3% and 16.4%, respectively), have shown antifungal activities against several fungal strains [47].

The leaf essential oil of *C. tonkinense* is one of the most larvicidal in this study (Tables 4 and 5). The major components in the essential oil likely account for the observed larvicidal activity. α-Pinene, has been shown to be larvicidal against *Ae. aegypti*, *Ae. albopictus* and *Cx. quinquefasciatus* [48]; sabinene and linalool have both demonstrated larvicidal against *Ae. aegypti* and *Cx. quinquefasciatus* [49]; and α-phellandrene has shown activity against *Ae. aegypti* and *Ae. albopictus* [50] as well as *Culex pipiens molestus* [51]. The leaf essential oil of *C. scortechinii*, rich in β-phellandrene (17.3%) and linalool (16.4%), had shown excellent larvicidal activity against *Ae. aegypti* and *Ae. albopictus* (LC50 = 21.5 and 16.7 μg/mL, respectively) [52].

### *3.3. Cinnamomum damhaensis*

The major components of *C. damhaensis* leaf essential oil were linalool (44.8%) and β-selinene (19.1%) (Table 2). The essential oil also showed pronounced larvicidal activity against *Ae. aegypti* and *Cx. quinquefasciatus* with 48 h LC50 values of 17.4 and 18.6 μg/mL, respectively (Table 5), which can be attributed to the high concentration of linalool (see above). Note that *Piper gaudichaudianum* and *Piper humaytanum* leaf essential oils, rich in β-selinene (10.5% and 15.8%, respectively), but devoid of linalool, showed only marginal larvicidal activity against *Ae. aegypti* [53].

### *3.4. Cinnamomum longipetiolatum*

The leaf essential oil of *C. longipetiolatum* was dominated by linalool (75.7%, Table 2), which likely accounts for the observed antimicrobial (Table 3) activity; linalool has shown broad antibacterial and antifungal activity [35,54]. Although linalool has shown larvicidal activity against *Ae. aegypti* (LC50 = 38.6 μg/mL) and *Cx. quinquefasciatus* (LC50 = 42.3 μg/mL) [49], the larvicidal activity of *C. longipetiolatum* leaf oil was less (24 h LC50 = 64.2 and 126.8 μg/mL against *Ae. aegypti* and *Cx. quinquefasciatus*, respectively, Table 4).

### *3.5. Cinnamomum polyadelphum*

The leaf essential oil of *C. polyadelphum* showed good activity against the Gram-positive organisms tested with MIC values of 32, 64 and 64 μg/mL on *E. faecalis*, *S. aureus* and *B. cereus*, respectively (Table 3). The essential oil also showed notable larvicidal activity against all three mosquito species with 48-h LC50 values of 17.3, 20.8 and 11.0 μg/mL against *Ae. aegypti*, *Ae. albopictus* and *Cx. quinquefasciatus*, respectively (Table 5). The major components in *C. polyadelphum* leaf essential oil were camphor (32.2%), neral (11.7%) and geranial (16.6%) (Table 2). The antimicrobial properties of camphor are relatively marginal [55,56]. Citral (mixture of neral and geranial), on the other hand, has shown greater antimicrobial activity on Gram-positive bacteria [57–59] and fungi [60,61]. Likewise, citral has exhibited mosquito larvicidal activity against *Ae. albopictus* [62] but camphor is inactive against larvae of *Ae. aegypti*, *Ae. albopictus* [52,62] or *Cx. pipiens* [63].

### **4. Materials and Methods**

### *4.1. Plant Material*

Leaves or stem bark of the *Cinnamomum* species were collected from locations in north central Vietnam (see Table 1). Plants were identified by Do N. Dai and voucher specimens (Table 1) have been deposited in the plant specimen room, Faculty Agriculture, Forestry and Fishery, Nghe An, College of Economics. The fresh plant materials (2.0 kg each) were shredded and hydrodistilled using a Clevenger apparatus for 4 h to give the essential oils. The essential oil yields are summarized in Table 1.

### *4.2. Gas Chromatographic Analysis*

Gas chromatography (GC) analysis was performed on an Agilent Technologies (Santa Clara, CA, USA) HP 7890A Plus Gas chromatograph equipped with a flame ionization detector (FID) and fitted with HP-5ms column (30 m × 0.25 mm, film thickness 0.25 μm, Agilent Technologies). The analytical conditions were—carrier gas H2 (1 mL/min), injector temperature (PTV) 250 ◦C, detector temperature 260 ◦C, column temperature programmed from 60 ◦C (2 min hold) to 220 ◦C (10 min hold) at 4 ◦C/min. Samples were injected by splitting and the split ratio was 10:1. The volume injected was 1.0 μL. Inlet pressure was 6.1 kPa.

An Agilent Technologies (Santa Clara, California, USA) HP 7890A Plus Chromatograph fitted with a fused silica capillary HP-5ms (30 m × 0.25 mm, film thickness 0.25 μm) and interfaced with a mass spectrometer (HP 5973 MSD) was used for the GC-MS analysis, under the same conditions as those used for GC-FID analysis. The conditions were the same as described above with He (1 mL/min) as carrier gas. The MS conditions were as follows—ionization voltage 70 eV; emission current 40 mA; acquisitions scan mass range of 35–350 amu at a sampling rate of 1.0 scan/s.

The identification of constituents was performed on the basis of retention indices (RI) determined with reference to a homologous series of *n*-alkanes, under identical experimental conditions, co-injection with standards (Sigma-Aldrich, St. Louis, MO, USA) or known essential oil constituents, MS library search (NIST 08 and Wiley 9th Version) and by comparing with MS literature data [64]. The relative amounts of individual components were calculated based on the GC peak area (FID response) without using correction factors.

### *4.3. Antimicrobial Screening*

The antimicrobial activity of the essential oils was evaluated using three strains of Gram-positive test bacteria, *Enterococcus faecalis* (ATCC299212), *Staphylococcus aureus* (ATCC25923), *Bacillus cereus* (ATCC14579), three strains of Gram-negative test bacteria, *Escherichia coli* (ATCC 25922), *Pseudomonas aeruginosa* (ATCC27853), *Salmonella enterica* (ATCC13076) and one strain of yeast, *Candida albicans* (ATCC 10231).

Minimum inhibitory concentration (MIC) and median inhibitory concentration (IC50) values were measured by the microdilution broth susceptibility assay [65]. Stock solutions of the oil were prepared in dimethylsulfoxide. Dilution series were prepared from 16,384 to 2 μg/mL (214, 213, 212, 211, 210, 29, 27, 25, 23 and 21 μg/mL) in sterile distilled water in micro-test tubes from where they were transferred to 96-well microtiter plates. Bacteria grown in double-strength Mueller-Hinton broth or double-strength tryptic soy broth and fungi grown in double-strength Sabouraud dextrose broth were standardized to 5 × 105 and 1 × 103 CFU/mL, respectively. The last row, containing only the serial dilutions of sample without microorganisms, was used as a positive (no growth) control. Sterile distilled water and medium served as a negative (no antimicrobial agent) control. Streptomycin was used as the antibacterial standard, nystatin and cycloheximide were used as antifungal standards. After incubation at 37 ◦C for 24 h, the MIC values were determined to be the well with the lowest concentration of agents completely inhibiting the growth of microorganisms. The IC50 values were determined by the percentage of microorganisms that inhibited growth based on the turbidity measurement data

of EPOCH2C spectrophotometer (BioTeK Instruments, Inc Highland Park Winooski, VT, USA) and Rawdata computer software (Brussels, Belgium) according to the following equations:

$$\%\_{\text{inluibition}} = \frac{OD\_{\text{control}(-)} - OD\_{\text{test agent}}}{OD\_{\text{control}(-)} - OD\_{\text{control}(+)}} \tag{1}$$

$$IC\_{50} = High\_{conc} - \frac{(High\_{init\%} - 50\%) \times (High\_{conc} - Low\_{conc})}{(High\_{init\%} - Low\_{init\%})} \tag{2}$$

where OD is the optical density, control(–) are the cells with medium but without antimicrobial agent, test agent corresponds to a known concentration of antimicrobial agent, control(+) is the culture medium without cells, Highconc/Lowconc is the concentration of test agent at high concentration/low concentration and Highinh%/Lowinh% is the % inhibition at high concentration/% inhibition at low concentration). Each of the antimicrobial screens were carried out in triplicate.

### *4.4. Larvicidal Screening*

Eggs of *Aedes aegypti* were purchased from Institute of Biotechnology, Vietnam Academy of Science and Technology and maintained at the Laboratory of Department of Pharmacy of Duy Tan University, Da Nang, Vietnam. Adults of *Culex quinquefasciatus* and *Aedes albopictus* collected in Hoa Khanh Nam ward, Lien Chieu district, Da Nang city (16◦03- 14.9"N, 108◦09- 31.2"E) and were identified by National institute of Malariology, Parasitology and Entomology, Ho Chi Minh City. Adult mosquitoes were maintained in entomological cages (40 × 40 × 40 cm) and fed a 10% sucrose solution and were allowed to blood feed on 1-week-old chicks and mice, respectively. Egg hatchings were induced with tap water. Larvae were reared in plastic trays (24 × 35 × 5 cm). The larvae were fed on Koi fish food. All developmental stages were maintained at 25 ± 2 ◦C, 65–75% relative humidity and a 12:12 h light:dark cycle at the Laboratory of the Faculty of Environmental and Chemical Engineering of Duy Tan University, Da Nang, Vietnam.

Larvicidal activities of the *Cinnamomum* essential oils were evaluated according to the protocol Liu and co-workers [66] with slight modifications. For the assay, 150 mL of water that contained 20 larvae (fourth instar) was placed in 250-mL beakers and aliquots of the *Cinnamomum* essential oils dissolved in EtOH (1% stock solution) were then added. With each experiment, a set of controls using EtOH only (negative control) and permethrin (positive control) were also run for comparison. Mortality was recorded after 24 h and again after 48 h of exposure during which no nutritional supplement was added. The experiments were carried out at 25 ± 2 ◦C. Each test was conducted with four replicates with five concentrations (100, 50, 25, 12.5 and 6 μg/mL). The data obtained were subjected to log-probit analysis [67] to obtain LC50 values, LC90 values and 95% confidence limits using Minitab® 19 (Minitab, LLC, State College, PA, USA).

### **5. Conclusions**

The essential oils of five species of *Cinnamomum* were collected from north central Vietnam and screened for antimicrobial and mosquito larvicidal activities. According to Duarte and co-workers [68], essential oils with MIC values between 50 and 500 μg/mL can be considered to have strong antimicrobial activity. Similarly, Dias and Moraes have concluded that essential oils with LC50 < 100 μg/mL are considered to be active [69]. Therefore, all of the *Cinnamomum* essential oils in this study can be considered to be active and show promise as antimicrobial agents and as alternative insecticidal agents against mosquito larvae.

**Author Contributions:** Conceptualization, D.N.D. and W.N.S.; methodology, D.N.D., L.T.H., N.H.H., W.N.S.; validation, D.N.D. and W.N.S.; formal analysis, L.T.H., W.N.S.; investigation, N.T.C., L.T.H., N.T.Y., D.T.M.C.; resources, D.N.D.; data curation, W.N.S.; writing—original draft preparation, W.N.S., D.N.D.; writing—review and editing, D.N.D., L.T.H., W.N.S.; supervision, D.N.D.; project administration, D.N.D.; funding acquisition, D.N.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number: 106.03-2018.02.

**Acknowledgments:** W.N.S. participated in this work as part of the activities of the Aromatic Plant Research Center (APRC, https://aromaticplant.org/).

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

**Sample Availability:** The Cinnamomum essential oils are no longer available.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Plant-Derived Pesticides as an Alternative to Pest Management and Sustainable Agricultural Production: Prospects, Applications and Challenges**

**Augusto Lopes Souto 1, Muriel Sylvestre 2, Elisabeth Dantas Tölke 3, Josean Fechine Tavares 1, José Maria Barbosa-Filho 1 and Gerardo Cebrián-Torrejón 2,\***

**Abstract:** Pests and diseases are responsible for most of the losses related to agricultural crops, either in the field or in storage. Moreover, due to indiscriminate use of synthetic pesticides over the years, several issues have come along, such as pest resistance and contamination of important planet sources, such as water, air and soil. Therefore, in order to improve efficiency of crop production and reduce food crisis in a sustainable manner, while preserving consumer's health, plant-derived pesticides may be a green alternative to synthetic ones. They are cheap, biodegradable, ecofriendly and act by several mechanisms of action in a more specific way, suggesting that they are less of a hazard to humans and the environment. Natural plant products with bioactivity toward insects include several classes of molecules, for example: terpenes, flavonoids, alkaloids, polyphenols, cyanogenic glucosides, quinones, amides, aldehydes, thiophenes, amino acids, saccharides and polyketides (which is not an exhaustive list of insecticidal substances). In general, those compounds have important ecological activities in nature, such as: antifeedant, attractant, nematicide, fungicide, repellent, insecticide, insect growth regulator and allelopathic agents, acting as a promising source for novel pest control agents or biopesticides. However, several factors appear to limit their commercialization. In this critical review, a compilation of plant-derived metabolites, along with their corresponding toxicology and mechanisms of action, will be approached, as well as the different strategies developed in order to meet the required commercial standards through more efficient methods.

**Keywords:** biopesticides; bio-based pesticides; chemical ecology; pest control; natural products

### **1. Introduction**

Pesticides may be defined as any compound or mixture of components intended for preventing, destroying, repelling or mitigating any pest [1]. Additionally, herbicides or weed-killers may also be considered as pesticides, and are used to kill unwanted plants in order to leave the desired crop relatively unharmed and well provided with nutrients, leading to a more profitable harvest [2].

Nevertheless, the world food production is constantly affected by insects and pests during crop growth, harvest and storage. As a matter of fact, there is an estimated loss of 18–20% regarding the annual crop production worldwide, reaching a value of more than USD 470 billion [3]. Furthermore, insects and pests not only represent a menace to our homes, gardens and reservoirs of water, but also, they transmit a number of diseases by acting as hosts to some disease-causing parasites. Therefore, the mitigation or control of

**Citation:** Souto, A.L.; Sylvestre, M.; Tölke, E.D.; Tavares, J.F.; Barbosa-Filho, J.M.; Cebrián-Torrejón, G. Plant-Derived Pesticides as an Alternative to Pest Management and Sustainable Agricultural Production: Prospects, Applications and Challenges. *Molecules* **2021**, *26*, 4835. https:// doi.org/10.3390/molecules26164835

Academic Editor: Francesca Mancianti

Received: 29 June 2021 Accepted: 4 August 2021 Published: 10 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

pests' activities may lead to a substantial reduction of the world food crisis as well as the improvement of human and animal health [2].

The great demand for food has led to the intensification of agricultural technology in order to achieve maximum productivity per hectare, through expansion of irrigation facilities, introduction of high-yielding varieties and application of increased amounts of agrochemicals, mostly synthetic [1] (for example, the 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (DDT) used from 1939 to 1962 [4] and the polychlorinatedbiphenyls (PCBs) used from 1926 to 1970 [5]). In spite of various technological achievements over the years, serious problems have come along, especially due to the indiscriminate use of synthetic pesticides. As they remain in our planet for an extremely long period, its long persistence in our biosphere allows insects to develop resistance against them; they are also known to contaminate indispensable resources, such as water, air and soil. This is illustrated by the problem of chlordecone (CLD), a pesticide widely used from 1972 to 1993 in the French West Indies (FWI) [6] which is often called as the "monster of the Antilles" because of the extent of soil and biomass contamination. Chemically, CLD is an organochlorine ketone, with high steric hindrance and high hydrophobicity, which allows it to adsorb strongly into soils rich in organic matter. At the same time, it is non-volatile and has a low biodegradability. It has also been demonstrated that CLD is a particularly persistent molecule, which remains almost perennial in soils, and the phenomenon of bioaccumulation in living organisms has been observed. Despite the ban on its use in the early 1990s, this molecule is still present today in the waters and soils of the French West Indies [7]. Extremely few pesticides used in the post-DDT era have long half-lives in the environment and none bioaccumulate in the way that the organochlorines did. Moreover, most of the synthetic pesticides act against non-target organisms (mammals, fish and plant species), becoming a potential health hazard to consumers. Furthermore, they are too expensive for the farmers in developing countries [8–11].

This problematic is illustrated as well by the neonicotinoid insecticides (such as imidacloprid, clothianidin, thiamethoxam, acetamiprid and thiacloprid). This more recent class of molecules is active against several pests (targeting the acetylcholine receptors) and can be applied to different cultures (such as tobacco, cotton, peach and tomato) [12].

The use of neonicotinoid insecticides was allowed in Europe in 2005 until 2013 (see Regulation (EU) No 485/2013) when the employ was restricted to protect wildlife, such as pollinators (honeybees), mammals, birds, fish, amphibians and reptiles, and the effects on vertebrates—mammals, birds, fish, amphibians and reptiles [13].

This awareness regarding pest problems and the environment has led to the search for powerful and eco-friendly pesticides that degrade after some time, avoiding pest resistance, which is also pest-specific, non-phytotoxic, nontoxic to mammals and relatively less expensive in order to obtain a sustainable crop production [14,15]. In addition to the awareness achieved in the various countries (such as China, United States, Brazil or Turkey) [16,17] and also in European Union (EU), the legislation has become increasingly stringent and binding with the consequences of a green choice in the use of pesticides and approbation of plant derivatives allowed in the biological control regulation (for example: clove (*Syzygium aromaticum* [L.] Merr. and L.M. Perry [Myrtaceae]) essential oil in the EU, the derived terpenes from *Chenopodium ambrosioides* L. (Amaranthaceae) in US, Ginkgo (*Ginko biloba* L. [Ginkgoaceae]) fruit extract and *Psoralea corylifolia* L. (Leguminosae) seed extract in China or the extract of *Tephrosia candida* DC. (Fabaceae) in Brazil).

In this context, biopesticides may meet those required standards and become the key to solve pest problems and promote sustainable production, once they are cheap, targetspecific, less hazardous to human health, bio-degradable and therefore environmentally friendly. Biopesticides are pest management agents based on biochemicals derived from living microorganisms, insects and plants. [2,18,19]. In this review, we will focus on biopesticides from plant origin.

Among the plant-derived pesticides, there are the insecticides (constituents that kill insects in any stage of development: adults, ova and larvae), which act by several different mechanisms affecting one or more biological systems, including nervous, respiratory and endocrine systems, as well as water balance. Additionally, insecticides can also be classified depending on the mode of its entry into the insect, namely: stomach poisons, contact poisons and fumigants [11]. A compilation of natural insecticides with their corresponding toxicity and mechanisms of action may be found in Table 1.

### **2. Plant Derived Insecticides That Affect the Nervous System**

The majority of insecticides, whether biological or synthetic, fit in this category, acting on several targets, such as: voltage-gated sodium channels, voltage-gated calcium channels, acetylcholinesterase enzyme (AChE), nicotinic acetylcholine receptors, GABA receptors and octopamine receptors.

### *2.1. Voltage-Gated Sodium Channels*

Pyrethrum, an oleoresin extracted from the dried flowers of the pyrethrum daisy, *Tanacetum cinerariifolium* (Trevir.) Sch. Bip. (Asteraceae), contains two major compounds, namely Pyrethryns I and II (cyclopropylmonoterpene esters) [20] that act as a modulator on voltage-gated sodium channels, which are essential for proper electrical signaling in the nervous system, causing a delay in sodium channel closing, resulting in over neuroexcitation, leading to loss of control of the coordinated movement, paralysis and death. This mode of actions is highly similar to other synthetic insecticides (such as synthetic pyrethryns). However, natural pyrethrins used to be more target-specific than synthetic ones [21]. It is defined as a contact and stomach poison that provides an immediate knockdown when applied, which has also demonstrated low toxicity to mammals and a particularly short residual activity, once it is rapidly degraded by sunlight, air and moisture; therefore, frequent application may be required [22]. Pyrethrins may be used against a wide range of insects and mites, including spider mites, flies, fleas and beetles [23,24]. Its activity may be enhanced by incorporating piperonylbutoxide (PBO) as a synergist [2].

Other natural products with a similar mechanism of action have already been reported, such as decalesides I and II, firstly isolated from the roots of *Decalepis hamiltonii* Wight and Arn. (Apocynaceae). They are classified as trisaccharides, and are toxic to a variety of insects by contact exposure, not orally, but through contact to the gustatory receptors located in the tarsi of the insect [25].

Sabadilla, an insecticidal preparation from pulverized seeds of *Schoenocaulon officinale* (Schltdl. and Cham.) A. Gray ex Benth. (Melanthiaceae), has been used by native people from south and central America for many years. Its alkaloid preparation contains mainly two major alkaloids: cevadine and veratridine at a proportion of 2:1, which have a mode of action similar to pyrethrins, although the binding site seems to be different [26]. Sabadilla is one of the least toxic plant extracts with pesticide activity, considered a contact and stomach poison with minimal residual activity. However, its major alkaloids, when isolated, are much more toxic to humans, and affect mostly stinks, squash bugs, thrips, leafhoppers and caterpillars [11,27].

### *2.2. Voltage-Gated Calcium Channels*

Ryanodine, an active component found in the roots and woody stems of *Ryania speciosa* Vahl (Salicaceae), native to Trinidad, activates the calcium channels from the sarcoplasmic reticulum of skeletal muscle cells. Once activated, the calcium channels release an excess of calcium ions into actin and myosin protein filaments, leading to skeletal muscle contraction and paralysis [28]. Ryanodine is a "fast act" poison, promoting its insecticidal activity either by contact or stomach, with a low mammalian toxicity and a long residual activity, providing up to two weeks of control after the first application. Ryania crude extracts insecticidal activity is synergized by piperonylbutoxide (PBO), and is reported to be most effective in hot waters, working efficiently against caterpillars, worms, potato beetles, lace bugs, aphids and squash bugs. [11,29]. Ryania is almost no longer used in the United States.

### *2.3. Acetylcholinesterase Enzyme (AChE)*

Acetylcholinesterase (AChE), an enzyme that hydrolyzes the neurotransmitter acetylcholine, plays an important role regulating the transmission of the cholinergic nervous impulse, and may also be a target for biopesticides. Coumaran (2,3-dihydrobenzofuran), an active ingredient found in *Lantana camara* L. (Verbenaceae), inhibits this enzyme, building up the concentration levels of acetylcholine in the synapse cleft, causing an excessive neuroexcitation due to the prolonged biding of the neurotransmitter to its postsynaptic receptor, leading to restlessness, hyperexcitability, tremors, convulsion, paralysis and death [30]. It presents low toxicity to mammals and works rapidly against houseflies and grain storage pests, in spite of its short residual activity. Regarding its mechanism of action, coumaran may also be compared to the monoterpene 1,8-cineole [31] or other synthetic pesticides such as organophosphates and carbamates [32]. Moreover, Khorshid, et al. [33] have presented the inhibitory activity of methanolic extract from *Cassia fistula* L. (Fabaceae) roots and proposed the indole alkaloids as new potential active agents. However, it should be noted that many essential oils and terpenes therefrom have demonstrated anti-AChE activity in vitro, but their contribution to insect mortality is questionable [34].

### *2.4. Nicotinic Acetylcholine Receptors*

In relation to nicotinic acetylcholine receptors, they are present in the insect nervous system, either on pre or postsynaptic nerve terminal, as well as the cell bodies of the inter neurons, motor neurons and sensory neurons [35]. Nicotine, an alkaloid firstly isolated from *Nicotiana tabacum* L. (Solanaceae), can mimic acetylcholine by acting as an agonist of the acetylcholine receptor, leading to an influx of sodium ion and generation of action potential. Under normal conditions, the synaptic action of acetylcholine is terminated by AChE. However, since nicotine cannot be hydrolyzed by AChE, the persistent activation caused by the nicotine leads to an overstimulation of the cholinergic transmission, resulting in convulsion, paralysis and finally death [35]. Nicotine is an extremely fast nerve toxin, most effective towards soft-bodied insects and mites. However, is the most toxic of all botanicals and extremely harmful to humans [2]. Alternatively, there is another class of insecticides inspired on nicotine chemical structure, called neonicotinoids, which may be represented by imidacloprid, acetamiprid and thiamethoxam. Similarly to nicotine, neonicotinoids interact with nicotinic acetylcholine receptors. However, they are more specific, being much more toxic to invertebrates such as insects than to mammals. Additionally, they have higher water solubility, which permits its application to soils and therefore, its absorption by plants, promoting a more efficient defense [36]. They may act as a contact or ingestion poison, leading to the cessation of feeding within several hours of contact followed by death shortly after [37].

### *2.5. GABA-Gated Chloride Channels*

GABA-gated chloride channels are potential targets for insecticides. Once they are blocked by its antagonists (such as the α-Thujone (isolated from *Artemesia absinthium* L. (Asteraceae)) or the picrotoxine (*Anamirta cocculus* (L.) Wight and Arn (Menispermaceae)), neuronal inhibition is reduced, leading to hyper-excitation of the central nervous system (CNS), convulsion and death. Previous research have reported the monoterpene thujone as a neurotoxin, since it acts on GABAA receptors as an allosteric reversible modulator, and as a competitive inhibitor of [3H]Ethynylbicycloorthobenzoate ([3H]EBOB binding) [38]. Additionally, the GABA receptor may be inhibited by the monoterpenoids carvacrol, pulegone and thymol through [3H]TBOB binding [39]. Similarly, the silphinene-type sesquiterpenes, plant-derived natural compounds, antagonize the action of aminobutyricacid (GABA), by stabilizing non-conducting conformations of the chloride channel [24,38]. As GABA is an endogenous ligand related to stimulate feeding and evoke taste cell responses on most herbivorous insects, the chemicals that antagonize GABA receptors may also be considered as antifeedant or deterrent compounds, affecting mostly aphids, lepidopterans and beetles [40,41].

### *2.6. Octopamine Receptors*

Octopamine is a multi-functional endogenous amine that acts as a neurotransmitter, neurohormone and neuromodulator on invertebrates [42]. Its receptors are widely distributed in the central and peripheral nervous systems of insects, comprising the octopaminergic system, constituting of several subtypes of octopamine receptors, which are coupled to different second messenger systems, therefore playing a key role in mediating physiological functions and behavioral aspects [43–45]. For instance, octopamine1 receptor modulates myogenic rhythm of contraction in locust extensor-tibiae through changes in intracellular calcium concentrations, whereas octopamine2A and octopamine2B receptors mediate their effects through the activation of adenylatecyclase. Moreover, octopamine3 receptors mediate changes in cyclic adenosine monophosphate (CAMP) levels in the locust central nervous system [46].

The rapid action of monoterpenes against some pests suggests a neurotoxic mode of action. This hypothesis was confirmed by Reynoso, et al. [47], who have demonstrated repellent and insecticidal activity of eugenol against the blood-sucking bug *Triatoma infestans* (Klug; Reduviidae) through activation of the octopamine receptor.

Previous studies have reported the presence of octopamine receptors in a large variety of insects, including, firefly, flies, nymphs, cockroaches and lepidopterans [46–48]. As these receptors do not conform to the receptor categories that have been recognized in vertebrates, agonists of octopamine receptors may be a valuable candidate for a commercial pesticide, once they are target-specific, less toxic to mammals and have a different mechanism of action when compared to the majority of pesticides currently in the market [47].

### **3. Plant Derived Insecticides That Affect Respiratory or Energy System**

Cellular respiration is a process that converts nutrient compounds into energy or adenosine triphosphate (ATP) at a molecular level. More specifically, this process is performed by the electron transport chain of the mitochondria, which comprises several important enzymes that are potential targets for insecticides. Rotenone is the most common natural product among rotenoids, a type of isoflavonoid and is usually found in species from Derris and Lonchocarpus (in Fabaceae) and Rhododendron (in Ericaceae), spread throughout East Indies, Malaya and South America [20].

Rotenone is defined as a complex I inhibitor of the mitochondrial respiratory chain, which works both as contact and stomach poison. It blocks the nicotinamide adenine dinucleotide (NADH) dehydrogenase, stopping the flow of electrons from NADH to coenzyme Q, therefore, preventing ATP formation from NADH, but maintaining ATP formation through flavine adenine dinucleotide (FADH2); therefore, it is one of the slowest acting botanical insecticide, and yet readily degradable by air and sunlight, taking several days to kill insects, affecting primarily nerve and muscle cells, leading to cessation of feeding, followed by death, from several hours to a few days after exposure. Moreover, this bio-based pesticide is constantly applied to protect lettuce and tomato crops as it has a broad spectrum of activity against mite pests, including leaf-feeding beetles, lice, caterpillars, mosquitoes, ticks, fire ants and fleas. Furthermore, its effects are substantially synergized by PBO or pyrodone (MGK 264).

Rotenone is highly toxic to mammals and fish [24,49]. Its activity and persistence are comparable to dichlorodiphenyltrichloroethane (DDT) [2]; moreover, previous studies have correlated a possible link between its exposure and Parkinson's Disease (PD) [50]. However, in spite of its high toxicity, rotenoids may be a potential source of novel complex I inhibitors, acting as a prototype for the development of safer and more efficient pesticide derivatives [51].

Acetogenins (annonins, asimicin, squamocin, annonacins) obtained from *Annona squamosa* L. (Annonaceae) are well known for their pest control properties. A botanical formulation based on annonins wherein asimicin is the major pesticidal compound has been patented [52].

### **4. Plant Derived Insecticides That Affect the Endocrine System**

Chemical constituents that interfere with the endocrine system of insects are classified as insect growth regulators (IGR). They may act either as juvenile insect hormone mimics or inhibitors, as well as chitin synthesis inhibitors (CSI). Normally, the juvenile hormones are produced by insects in order to keep its immature state. When a sufficient growth has been reached, the production of the hormone stops, triggering the molt to the adult stage [53]. Triterpenes from *Catharanthus roseus* (L.) G. Don (Apocynaceae), such as α-amyrin acetate and oleanolic acid, have demonstrated interesting growth regulator activity [54]. Acyclic sesquiterpenes such as davanone, ipomearone and the juvenile hormone from silkworm are perfect examples of natural products with IGR activity as well. Therefore, the constant application of IGR towards the crops will maintain the insects in its larvae state, preventing a successful molting and resulting in an efficient pest control [55]. On the other hand, it has been reported the antijuvenile hormone activity of two chromenes found in *Ageratum conyzoides* L. (Asteraceae), precocene I and II promotes a precocious metarmophosis of the larvae and production of sterile, moribund and dwarfish adults after exposure [56]. Although, resistance to azadirachtin has been demonstrated [57], indicating that insects can develop resistance to natural hormones or hormone-related compounds; however, this class of compounds remains a natural potential for commercial bio-based pesticides [55]. Additionally, complex polyphenolic fractions also present a wide range of insecticidal activities, interfering with the fecundity and inducing the disruption of the oogenesis [58,59] (WO 94/13141).

Moreover, previous researches have reported a natural insecticide of broad-spectrum activity, which has low mammalian toxicity and is the least toxic among botanical insecticides. It is called azadirachtin, a complex tetranortriterpenoid limonoid, majorly found in the seeds of *Azadirachta indica* A. Juss. (Meliaceae), a plant species commonly known as the Neem tree which originated from Burma, but is currently grown in more arid, tropical and subtropical zones of Southeast Asia, Africa, Americas and Australia [24,26,60]. Azadirachtin is considered a contact poison of systemic activity, which may be categorized in two ways: direct effects towards cells and tissues, or indirect effects, represented by endocrine system interference. It is a powerful compound that acts mainly as a feeding deterrent and insect growth regulator, comprising a wide variety of insect taxa including Lepidoptera, Diptera, Hemiptera, Orthoptera, Hymenoptera [60]. As for its growth regulatory effects, azadirachtin affects the neurosecretory system of the brain insect, blocking the release of morphogenetic peptide hormones (e.g., prothoracicotropic hormone (PTTH) and allatostatins). These hormones control the function of the prothoracic glands and the corpora allata, respectively. Therefore, as the moulting hormone (which controls new cuticle formation and ecdyses) and the juvenile hormone (JH) (which controls the juvenile stage at each moult) are regulated by prothoracic glands and the corpora allata, any disruption on this biochemical cascade may lead to moult disruption, moulting defects or sterility. The effects on feeding, developmental and reproductive disruption are caused by effects of the molecule directly on somatic and reproductive tissues and indirectly through the disruption of endocrine processes [60].

Neem-based non-commercial products are normally found as neem oil, obtained from the cold pressing of its seeds, in order to control phytopathogens (including insects). The other product is a medium-polarity extract containing azadirachtin (0.2–0.6% of seed/weight) [2], whereas the actual commercial product is a 1 to 4.5% azadirachtin solution [61]. Despite its 20 h half-life, it ensures a reasonable persistence in field applications due to its systemic action [2].

In relation to CSIs, they inhibit the production of chitin, a β-(1,4)-linked homopolymer of N-acetyl-D-glucosamine, one of the most important structural components of nearly all fungi cell walls, and also a major component of the insect exoskeleton, which provides physical protection and osmoregulation. As chitin is absent on plant and mammalian species, while it is abundant in arthropods and most fungi, chitin biosynthesis has become an important target for developing more specific insecticides and antifungal agents. Previous

research has reported chitin synthase inhibition activity of 2-benzoyloxycinnamaldehyde (2-BCA), a natural product isolated from the roots of *Pleuropterus ciliinervis* Nakai (Polygonaceae), which is a plant species traditionally used in Chinese folk medicine to treat inflammation and several types of infection [62].

### **5. Plant Derived Insecticides That Affect the Water Balance**

Insects have a thin layer of wax covering their body, which provides the ecological function of preventing water loss from the cuticular surface. For instance, vegetable crude oils of rice bran, cotton seed and palm kernel, as well as saponins (natural soaps) may act by disrupting this protective waxy covering, affecting the water balance of insects through a rapid water loss from the cuticle, therefore leading to death by desiccation. Interestingly, the action of soaps affects the wax covering of insects [63]. The action of soaps on the wax covering of insects is influenced by the temperature [64]. Additionally, the crude oils may also act by interfering with insect respiration by plugging the orifices called spiracles, resulting in death by asphyxiation, controlling several types of insects such as whiteflies, mites, caterpillars, leafhoppers and beetles [1].

### **6. Other Classes of Pesticides**

The botanical pesticide agents may also be categorized into repellents, attractants, antifeedants or deterrents, molluscisides, fungicides, phytotoxins (herbicides) and phototoxins [15]. These classes are less common in plant sources than the insecticides [65]. Sometimes, a given compound may act as an insecticide and/or as a repellent. The major difference between those two is that the repellent does not kill insects, but only keeps them away by releasing pungent vapors or exhibiting a slight toxic effect [66].

### **7. Repellents**

There are several essential oils which are majorly constituted of monoterpenes and are considered extremely effective repellents, including lemongrass (*Cymbopogon flexuosus* (Nees) Will. Watson (Poaceae)), eucalyptus (*Eucalyptus globulus* Labill. (Myrtaceae)), rosemary (*Rosmarinus officinalis* L. (Lamiaceae)), vetiver (*Vetiveria zizanioides* (L.) Nash (Poaceae)), clove (*Eugenia caryophyllus* (Spreng.) Bullock and S.G. Harrison (Myrtaceae)) and thyme (*Thymus vulgaris* L. (Lamiaceae)) [67]. Catnip oil, for example, extracted from *Nepeta cataria* L. (Lamiaceae), is considered a highly effective repellent of mosquitoes, bees and other flying insects. As a matter of fact, this oil repels *Aedes aegypti* L. (Culicidae) ten times more than DEET, which is probably related to its most effective constituent, nepetalactone, a monoterpene lactone [68], which is also reported as a repellent for lady beetles, cockroaches, flies and termites [69,70]. The anthraquinone tectoquinone was also described as a repellent against termites [71,72], and alstonine alkaloid has a repellent and larvicidal activity against *Anopheles gambiae* Giles (Culicidae) [73].

### **8. Attractants**

In relation to attractants, they are considered semio-chemicals or communication compounds, released by plants in order to attract insects or to attract natural predators of the insects that feed on the plant [74]. Miller [75] have related the release of (−) and (+) limonene from white pine (*Pinus strobus* L. (Pinaceae)) to the attraction of the white pine cone beetle, *Conophthorus coniperda* Schwarz (Curculionidae), as well as the attraction of the predator beetle, *Enoclerus nigripes* Say (Cleridae), through the release of (−)-α-pinene, as well as the sesquiterpene caryophyllene [76].

### **9. Antifeedants or Deterrents**

Previous studies have correlated antifeedant activity to a chemoreception mechanism, consisting in the blockage of receptors that normally respond to phagostimulants or through stimulation of deterrent cells (primary antifeedancy). According to Qiao et al., [77] azadirachtin reduces the cholinergic transmission of neurons related to the

suboesophageal ganglion (SOG) of *Drosophyla melanogaster* Meigen (Drosophylinae), which are strongly related to feeding behavior. Additionally, food consumption may also be reduced due to its toxic effects after the first intake (secondary antifeedancy), promoting astringency, bitter taste or anti-digestive activity to certain herbivores [78,79]. For instance, Okwute and Nduji [80] have reported that schimperii, a gallotannin isolated from *Anogeissus schimperi* (Hochst. ex Hutch and Dalziel) (Combretaceae) was responsible for conferring this unattractive taste to herbivores. Similar effects were reported to, isoflavonoids [81], acetogenines [82,83] or cyanogenic glycosides, such as linamarin [84].

Moreover, Lajide, Escoubas and Mizutani [66] have reported feed deterrent activity of ent-kaurane diterpenoids isolated from *Xylopia aethiopica* (Dunal) A. Rich. (Annonaceae), among which, (−)-kau-16-en-19-oic acid has demonstrated the strongest antifeedant activity. According to Okwute [2], 15-epi-4E-jatrogrossidentadione, a diterpene from *Jatropha podagrica* L. (Euphorbiaceae) have also demonstrated its antifeedant activity towards *Chilo partellus* Swinhoe (Crambidae). Moreover, silphinene sesquiterpenes (*Senecio palmensis* C. Sm. (Asteraceae)) and thymol (*Thymus vulgaris* L. (Lamiaceae)) have been described as model of insect antifeedants [40].

However, as demonstrated by Huang et al., in spite of numerous natural plant natural products acting as antifeedants, no commercial product based on this mode of action have been produced. Insect habituation to feeding deterrents considerably limits their utility in crop protection [85].

### **10. Phytotoxines or Herbicides**

Regarding phytotoxins, they may be defined as natural herbicides that are naturally released by plant species in order to interfere with the growth or germination of specific targets around them, such as weeds, leaving the emitting plant with more chances to survive. In nature, such action is called allelopathy, and the compounds that promote this action are defined as allelopathic agents [86–88]. Clay, et al. [89] have reported a study regarding herbicidal activity of citronella oil against different weed species: the oil at a dose of 504 kg a.i. ha-1 largely killed the foliage of the weed species within one application. However, most species have regrown substantially after two months, except for *Senecio jacobaea* L. (Asteraceae), which was the most susceptible one. According to Ismail, et al. [76], its herbicidal activity occurs through inhibition of photosynthesis. Besides essential oils herbicidal activity, Ismail, Hamrouni, Hanana and Jamoussi [90] have also reported plant-derived isolated compounds, such as eugenol and 1,8-cineole, with herbicidal activity promoted through inhibition of DNA synthesis and mitosis. Furthermore, several classes of secondary metabolites have been already described as phytotoxins, including naphtoquinones, such as juglone [91,92], amino acids such as m-tyrosin e [93] and L-tryptophane [94], terpenoids as 5,6-dihydroxycadinan-3-ene-2,7-dione [2,95] and citronnellol [90], catechins [2,96], polyphenols [97] and alkylamides [98].

### **11. Phototoxins**

There is a class of phytochemicals called phototoxins or light-activated compounds that instead of losing their efficiency due to sunlight degradation, they are actually increased or activated by two different mechanisms. In the first mechanism (less common), molecular oxygen from the phototoxin absorbs the energy from the light, generating activated species of oxygen which ultimately damage important biomolecules [99]. The other mechanism of action is photogenotoxic, where phytochemicals cause damage to DNA, triggered by sunlight activation, regardless of the presence of oxygen in the phototoxin. In actuality, thephototoxin on its ground state, absorbs the photon, reaching its excited state, which interacts with ground state O2 located in the tissue of its target, generating singlet oxygen and enabling insecticidal activity. This peculiar mode of action of photoxins is so different from conventional synthetic pesticides that cross resistence among them is unlikely [100,101].

Light-activated phototoxins may be exemplified by several classes such as quinones, furanocoumarins, substituted acetylenes and thiophenes. For instance, Marchant and Cooper [102] have reported several phototoxins, such as 3-methyl-3-phenyl-1,4-pentadiyne, an oil constituent from *Artemisia monosperma* Delile (Asteraceae), which under sunlightinduced conditions exerts an activity similar to DDT against the housefly *Musca domestica* L. (Muscidae) and cotton leaf worm *Spodoptera littoralis* Boisduval (Noctuidae) larvae. They have also discovered that an acetylenic epoxide from *Artemisia pontica* L. (Asteraceae), called ponticaepoxide, exhibits an LC50 of 1.47 ppm against mosquito larvae when submitted to UV light. Additionally, Nivsarkar, et al. [103] have also found that the major compound from the roots of *Tagetes minuta* L. (Asteraceae), a thiophene called terthiophene or α-terthienyl (αT), is highly toxic against several organisms when co-submitted to near UV light radiation, such as nematodes, red flower beetles (*Tribolium casteneum* Herbst (Neoptera)), blood-feeding insects such as *Manduca sexta* L. (Sphingidae) and mosquito larvae (dipteres): *Aedes aegypti* L. (Culicidae), *Aedes atropalpus* Coquillett (Culicidae), *Aedes intrudens* Dyar (Culicidae) and yet, to our knowledge no commercial product has been generated. Plant-based natural product chemical structures with their corresponding pesticide activity and targets may be found in Table 2.

### **12. Discussion**

Since ancient times, efforts to protect the agricultural harvest against pests have been reported. The use of inorganic compounds to control pests was reported between 500 B.C and the 19th century. They included products based on sulphur, lead, arsenic and mercury [2]. On the other hand, plant biodiversity has proved to be an endless source of biologically active ingredients, used for traditional crop and storage protection. Egyptian and Indian farmers used to mix the stored grain with fire ash [104]. The ancient Romans used false hellebore (*Veratrum viride* Aiton (Melanthiaceae)) as a rodenticide. Moreover, pyrethrum (extract from *Tanacetum cinerariifolium* (Trevir.) Sch.Bip (Asteraceae)) was used as an insecticide in Persia and Dalmatia, whereas the Chinese have discovered the insecticidal properties of *Derris* spp. (Fabaceae) [105].

Previous studies have already reported more than 2500 plant species belonging to 235 families, which have demonstrated their biological activity against several types of pests [1]. However, in spite of the remarkable potential as natural sources for commercial botanical pesticide development, not many have been found on the market, remaining in use only for small organic crops and commonly classified as so-called farming products [106].

Plant-derived pesticides can be processed in various ways: as crude plant material in the form of dust or powder; as extracts or as pure plant natural products, formulated into solutions or suspensions [2]. Several different classes of natural compounds that promote pesticide activity have already been reported, namely: fatty acids, glycolipids, aromatic phenols, aldehydes, ketones, alcohols, terpenoids, flavonoids, alkaloids, limonoids, naphtoquinones, saccharides, polyolesthers, saponins and sapogenins [20,107–110]. However, several factors appear to limit the commercialization of botanical pesticides, such as: problems in large scale production, non-availability of raw materials, poor shelf life, diminished residual toxicity under field conditions, limitations regarding standardization and refinement of the final product. Additionally, as the phytochemical profile of plant species may vary according to its genome/transcriptome/proteome/metabolome, and this variation depends on several edaphic-climatic factors (i.e., temperature, relative humidity, level of sunlight radiation, altitude, photoperiod and type of soil) as well as ecologic interactions, (i.e., herbivory or mutualism), manufacturers must take additional care in order to maintain efficiency and ensure that their products will perform consistently (standardization). Finally, even if all these issues are addressed, regulatory approval remains as the major barrier. A serious drawback to commercialization of botanicals is the high cost of processing plant materials to meet the standards of pesticide regulatory authorities [111]. Marrone [112] provided an overview of the current state of biopesticides and offered some

ideas for improving their adoption, including conducting on-farm demonstrations and more education and training on how the products work and how to incorporate them into integrated pest management. In many jurisdictions, no distinction is made between synthetic pesticides and biopesticides. Simply because a compound is a natural product, it does not mean that it is safe, since most of the toxic poisons are natural products or inspired by them. Furthermore, if biopesticides are used indiscriminately, as wells as the synthetics, they may also lead to the development of pest resistance [113].

In this context, only a few new sources of botanicals have reached commercial status in the past twenty years. Thus, the major commercial botanic pesticides currently in use include: pyrethrin, rotenone, azadirachtin and essential oils in general [111], along with three other products, commercialized in a more limited way: ryania, nicotine and sabadilla or veratrine alkaloids [20].

Therefore, the best strategy for a botanical pesticide to meet all the standards required and reach commercial status in a more efficient and pragmatic way is by performing bioassay-guided fractionation in a high scale [82,109,114]. In other words, the bioassays assessment of several plant extracts and its fractions, obtained whether by sophisticated or unsophisticated purification procedures, may lead to the discovery of the most effective compound or mixture of compounds correlated to the pesticide activity of each corresponding species. The isolated compound may act as a lead compound or prototype for the synthesis or semi synthesis of pesticide derivatives, which, by structure–activity relationship (SAR) techniques may result in more effective and safer products. However, sometimes, when the compound is presented on its isolated form, it may promote no activity at all, proving that extracts or fractions from a certain plant species are more effective than its isolated compounds, due to synergic effect of compound mixture, which may also lead to the manufacture of potential raw material for commercial biopesticides [114,115].

**Table 1.** Toxicity and mechanism of action of bio-based natural insecticides.

C—Contact. S—Stomach. F—Fumigant. IGR—Insect Growth Regulator. R—Repellent.

**Table 2.**

*Cont.*

**Table**

**2.**

*Cont.*

**Table 2.**

*Cont.*

**Table 2.** *Cont.*

**Table 2.**

*Cont.*

 Purchased from Sigma–Aldrich Chemical Co.

\*\*

224

**Table 2.** *Cont.*

### **13. Conclusions**

Despite several factors that appear to limit botanical pesticide commercialization, such as problems in large scale production, non-availability of raw materials, poor shelf life, diminished residual toxicity under field conditions and lack of extract standardization, a multidisciplinary approach, comprising bioassay-guided fractionation, combined with structure-activity relationship (SAR) and analytical techniques, has revealed to be an extremely efficient strategy in order to develop bio-based pesticides that meet all the commercial standards required. In summary, plant-derived pesticides have indicated their potential as a great alternative for pest management, once they become cheap, targetspecific, less hazardous to human health, biodegradable and ecofriendly; therefore, they may improve crop efficiency and reduce food crisis while maintaining sustainability.

**Funding:** This research was funded by "Plan Chlordecone III en Guadeloupe et Martinique" in the framework of DICHOAL-MAREC (DIREC) project.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not Applicable.

**Acknowledgments:** Special thanks are due to Tara Massad (expert in insect chemical ecology from Gorongosa National Park (Mozambique)) for her attentive lecture and fruitful discussion.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

### *Review* **Diatomaceous Earth for Arthropod Pest Control: Back to the Future**

**Valeria Zeni 1,†, Georgia V. Baliota 2,†, Giovanni Benelli 1,\*, Angelo Canale 1 and Christos G. Athanassiou 2**

**Abstract:** Nowadays, we are tackling various issues related to the overuse of synthetic insecticides. Growing concerns about biodiversity, animal and human welfare, and food security are pushing agriculture toward a more sustainable approach, and research is moving in this direction, looking for environmentally friendly alternatives to be adopted in Integrated Pest Management (IPM) protocols. In this regard, inert dusts, especially diatomaceous earths (DEs), hold a significant promise to prevent and control a wide range of arthropod pests. DEs are a type of naturally occurring soft siliceous sedimentary rock, consisting of the fossilized exoskeleton of unicellular algae, which are called diatoms. Mainly adopted for the control of stored product pests, DEs have found also their use against some household insects living in a dry environment, such as bed bugs, or insects of agricultural interest. In this article, we reported a comprehensive review of the use of DEs against different arthropod pest taxa, such as Acarina, Blattodea, Coleoptera, Diptera, Hemiptera, Hymenoptera, Ixodida, Lepidoptera, when applied either alone or in combination with other techniques. The mechanisms of action of DEs, their real-world applications, and challenges related to their adoption in IPM programs are critically reported.

**Keywords:** urban pests; agricultural pests; aphids; cockroaches; kissing bugs; insect vectors; green insecticides; mosquitoes; moth pests; non-target toxicity; stored product pests; termites

### **1. Introduction**

Among different types of inert materials currently adopted in pest control, diatomaceous earths (DEs) hold a prominent position, as they are apparently the most often tested material for this purpose. A search in *Journal of Stored Products Research* for published papers between January 2019 and January 2021 revealed the publication of 13 papers with "diatomaceous earth" on their title, emphasizing the utilization of DEs in stored product protection. DEs are not only used for the management of insects and other arthropods, but they also have multiple uses including the control of different pathogens, such as fungi and bacteria [1–4]. Other types of inert dusts, such as zeolites [5] or kaolin [6], have been also investigated for pest control. This work will be focused solely on the use of DEs in crop protection but also in post-harvest and urban pest control, highlighting their wide applicability.

In a recent review paper, Athanassiou et al. [7] categorized the materials that can be used in pest control and fall into the category of "nano" under the general term of nanoparticles. Although there are cases where DE particles can touch the "nano" scale, DEs are generally classified in the "micro" category and can be considered as "microparticles" in contrast with nanoparticles.

DEs are the fossilized remains of phytoplankton, which are diatoms that occurred mostly during the Miocene and Eocene periods [1]. Diatoms are unicellular eukaryotic

**Citation:** Zeni, V.; Baliota, G.V.; Benelli, G.; Canale, A.; Athanassiou, C.G. Diatomaceous Earth for Arthropod Pest Control: Back to the Future. *Molecules* **2021**, *26*, 7487. https://doi.org/10.3390/ molecules26247487

Academic Editor: Baoan Song

Received: 16 October 2021 Accepted: 3 December 2021 Published: 10 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

algae that are characterized by an external skeleton (frustule) rich in silicon dioxide whose fossilized remains constitute DEs [1,2,8]. These diatoms are abundant either in fresh-water or marine environments, but they are also present in terrestrial ecosystems.

The present review provides a focus on the utilization of DEs to manage different arthropod pest categories when applied either alone or in combination with other techniques.

### **2. Which Is the Mode of Action of DEs?**

There are different theories about the insecticidal effect of DEs [2]. It is generally considered that DE particles attach to the insects' cuticle, causing death through desiccation [1,2,9], although the abrasion is also a complementary action, i.e., through cuticular micro-wounds [2]. The shape of DEs may be a critical factor in this sense, as round-shaped diatom may lead to more rapid water absorption, while sharp-shaped DE acts more as an abrasive factor [1,2,9,10]. Nevertheless, the shape of the diatom, and probably its action (i.e., sorption vs. abrasion) can be chanced through different processing techniques [11].

### **3. Why Use DEs for Arthropod Pest Control?**

Thanks to their characteristics, the use of DEs is advantageous for several types of applications [2,8]. First, DEs are natural substances, and given their low toxicity to mammals and the environment, the registration process is greatly simplified. In addition, being inert materials, DEs have no interaction with the commodity and can be easily removed through standard processing, such as sieving [1,10,12–14], while their presence in the final product, such as flour or semolina, does not alter baking or pasta-making properties [1,12]. For more than two decades, DEs have been used as feed additives and in veterinary pest control [1]. Moreover, DEs are easily accessible [8,15]. The natural deposits from where DEs are extracted are found almost everywhere. Following their extraction, these powders are sieved to obtain a homogeneous mixture of particle sizes and dried at approximately 2–6% moisture content [1,11,15]. Finally, due to their mechanism of action, no physiological pest resistance is expected to occur, while tolerance may be exhibited through reduced contact with the DE particles [16–18].

### **4. Any Dark Facets for DEs Use in Pest Control?**

In general, to be effective, DEs must be applied at elevated concentrations, which are much higher than those of conventional insecticides and often exceed 1000 ppm [2,8,19,20]. In this way, they create a "dusty" appearance on the products and might cause health problems to workers, such as respiratory disorders [1,2,8,10]. In addition, their application on stored products results in the reduction of the test weight (weight to volume ratio), which is a critical characteristic in the international grain market [1,12].

In this scenario, we focused on current knowledge and challenges on the use of DEs in stored products as well as for managing arthropod pests of agricultural importance, urban pests, and vectors of public health relevance. The potential impact of DEs on non-target species is also discussed.

### **5. DEs to Control Stored Product Pests**

Currently, most studies assessing the toxicity of DEs on arthropods of economic importance are focused on stored product pests. Storing durable commodities is significant since it ensures stable food and feed production all year long and on a global scale. However, the storage environment, which may range from warehouses to retail shelves, is also a prosperous place for a range of insects to thrive [21]. Insect infestations have multiple effects on stored food, feed commodities, and seeds. Beyond the direct damage caused by food consumption, insects also pose a quarantine threat. Insect fragments within durable edible products provoke allergic reactions, alter the organoleptic characteristics, and potentially carry disease-causing pathogens [22]. Therefore, even a small percentage of damage may result in profound monetary losses. Despite the technological advantages over the years, most segments of the food industry are very susceptible to insect infestations, especially

when it comes to stored grains [23]. On the other hand, pest management currently depends mostly on chemical methods, but such approaches must be at least improved by adopting more sustainable and eco-friendly treatments for raw and processed commodities [24]. Herein, we analyze the various factors routing the efficacy of DEs against stored product pests and their real-world use, even in combination with fungal and plant-borne pesticides.

### *5.1. Biotic and Abiotic Factors That Influence the Efficacy of DEs*

Given their high absorptive power, the efficacy of DEs is highly determined by the levels of relative humidity (R.H.)/moisture content (m.c.). Hence, in humid conditions, some types of DEs may not be as effective as in dry conditions. For instance, Vayias and Athanassiou [25] tested larvae of the confused flour beetle, *Tribolium confusum* Jacquelin du Val (Coleoptera: Tenebrionidae) for their susceptibility to DEs, and they found that the efficacy of commercial DEs was reduced as the R.H. level rose from 55% to 65%. This is particularly important for grain protection, as R.H. levels between 55% and 75% correspond with an equivalent of 10.5% to 14% m.c., which are realistic ranges for long-term storage [26,27]. However, there are studies where the efficacy of DEs was not much affected by the increased R.H., suggesting that certain DE types do not interact much with moisture. A slurry formulation of DE, i.e., a mixture of DEs and water, may not be as effective as dust (powder) formulations [28]. However, a slurry formulation can be more practical in terms of direct application in the commodity with the same technology as traditional grain protectants [28,29].

The temperature might act indirectly on the efficacy of DEs, since at a higher temperature, the water loss occurs faster. In addition, insect mobility is increased at elevated temperatures, causing an increase in the contact with the DE particles. Athanassiou et al. [20] tested a commercially available DE on wheat for the control of adults of *T. confusum* and the rice weevil, *Sitophilus oryzae* (L.) (Coleoptera: Curculionidae), and noticed that there was a positive correlation between the mortality rates and the temperature. Indeed, by increasing the temperature by 10 ◦C, Athanassiou et al. [20] reported that the mortality rates were raised from approximately 45% at 22 ◦C to 100% at 32 ◦C. Other studies show similar results for a wide range of species [2,25,30,31], but some reports show that the increase in temperature decreases mortality [30,32]. The type of commodity on which DEs are applied is another critical aspect that should be considered. In the case of stored grain protection, not all grains are equal in terms of their response to DEs, suggesting that there are specific interactions with the external parts of the grains mass that may partially inactivate the DE particles. In a series of studies [13,14,19] it was shown that DEs are less effective on maize than on small grains, such as wheat, rice, and barley. Kavallieratos et al. [13] used sieves to remove two different DEs from eight grains, and the percentage of DEs removed was always higher on maize and minimal on wheat or barley. In addition, DE adherence was much lower in peeled barley than in non-peeled barley, which is a clear indication that the shape of the external kernel part is critical in maintaining the DE particles [13]. Still, these adherence differences among the different grains did not correlate with adult mortality in the lesser grain borer, *Rhyzopertha dominica* (F.) (Coleoptera: Bostrychidae) [13,14].

Different target species have different levels of susceptibility to DEs. It is generally expected that soft-bodied insects are more vulnerable to DEs, as their cuticula can be easily damaged, causing rapid desiccation [29]. However, this is not always true. For instance, stored product mites, such as Astigmata, are extremely vulnerable to DEs, which is considered as a direct consequence of their sensitivity to water loss and their thin cuticles [29,33]. Nevertheless, another category of soft-bodied stored product pests, psocids (Psocoptera), are extremely tolerant to DEs [34]. Psocids have a certain mechanism that can moderate water loss and absorb moisture from the air to compensate losses [35,36]. Larvae are considered more susceptible to DEs than adults [29]. For instance, Vayias and Athanassiou [25] found that *T. confusum* larvae were more susceptible to DE than adults, with early-stage larvae being the most vulnerable larval instar. However, this is not true for the yellow mealworm, *Tenebrio molitor* L. (Coleoptera: Tenebrionidae), where adults are

susceptible to DEs, but larvae remain unaffected due to the occurrence of a mechanism that moderates water loss [37]. The adults of the red flour beetle, *Tribolium castaneum* (Herbst) (Coleoptera: Tenebrionidae) and *T. confusum*, are being considered as the least susceptible beetle species to DEs, with the latter slightly more tolerant [19,25,29,38,39]. On the other hand, adults of the rusty grain beetle, *Cryptolestes ferrugineus* (Stephens) (Coleoptera: Laemophloeidae), are very susceptible to DEs, as they are flat-bodied, and water loss can rapidly occur [1,9,30]. Still, there are dissimilar and not directly comparable results for different species of stored products [15,29,30], but some general conclusions can be drawn based on the above observations. Apart from body size, shape, and characteristics, insect mobility is a critical parameter, as slow-moving insects may have a lower DE particle uptake. This is considered a key feature for the reduced susceptibility of *R. dominica* to DEs [1,30], although some reports show that this species is particularly susceptible to different DEs [13,14].

Some additional parameters that influence the efficacy of DEs have to do with their physicochemical characteristics. For instance, it has been shown that particle size is an important parameter, and the smaller the particles, the highest the DE efficacy against insects [1,8,9,11]. Vayias et al. [9] have shown that DEs with particles that were smaller than 45 μm were more effective than DEs with larger particles against *R. dominica*, *S. oryzae,* and *C. ferrugineus*. Nonetheless, Baliota and Athanassiou [11] have shown that it is the particle shape, rather than the size, that had a certain effect on the insecticidal value of DEs, and that smaller particles do not necessarily mean higher efficacy. Moreover, very small particles may not be desirable for safety issues [2].

Several physicochemical characteristics can be further utilized toward the prediction of the expected insecticidal value of DEs. Koruni´c [10] summarized these characteristics in standardized testing, which can be carried out for rapid screening of DE samples, without the need to conduct bioassays with insects, which is a time-consuming procedure. The silicon dioxide content and pH are important factors, while clay and other impurities are not desirable [10]. Even more important parameters are the tapped density, the bulk density reduction, and the adherence to grain kernels [1,10]. Diatom species, origin, and other characteristics may be less important [1,2,9,11,15,40].

### *5.2. Combinations with Contact Synthetic Insecticides*

One of the possible solutions to the implications caused by the high doses of DEs is the combination of DEs with other substances thanks to the adsorptive nature of the DE particles. Indeed, the utilization of DEs as a carrier is a promising solution not only for the application of insecticides in reduced concentrations but also to combine at least two different modes of action, i.e., desiccation through the inert dusts and an additional action depending on the type of chemical (e.g., neurotoxic, etc.). Several studies have indicated a significant potential and even synergism of combinations of commercial DE formulations with residual insecticides. Wakil et al. [41] reported high mortality rates of *R. dominica* in wheat, rice, and maize treated with a combination of thiamethoxam and a commercial DE formulation, SilicoSec® (Biofa GmbH, Munsingen, Germany), in relatively low doses (0.25, 0.5, and 0.75 ppm for thiamethoxam and 100 ppm for SilicoSec®). The combination of 150 ppm of Protect-It® (Hedley Technologies Inc., Mississauga, ON, Canada) with 1.25, 2.5, or 5.0 ppm of imidacloprid resulted in higher mortality rates of different stored product insects than applications of these insecticides alone at almost all exposure intervals and commodities tested [42]. Ceruti and Lazzari [43] used 500 and 1000 ppm of Keepdry® (Irrigação Dias Cruz ME, Brazil) in combination with 0.5 or 1.0 g a.i./t of deltamethrin powder, which may represent an efficient control measure against the maize weevil *Sitophilus zeamais* Motschulsky (Coleoptera: Curculionidae) in stored corn, highlighting the potentials of having reduced residues of deltamethrin, as compared with using this active ingredient alone. Arthur [44] stated that an insecticide formulation (F2) containing 0.03% deltamethrin, 0.37% piperonyl butoxide, 0.95% chlorpyriphos-methyl, 10% mineral oil, and 88% Protect-It® was extremely effective in wheat, maize, and paddy

rice at the rate of 100 ppm against *S. oryzae*, *S. zeamais*, *R. dominica* and *T. castaneum*. Awais et al. [45,46] tested three different doses of the DE formulation Concern (Wood StreamTM Corporation, Lititz, PA, USA) combined with the Insect Growth Regulators (IGRs) lufenuron and tebufenozide against *T. castaneum* and the khapra beetle, *Trogoderma granarium* Everts (Coleoptera: Dermestidae) respectively, with the overall conclusions to specify that the combined use of DEs and IGRs is highly operative and beneficial for stored product insect control. A combination of the IGR S-methoprene and Protect-It® could also be a promising mixture as reported by Arthur [47]. In that study, the mixture had an additive effect and reduced the concentrations of both components required to suppress the progeny of *R. dominica* compared to the application of each insecticide alone [47]. In addition, SilicoSec® (25 ppm) and beta-cyfluthrin (0.125 or 0.25 ppm) acted synergistically for the control of *T. castaneum* and, especially, *S. oryzae* [48]. The long-term protection of a given insecticide is one of the key elements in stored-grain pest management, aiming to prevent new infestations and control the reproduction of the already existing individuals. Mixtures with DEs have the potential to enhance the residual efficacy of an insecticide. Wakil et al. [49] reported an increased mortality of adults of *R. dominica* over 9 months of wheat storage with applications of 200 ppm of SilicoSec® and 0.5 ppm thiamethoxam in comparison with the residual efficacy of thiamethoxam alone, which was decreased significantly 2 months after its application. Koruni´c et al. [50] applied a formulation containing a low quantity of DE and small amounts of deltamethrin and reported a high residual efficacy against *S. oryzae*, *R. dominica*, and *T. castaneum* even 12 months after the treatment. Wakil and Schmitt [51] also found that applications with 150 ppm of DEBBM (DE + bitterbarkomycin) plus 5.0 ppm imidacloprid were more effective than single insecticidal treatments for a period of five months, against all tested species on stored wheat.

### *5.3. Combination with Fungal Agents*

Recently, extensive research focused on the adoption of entomopathogenic fungus species as an alternative approach to control insect pests of stored grain [52–57]. Fungal species such as *Beauveria bassiana* (Balsamo) Vuillemin (Ascomycota: Hypocreales), which is probably the most examined entomopathogenic fungus for stored product insects [58–64] has a complex interaction with cuticular lipids [65]. Results exalted the suitability of fungi as stored-product protectants but also pointed out their need for peculiar humid conditions to achieve satisfactory conidial adherence, germination, and penetration through the cuticle [66–68]. Increased humidity in stored commodities should be avoided [29], and hence, the fungal strains should be effective at drier conditions. Since DEs best perform under low humidity levels [2,25], the combination of fungi with DEs is very promising. The synergistic effect between DEs and entomopathogenic fungi expands the area for fungal spore penetration, increasing insect mycosis [40,62,69–73]. In addition, Batta [71] reported that the utilization of two different formulations of DE dusts, i.e., The Fossil Shield 90.0® (The Fossil Shield Co., Eiterfeld, Germany) and SilicoSec® (Agrinova GmbH, Obrigheim/Muhlheim, Germany), had a negligible effect on the viability of conidia of two fungal species. Dal Bello et al. [74] indicated the DE–fungal combinations to overcome some of the constraints in the use of fungi as biocontrol agents.

Applications of mixtures with these two ecologically compatible agents is a very appealing approach to IPM and can grant a more consistent management of multiple pest species under a wider range of environmental conditions.

The study of Athanassiou and Steenberg [70] demonstrated the potentials of these two agents together. The authors tested the insecticidal effect of *B. bassiana* combined with relatively low doses of Insecto® (Insecto Natural Products Inc., Costa Mesa, CA, USA), SilicoSec®, and PyriSec® (Biofa Gmbh, Germany), reporting a high level of control against the granary weevil *Sitophilus granarius* (L.) (Coleoptera: Curculionidae) under a broad range of temperatures and relative humidity levels [70]. In another published work by Wakil et al. [73], the application of 15 and 30 ppm of DEBBM combined with three doses of *B. bassiana* considerably increased adult mortality of *R. dominica*, especially at increasing

temperatures and longer exposure intervals compared with DEBBM and *B. bassiana* alone. The synergistic interaction between Protect-It® and *B. bassiana* against several major storedproduct insect species was also proved in laboratory bioassays [65,75]. Shafighi et al. [76] mentioned the high "speed of kill" of the combination of low doses of SilicoSec® when combined with entomopathogenic fungi against *T. castaneum.* Rizwan et al. [77] reported that the combination of the commercial DE formulation Diafil 610 (Celite Corporation, Lompoc, CA, USA) with *B. bassiana* had a suppressive effect on progeny (F1) production of the same beetle species. In field trials conducted on small farms, the treatment with mixtures of DE and *B. bassiana* outperformed the analogous combinations with imidacloprid after six months of storage [51].

*Metarhizium anisopliae* (Metschnikoff) Sorokin (Deuteromycotina: Hyphomycetes) and *Paecilomyces fumosoroseus* (=*Isaria fumosorosea*) (Wise) Brown & Smith (Ascomycota: Hypocreales) have also become a test subject for their insecticidal efficacy when combined with DEs, with reports to be in accordance with their potentials as control agents against several insect species, providing also long-term protection when applied in a variety of stored grains [40,61,76,78,79]. The virulence of *P. fumosorosea* integrated with DEBBM was shown to be an effective control measure for *R. dominica* in stored wheat [80]. *Nomuraea rileyi* (Farl.) Samson (Ascomycota: Hypocreales) and *Lecanicillium lecanii* (Zimm.) Zare & W.Gams (Ascomycota: Hypocreales) along with natural or modified DE formulations have been reported to show insecticidal, repellent, and ovicidal effects against *Bruchidius incarnatus* (Boheman) (Coleoptera: Chrysomelidae) and *R. dominica* under a variety of temperature and relative humidity conditions [81].

### *5.4. Combination with Botanicals*

Plant extracts, essential oils, and other plant-based products are all ingredients with the potential to control stored-product insects [82,83]. However, their utilization is sometimes challenging due to their instability and high recommended doses. Combining them with DEs may enhance their properties, pursuing better insecticidal performances at lower doses and under a wide range of conditions. Several studies have been conducted toward this direction, using compounds from several sources. Bitterbarkomycin (BBM), a plant extract from the roots of *Celastrus angulatus* Max (Celastraceae), is known for its strong insecticidal and antifeedant activity against several insect species; low doses of DEBBM led to high mortality rates of *S. oryzae, S. zeamais*, *T. castaneum, R. dominica,* and *C. ferrugineus* in stored wheat [28,34]. Two DE formulations enhanced with abamectin, a macrocyclic lactone produced either directly by the actinomycete *Streptomyces avermitilis* or generated through semisynthetic modifications [84], were found to have high insecticidal properties against stored-product insects at rates as low as 75–125 ppm [28].

Constraints of the use of essential oils, such as their poor penetration, strong odor, lack of persistence, and high concentration requirements could be reduced if combined with DEs. Yang et al. [85] tested a combination of essential oil derived from *Allium sativum* L. (Amaryllidaceae) with 250 ppm of a DE formulation, reporting a strong synergistic effect and high initial efficacy against *S. oryzae*. Ziaee et al. [86] examined the synergistic/antagonistic interaction between *Carum copticum* (L.) (Apiaceae) essential oil with natural DE formulations of Iranian deposits against *T. confusum* and *S. granarius*, reporting the potentials of the combination for use in IPM programs. The same authors also stated that the essential oil increased the DE efficacy by increasing insect's locomotion activity through the particles and, at the same time, DEs reduced the oil concentration for the satisfactory protection of stored products. A new insecticide formulation using Celatom MN 23 (Celatom Diatomaceous Earth Functional Additives Technical Data Sheet, EP Minerals, Reno, NV, USA) enhanced with essential oil extracted from *Anethum graveolens* L. (Apiaceae) has been also examined by Koruni´c and Fields [87] and found to be effective in controlling four stored-product beetle species at lower doses and with far fewer negative effects on bulk density than using the DE alone. On the contrary, Campolo et al. [88] reported an antagonistic effect of *Citrus sinensis* (L.) Osbeck (Rutaceae) peel essential oil

when admixed with the DE formulation Protector (Intrachem Bio, Grassobbio, Lombardy, Italy). Paponja et al. [89] developed an enhanced DE formulation admixing SilicoSec® with several botanicals (essential oil lavender, corn oil, and bay leaves dust) and silica gel, reporting higher mortality of all three insect species tested. Successful formulations of DEs and botanicals for the control of storage pests may be expected soon, but further testing is required to determine the duration of efficacy, cost of formulations, testing for their effect on non-target organisms, human safety, and effects on end-use quality.

### **6. DEs and Their Application in Urban, Agricultural, and Medical Environments**

In the following paragraphs, we reviewed the studies that have investigated the efficacy of DEs against urban, medical, and agricultural pests [90–92]. Against these important pest groups, the insecticidal activity of DEs has been examined both when applied alone or in combination with entomopathogenic fungi or botanicals [42,93], following the same approach shown in the above-reported paragraphs dealing with stored product pest control. As a general trend, it has been noted that the biological activity of DEs increased when combined with entomopathogenic fungi [42,94].

### *6.1. DEs to Control Urban Pests*

Insects and mites have successfully adapted over the years to the urban environment thanks to their ability to utilize food resources and harborages with humans [91]. These arthropod species can also transmit pathogenic organisms to food, as well as damages to house structures [91,95–97]. The control of arthropod vectors and pests, including urban ones, is challenging because of their strong reproductive ability, adaptability, and growing resistance to insecticides [98]. In addition, the adoption of insecticides in indoor areas is hazardous for human health [91]. Recently, several studies investigated the adoption of DE as an alternative to insecticides, highlighting their efficacy on different urban pests through different application scenarios [91,96,99–103] (Table 1).

The efficacy of DEs has been widely investigated on cockroaches, which are a worldwide public health pest that causes water and food contamination through transmitting pathogens mechanically, such as different forms of gastroenteritis [91,96]. A study compared the mortality of adult males and nymphs of the German cockroach, *Blattella germanica* L. (Blattodea: Ectiobidae), when DEs are applied as dry formulations or with the addition of water [96]. Mixing DE with water reduced the DE effectiveness, and the LC50 value was found to be 10 times lower if compared with dry DEs [96]. Similar results have been also found in stored product pests treated with dry DEs or with DEs formulated in water [30]. Overall, the bioactivity of DEs is inversely proportional to the water content and relative humidity [1]. To overcome the limitations related to high relative humidity conditions, mixing DEs with highly hydrophobic silanes may be a solution [99]. As reported by Faulde et al. [99], when DEs are mixed with hydrophobic silanes, a complete control of *B. germanica* could be achieved under humid conditions (R.H. > 80%) within 11 days [99]. In this work, it has been reported that the highest mortality rate of *B. germanica* (100% after 110 h) was achieved with the commercial DE Fossil-Shield® 90.0 S White, whose hydrophobicity increased by 3% Aerosil® with 1,1,1-trimethyl-*N*-trimethyl silane [99]. The same modified DEs led to the complete eradication of American cockroach, *Periplaneta americana* L. (Blattodea: Blattidae), and the silverfish, *Lepisma saccharina* L. (Thysanura: Lepismatidae), within 10 days, but the complete population suppression was not achieved in the case of the oriental cockroach, *Blatta orientalis* L. (Blattodea: Blattidae) [100]. These results highlight that cockroach susceptibility to DEs not only varies according to its formulations and their modifications, but it is also species-dependent [99,100]. Overall, hydrophobized DEs are more effective on certain cockroach species because of the higher absorption capacity of their cuticular waxes and the subsequent death by desiccation [99].

Thanks to their properties, DEs may act as physical barriers for arthropod pest intrusions and can be used to forecast the occurrence of subterranean termites that threaten housing construction and forest trees [101]. A study conducted by Gao et al. [101] showed that *Reticulitermes chinensis* Snyder (Rhinotermitidae: Blattodea) adult workers were not able to penetrate a 3 mm layer of dry DEs, suppressing their tunneling behavior, and died as a consequence of their movement. As reported by Ahmed et al. [103], mixing the soil with biofertilizers and DE increased the mortality and reduced the gallery length of another subterranean termite species, *Coptotermes heimi* (Wasmann) (Rhinotermitidae: Blattodea). On the other hand, DEs cannot be used as a barrier to prevent penetration of the soil surface by *Coptotermes formosanus* Shiraki (Rhinotermitidae: Blattodea), who was fully able to penetrate a DEs layer in laboratory bioassays [104]. Interestingly, although highly effective for the control of subterranean termites and cockroaches, DEs do not seem to be the most efficient inert dusts to control the pharaoh ant, *Monomorium pharaonis* (L.) (Hymenoptera: Formicidae)—a notorious domestic pest, for which the adoption of chemical-based insecticides is not recommended, particularly when ants infest crowded buildings such as hospitals [105]. Van Den Noortgate et al. [106] highlighted that the efficacy of DEs was lower if compared with various porous powders. For instance, zeolites ordered mesoporous silica material, and carbon black performed better than the DE benchmark material, especially the activated carbon powder (ACP) whose survival median time was almost four times shorter than that of the DEs (LTDE: 95 min; LTACP: 25 min) [106].

**Table 1.** Local and commercial

diatomaceous

 earths (DEs) evaluated against immature and adult stages of arthropods of urban interest. In addition to the mortality

calculated at 24 h; (\*\*) LC50 calculated at 48 h; (\*\*\*) LC50 calculated at

 72 h.

(\*) LC50

#### *Molecules* **2021**, *26*, 7487

**Table 1.** *Cont.*

### *6.2. DEs to Control Arthropod Pests and Vectors of Medical and Veterinary Importance*

In recent years, several studies have investigated the use of DEs for managing pest and vector species of medical and veterinary importance [90,108–112] (Table 2). Many arthropods can play a pivotal role in the transmission of pathogens and eventually cause diseases in a wide range of vertebrates, including humans, livestock, pets, and wildlife [97]. Herein, we focus on some studies carried out to prove the efficacy of DEs alone or in combination with entomopathogenic fungi against arthropod pests and vectors of public health importance [90,108–112]. Many studies have investigated the adoption of DE to control bed bugs, *Cimex lectularius* L. (Hemiptera: Cimicidae), which are obligatory hematophagous insects that feed commonly on humans [95,97]. Apart from blood sucking, bed bugs are responsible for a range of emotional problems, anxiety, and sleeplessness [90]. As for other urban pests, there is no longer an absolute method to control/eradicate bed bugs, and the management relies either on the use of chemicals, such as pyrethroids, or on non-chemical tools, such as steam [95]. Given their low impact on mammals, DEs recently seem to be more of an option in bed bug control [90,113]. Several commercial DEs are known to be effective in the control of bed bugs, such as Bed Bug Kill [113], DE 51 [108], Mother Earth® D [90,108,114], Alpine® [90,114], Pro-Active®, DX13TM-dust, and aerosol [90]. As reported by Akhtar and Isman [90], who evaluated the efficacy of several commercial DEs, their activity depends on the content of amorphous silicon dioxide and the dimension of the particles, resulting in the higher effectiveness of one DE to another. The efficacy of DEs may be increased by the addition of a dispersal agent, such as a bed bug alarm pheromone, which enhances bed bug crawling activity, increases bed bug locomotor activity, and thereby causes a higher contact with DE [115]. In addition, DE can be horizontally transferred from a treated bed bug to an untreated one [108]. This phenomenon is typical of gregarious insects and can facilitate the spread of DEs toward spaces that are hard to reach, contributing to the management of public health pests [108].

DEs have a recent use in the control of kissing bugs, *Triatoma infestans*(Klug) (Hemiptera: Reduviidae), which is a vector of *Trypanosoma cruzi*, causing Chagas' disease [97]. To date, several studies emphasize the efficacy of entomopathogenic fungi to control this species [116,117], but low humidity seems to be a limiting factor for fungal infection [117]. In addition, entomopathogenic fungi do not induce quick and high mortality as synthetic insecticides [117]. For these reasons, combining entomopathogenic fungi with oils and DEs may be a solution. The combination of DE + oil eventually enhances the adhesion and spread of particles (DE and conidia) on the lipophilic cuticle. In addition, the fungal development may be favored by the higher moisture provided by the abrasive action of DE and the subsequent trapping of moisture, and lastly, the oil serves as a nutrient source for the fungi [117]. The efficacy of the mixture is already well established, especially against stored-product pests [116–120], as detailed above. In laboratory bioassay, the mixture of *B. bassiana* and a commercial DE caused high mortality rates in all nymph instars and adults of *T. infestans*, ranging from 82% to 100% [120], but the same mixture elicited only 52.4% of *T. infestans* death in a field test in Northern Argentina [116]. Another study highlighted that the efficacy of the commercial DE KeepDry® toward *T. infestans* nymphs is highly increased when combined with vegetable oil and *M. anisopliae* (IP 46)*,* even at a R.H. level of 75% [117]. The same combination was also effective in the control of the yellow fever mosquito, *Aedes aegypti* (L.) (Diptera: Culicidae), a vector or dengue, chikungunya, and Zika virus in the tropical and subtropical regions [109]. Overall, the combination of DE, entomopathogenic fungi, and a mineral/vegetable oil may represent a promising tool for the development of effective management strategies against *T. infestans* and *A. aegypti*. The combination of *M. anisopliae* (IP 119) with the commercial DE KeepDry® has been also successfully evaluated toward the cattle tick, *Rhipicephalus microplus* (Canestrini) (Ixodida: Ixodidae) [121]. The microsclerotia of *M. anisopliae* were incorporated in pellets containing inorganic materials, such as vermiculite, DE, and SiO2 [121]. Overall, the pellets formulated with *M. anisopliae* microsclerotia effectively suppressed *R. microplus* in laboratory tests, demonstrating a promising pellet formulation for targeting the non-parasitic stage

of this tick on the pasture [121]. Pellets can represent a possible upgrade of conventional granules thanks to their properties: a higher dose uniformity, higher mechanical resistance, narrower particle size distribution, and higher capacity of active incorporation [121]. The combination of fungal spores of *B. bassiana* and commercial DE resulted in significantly increased efficacy against blood-sucking poultry red mite *Dermanyssus gallinae* (De Geer) (Mesostigmata: Dermanyssidae) [94], which is a worldwide hematophagous ectoparasite in poultry farming [122] that is also responsible for the transmission of avian influenza viruses and *Salmonella enterica* ssp. *enterica* (S.) ser. Enteritidis and other important enterobacteria [123,124]. Although DEs were highly effective in the control of *D. gallinae*, Kilping and Steenberg [93] highlighted that four commercial DEs (SilicoSec®, Diamol®, Protect-It®, and Fossil Shield 90.0®) elicited avoidance behavior and repellence of the mites on the treated substrate. The authors noticed that the more effective the DE is, the greater the repellent activity. Consequently, the repellent activity had an impact on the efficacy of the inert dusts since mites will avoid treated surfaces. Furthermore, the dry conidia of *B. bassiana* also elicited a repellent response to poultry red mites both when applied on its own and when admixed with a low dose of the commercial DE Diamol [93].

In field bioassays, a liquid formulation of DEs elicited high mortality rates of the poultry red mite population [108]. A gradual reduction of the mite population (34% on day 7 to 53.5% on day 14; over 90% on days 21–28) was observed when the application of DE was combined with the mechanical cleaning [108]. The cleaning physically removes the mites and might also help the liquid formulation to stick better to surfaces than when covered with dust [108]. Overall, the adoption of liquid DEs is advisable mainly because product wastes are reduced, and an easier and safer application is provided [125]. Interestingly, DEs were found not to be as efficient as other products, such as kaolin and sulfur, to control the northern fowl mite, *Ornithonyssus sylvarium* (Canestrini & Fanzago) (Mesostigmata: Macronyssidae), which is another threat for hens [125,126]. Testing out the liquid formulations of DE, Martin and Mullens [126] noticed that a significant reduction of the northern fowl mite population occurred when DE was applied for two consecutive weeks, and the highest reduction of mite population was achieved with high concentrations of sulfur (≥5.3%). Although DE effectiveness was found to be lower, in general, their use in dust boxes seems to enhance bird natural dustbathing behaviors, which translates into an increase in animal welfare and a reduction in the use of pesticides [127].

**Table 2.** Local and commercial

diatomaceous

 earths (DEs) evaluated against immature and adult stages of arthropods of medical and veterinary relevance. In addition

### *6.3. DEs to Control Crop Pests*

Although DEs are not widely used by farmers to control arthropod pests, several studies reported their efficacy to control pests of agricultural interest such as soft-bodied insects, ants, and moths [42,131–134] (Table 3). It is generally advisable to use DE as an adjuvant rather than an active ingredient alone, considering the wide range of environmental conditions during the application [135]. Indeed, studies about DE effectiveness toward crop-damaging arthropods mainly focused on their use in combination with other products such as essential oils or entomopathogenic fungi [42,92,132,136,137]. For instance, a study conducted on the green peach aphid, *Myzus persicae* (Sulzer) (Hemiptera: Aphididae), reported that a DE + the essential oil of *Thymus capitatus* (L.) (Lamiaceae) caused mortality higher than 95% through in vitro bioassays [136]. The adoption of DE in solid form or suspended in water with neem oil extracted from *Azadirachta indica* A. Juss. (Meliaceae), also protects maize and tomato plants, causing a decrease in the number of larvae of the southern armyworm, *Spodoptera eridania* Stoll (Lepidoptera: Noctuidae) and fall armyworm, *Spodoptera frugiperda* Smith & Abbot (Lepidoptera: Noctuidae) [132]. In addition, Fossil Shield®, already adopted to control the red poultry mite [130], was proved to increase the efficacy of neem oil extract against cowpea aphid, *Aphis craccivora* Koch (Hemiptera: Aphididae) on the yardlong beans, *Vigna unguiculata* subsp. *sesquipedalis* L. (Fabaceae) [138]. Evaluating side effects on the aphid predator *Menochilus sexmaculatus* F. (Coleoptera: Coccinellidae), the toxicity of DE + neem oil was lower than that of the recommended chemical insecticide [138]. The same mixture, i.e., Fossil Shield® + neem oil, was evaluated against *M. persicae* (Sulzer) on globe artichoke *Cynara cardunculus* var. *scolymus* (L.) (Asteraceae) with promising results. The aphid population was reduced by 97% the day after the second spray [139]. Interestingly, the combination had a low impact on *M. persicae* common predators *Chrysoperla carnea* (Stephens) (Neuroptera: Chrysopidae), *Orius* spp. (Hemiptera: Antochoridae), *Coccinella* spp. (Coleoptera: Coccinellidae), and *Scymnus* spp. (Coleoptera: Coccinellidae), while *C. carnea* and *Orius* spp. were found to be more susceptible than the two coccinellids to that combination [139]. These results substantiated the findings by Ulrichs et al. [138], outlining the lower susceptibility of coccinellid predators to neem oil and DEs. In contact bioassays, DEs were also low risk toward predators of the spider mite, *Tetranychus urticae* Koch (Trombidiformes: Tetranychidae), such as *Phytoseiulus persimilis* Athias-Henriot (Mesostigmata: Phytoseiidae), *Neoseiulus fallacis* Garman (Mesostigmata: Phytoseiidae), and *Stethorus punctillum* (Weise) (Coleoptera: Coccinellidae), when DEs were tested in contact bioassay [140].

The synergistic interaction between DE and entomopathogenic fungi has been also evaluated toward an extremely wide range of agricultural pests [42,92]. For instance, a study on the western flower thrips, *Franklinella occidentalis* Pergande (Thysanoptera: Thripidae) highlighted that combining the entomopathogenic fungi, *Metarhizium flavoviride* (Gams and Rozsypal) (syn. *Metarhizium anisopliae* var. Acridum, pro parte) (Hyphomycetes: Deuteromycotina), with a commercial DE resulted in higher mortality of the thrips compared to the efficacies of each compound alone [92]. Synergistic interaction between DE and entomopathogenic fungi has been also reported for *T. infestans* [117], the silverleaf whitefly *Bemisia tabaci* (Gennadius) (Hemiptera: Aleyrodidae) [137], the cotton aphid, *Aphis gossypii* Glover (Hemiptera: Aphididae) [42], the indianmeal moth, *Plodia interpunctella* (Hübner), the almond moth, *Ephestia cautella* (Walker), and the Mediterranean flour moth, *Ephestia kuehniella* Zeller (Lepidoptera: Pyralidae) [141]. The insecticidal activity of DEs + fungi was evaluated toward the fire ant, *Solenopsis invicta* Buren (Hymenoptera: Formicidae) [131], which is responsible for a decline in production through direct predation on different plant parts (e.g. roots, fruits, flowers, stems), with reduction estimated to 15 to 33% in soybean, 20 to 35% in potato crops, or 50% in eggplant [141]. The effects of the combination of DE + *B. bassiana* toward healthy fire ants do not greatly increase the effect of *B. bassiana* alone, but testing DE alone in ants infected by *Thelohania solenopsae* (a common intracellular pathogen of fire ants) led to high insecticidal activity of DE, suggesting the synergistic interaction between *T. solenopsae* and DE [131].

### **7. DEs in Real-Scale Pest Management**

The gradual withdrawal of active ingredients from the chemical-based pest management and the necessity to place insect control under the principles of IPM [142] led to the "re-evaluation" of inert dusts as a novel, effective, and sustainable management of arthropod pests and vectors all over the world. This is the main reason behind the recent popularity of DE formulations in pest management strategies [2], although commercial products ("Naaki" in Germany and "Neosyl" in England) have been available for stored-product protection since the 1930s [143]. In the following years, various enhanced or modified DE formulations have been created and evaluated, under extensive laboratory research, and some of them have reached the market as commercial formulations.

However, what are their potentials in praxis? Since several reports have questioned the compatibility of DEs with modern pest management programs, in this chapter, factors in terms of their potential impact in real scale applications (particularly as stored product protectants), including their utilization to reduce the standard application doses of residual insecticides and the role that DEs could have in resistance management will be discussed. Additional data of other relatively promising substances will be evaluated, aiming to expand the list of viable alternatives to hazardous chemicals that can be used as protectants in the food industry and beyond.

Virtually all the "classic" papers on the insecticidal efficacy of DEs examine formulations at a laboratory scale, with scarce data to be available in the literature regarding applications in large-scale scenarios. DEs have been approved for arthropod pest control, and commercial formulations are currently available as effective grain protectants. However, the grain industry is reluctant to use them for direct mixture with grains, as DE particles can adversely affect some physical and mechanical properties of the treated grain, obstructing their wider use as grain protectants. Indeed, for a satisfactory level of efficacy, the commercially available DE formulations should be applied at doses between 400 and 1000 ppm, but even in this case, adverse effects cannot be avoided [2,30]. However, several reports suggest that using DEs at concentrations lower than those indicated on the label could cause a sufficient reduction in the bulk density (test weight) of the grain [1,10,12,15]. Bulk density refers to a grading factor extensively used by the industry to determine the grain price, and its reduction through DE applications is of major importance. Koruni´c [10] examined 42 DE dusts from around the world and found significant correlations between DE insecticidal efficacy and adherence to kernels with bulk density reduction. In a later report, Koruni´c et al. [144] stated that the insecticidal efficacy and bulk density reduction could be linked by the capacity of a given DE to adhere to surfaces, which, eventually, is positively correlated with the insecticidal value of a given DE. Furthermore, when the DE particles are attached to the surface of the kernels, the spaces among the kernels increase, affecting their flowability, especially in mechanized handling systems. Jackson and Webley [145] found that when 0.5 g/kg of DE was applied on maize, the flow rate was reduced by about 39%. Apart from the grain industry, the milling industry has also expressed concerns about using DE formulations, as the presence of DE particles in the grain can damage the milling machinery through abrasive action. To overcome these limitations, new ways of DE applications have been proposed, intending to make the most of their advantages.

 interest. In addition to the

**Table 3.** Local and commercial

diatomaceous

 earths (DEs) examined against immature and adult stages of arthropod pests of agricultural

### **8. DE Applications for Structural Treatments**

DEs leave no harmful residues in the surfaces applied and hence, applications could be carried out in food and processing facilities. Koruni´c et al. [12] reported that the treatment of hard wheat with either 50 or 300 ppm of Protect-It® had no significant effect on the milling, analytical, rheological, or baking quality, and these doses did not affect the properties for pasta production, while 100 to 900 ppm on barley showed no differences in malting quality characteristics. Desmarchelier and Dines [151] reported that treatments with Dryacide® did not affect flour quality, as determined by the volume of sponge cakes and the production of carbon dioxide by fermenting dough. Aldryhim [152] found no evidence of an adverse effect on wheat seed germination, wheat flour, and baking quality, using Dryacide®.

Another advantageous feature of DEs is their persistence and stability in a wide range of temperatures [153,154], as compared with contact insecticides [155,156]. Arthur [39] exposed adults of *T. castaneum* and *T. confusum* to filter papers containing 0.5 mg/cm2 of Protect-It® and reported a positive effect of temperature and exposure interval on insect mortality, along with a negative effect of humidity.

The utilization of DEs has been addressed as a good way to strengthen the effects of heat treatments, as the exposed insects are expected to die earlier due to increased desiccation. According to Fields et al. [157], the complete control of *T. confusum* in an oat mill could be achieved after treatment at 41 ◦C for 13–22 h, when DEs are combined with heat. In contrast, heat alone caused the same results after 32–38 h exposure at a sufficiently higher temperature, 47 ◦C [157]. Additional data by Dowdy [158] and Dowdy and Fields [159] indicated that DEs appear to be of value in areas where lethal temperatures cannot be reached during heat treatment applications. Moreover, even after the treatments combining heat with DEs, delayed mortality may occur after a while for the remaining insects due to the residual toxicity of DEs [159].

Laboratory studies have been conducted to evaluate the insecticidal efficacy of different DE formulations when applied directly in different types of surfaces, such as concrete, ceramic, plywood, plastic, metal, etc. [39,160–164]. In general, lower doses are required on some surfaces, such as metal and glass, compared with surfaces with a rougher construction, such as wood and concrete [160,163–165]. Collins and Cook [162] reported that 5 g/m2 of SilicoSec® was just as effective as 20 g/m2 to achieve mortalities above 86% of different stored product insect and mite species after one week of exposure to glass and plastic surfaces. This observation is in accordance with the reports of Cook [166], Mewis and Ulrichs [37], and Athanassiou et al. [15], indicating that there is a limitation in the amount of DE particles the insects can pick up. In general, insects seem to pick up DE particles more easily if the formulation is equally applied onto the surface (e.g., on a Petri dish) and not adhered on the grain kernels [15]. In the latter case, DE particles are also likely to lose effectiveness by lipid absorption from the external part of the kernel [2].

Cleaning and sanitation before DE structural treatments is a key element in pest management practices since the presence of food may increase insect survival rates [159,167]. Arthur [39] using 0.5 mg/cm2 of Protect-It® in plastic surface against *T. castaneum* and *T. confusum* emphasized the importance to eliminate the presence of food materials within the storage environment to maximize the effectiveness of the treatments. Dowdy [158] also addressed the impact of food in the effectiveness of treatments combining heat with some commercial DEs: Insecto®, Protect-It®, Concern®, and Natural Guard® (VPG Co-op Gardening Group, Inc., Bonham, TX, USA). Access to food significantly decreased insect mortality, providing an average between 21 and 88% of individuals fed and not fed respectively, 7 d after the treatment. Similar results have been published using other inert dust formulations, as food may provide water and nutrition that can lead to increased and prolonged insect survival, which may allow the continuance of the infestation for a certain period and the concomitant progeny production [168–170].

Although most laboratory studies tended to prefer dry DE applications over the use of slurry solutions [15,28,153,161,171], the reverse is probably more desirable in commercial practice. Slurries may be used easier in their application by the personnel, as there is a need to avoid exposure to the very dusty atmospheres created by dry-blown methods [153,165].

### **9. Other Relative Promising Substances**

A plethora of other inert dusts has been also tested for their toxicity against arthropod species, with special reference to stored product pests. In general, inert dusts/materials can be categorized according to their chemical composition or level of activity in four wide groups: (*a*) clays, sand, kaolin, paddy husk ash, wood, and volcanic ash, (*b*) katelsous (rock phosphate and ground sulfur), lime (calcium hydroxide), limestone (calcium carbonate), and salt (sodium chloride), (*c*) synthetic silica aerogels produced by drying aqueous solutions of sodium silicate and (*d*) dusts containing natural silica, including DEs [1,2,172,173]. Zeolites (alkali metal aluminum silicates) have been also included in this group by Subramanyam and Roesli [2], since these substances have similar physical properties with DEs. In addition, Golob [173] divided the DE formulations into two groups, addressing the modified DE formulations that contain over 98% silicon dioxide (compared to the 90% silicon dioxide of the natural dusts) as the fifth group of inert dusts.

Zeolites are among the most promising alternatives to DEs, and their potentials in food and agriculture are well described by Eroglu et al. [5]. Nevertheless, regarding stored product protection, there are disproportionally few data as compared with DEs, although the interest for zeolites in stored product protection has been increased [174–178]. Zeolites' particle size effect, adherence to kernels, and influence on the test weight of grains have been examined by Rumbos et al. [177], showing similar trends with DEs. The results of these studies encourage further research to evaluate the use of zeolites as grain protectants but also to surface treatments or "crack and crevice" applications. Attempts have been also made to evaluate the insecticidal efficacy of other inert dusts, to use them in modern pest management at the post-harvest stages of durable agricultural commodities [2]. Even some of the currently existing DEs cannot be considered as pure DE formulations, as they contain additional inert materials that have a certain insecticidal action and can be drastically modified to obtain increased efficacy [11].

### **10. Conclusions and Future Challenges**

The need to gradually withdraw from the chemical-based pesticide policies to more sustainable and ecological approaches is, at the present, one of the most challenging aspects of pest management. The current decrease in the registered pesticides will undoubtedly continue, increasing simultaneously the need to develop novel, effective but also ecologically compatible substances. On the other hand, the introduction of a new pesticide is a costly and long process, making the total overdrawn from the traditional protectants an unrealistic scenario. Thus, inert dusts such as DEs might have an important role to play in future pest management strategies, ensuring an abundant supply of safe and healthy food and feed. DEs hold great potential as carriers of common insecticides, minimizing the required application doses of the latter. In addition to synergistic effects, combined applications may also alleviate the negative effects of the substances and can be more compatible with the desired criteria for food safety and protection of human health and the environment. However, the introduction of other agents must be always appraised under the prism of the potential negative effects they may hold, such as for instance the adoption of entomopathogenic fungus agents [179]. Nevertheless, the application of insecticides and acaricides with a different mode of action may be a solution for the control of resistant arthropod populations, which is a hot topic in modern Integrated Pest Management [180–182]. Although there are plenty of data for their insecticidal and acaricidal properties, little progress has been made regarding the optimal processing of DE dusts used as insecticides and acaricides. Today, most commercial DE formulations are prepared through a basic process of quarrying, drying, and milling the mined heterogeneous rocks. This simplistic treatment leads eventually to formulations with great variability in their physicochemical characteristics, influencing simultaneously their insecticidal/acaricidal

properties but also some properties of the commodity itself. Therefore, more specific methods of processing must be found to standardize the production of dusts bearing the most desirable features for increased insecticidal efficacy.

Application methods and systems of DEs are also an issue of major importance, requiring additional investigation. Even with the current DE formulations, different application techniques, such as using slurries or treating only partial layers of the food, should be explored. Thus, research should be conducted under a range of food-handling establishments to design effective protocols for pest management but also to determine the effects of sanitation on the performance of DE dusts. Such real-scale applications may highlight the potential of DEs and explore ways of integrating DE applications within the total pest/vector management program in food industry, agricultural and urban settings.

The data from laboratory studies underline the insecticidal and acaricidal value of DEs under a wide range of arthropods. Further analysis must be conducted toward this direction not only to identify all the target species but also to investigate the overall outcome of DEs in non-target species. Indeed, by examining the current literature, we observed that non-target effects of DE have been evaluated only on a limited number of natural enemies of crop pests, with special reference to aphidophagous coccinellids, lacewings, anthochorids, and Phytoseiidae mites, showing limited consequences for these important biocontrol agents. In this promising scenario, further research should be devoted in understanding the potential non-target effects of DE-based formulations.

**Author Contributions:** Conceptualization, G.B. and C.G.A.; Literature collection and analysis, V.Z., G.V.B., G.B., A.C. and C.G.A.; writing—original draft preparation, V.Z. and G.V.B.; writing—review and editing, V.Z., G.V.B., G.B., A.C. and C.G.A.; visualization, G.B., A.C., C.G.A.; supervision, G.B., A.C., C.G.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** Valeria Zeni is partially funded by PRIMA iGUESSmed Project. This article has been co-financed by the European Regional Development Fund of the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship, and Innovation, under the call RESEARCH-CREATE-INNOVATE (project code: T2EΔK-03532).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** We are grateful to Carey Yuan for her kind assistance during the preparation of this Invited Review for *Molecules*.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Not applicable.

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