Chlorpyrifos (CPF) represents one of the most used organophosphorus pesticides. Many recent studies have investigated the effect of this toxicant in animal models, mainly rats and mice. Chronic exposure seems not only to affect the gut but also influence serum gonadotropins levels (follicle-stimulating hormone, luteinizing hormone and testosterone) and raise proinflammatory cytokines (such as IL-6, monocyte chemoattractant protein-1, and TNF-α) in rats, suggesting a potential role in the development of infertility and colitis [50]. Rats born from an exposed mother to oral CPF administration were smaller and lighter than non-exposed. Moreover, gut histological structures and the microbial community were altered; intestinal villi were shorter and thinner, and probiotic bacteria resulted as being lower in the ileum, caecum and colon [24]. It has been hypothesized that CPF could modify the gut microbiota composition in a diet-specific manner [17], even if authors observed differences only at the taxonomic level of genus and species but not at phylum [51]. Long-term exposure to CPF in mice could raise gut absorbency, reducing the gene expression of tight junction proteins such as occludin, claudin 1, and ZO-1, both in ileum and colon. It was observed that exposed mice gut microbiota showed an increment in Proteobacteria phylum and a decrement in Bacteroidetes phylum, suggesting that these phyla are mainly affected by CPF exposure [52]. Still, long-term exposure seems to amplify spontaneous vertical activity and female activity after acute stress in Wistar rats [53]. The host’s genetic profile can change the gut microbial response to CPF exposure. Guardia-Escote et al. orally exposed mice to CPF apoE3 and apoE4, observing differences between the two genotypes at different taxonomic levels; the phylum Verrucomicrobia resulted as highly represented only in apoE4 mice [54]. Subsequently to a daily dose of 1 mg/kg for 30 days in mice, besides altered gut microbiota, CPF also induced the alteration of urine metabolites, amino acids and short-chain fatty acids (SCFAs). As a result, the GM abundance of Firmicutes resulted as weakened, unlike Bacteroidetes, which resulted as higher [55]. Gut microbiota is supposed to be responsible for pesticide resistance [56]. A model organism silkworm, ‘Bombyx mori’, was fed with different antibiotics to induce gut dysbiosis and then exposed to CPF. It was observed that the antibiotic-fed silkworm larvae showed a significant amount of Firmicutes and Actinobacteria but a minor amount of Proteobacteria. Additionally, the antibiotic-treated silkworms seemed to be more vulnerable to CPF, supporting the hypothesis that the gut microbiota plays a crucial role in pesticide resistance [57]. Though, other authors did not demonstrate a shift in a mosquito gut microbial community, despite the pesticide-degrading microbiota resulting enriched [58]. In a Drosophila melanogaster model, Lactobacillus plantarum (Firmicutes) showed a greater capacity compared to Acetobacter indonesiensis (Proteobacteria), in the metabolization of CPF to the more effective metabolite chlorpyrifos oxon (CPO) [59].

Other authors highlighted CPF effects on oxidative stress and gut microbiota dysbiosis in zebrafish. They found an abundance of Proteobacteria with an alteration of α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria in CPF-treated zebrafishes compared to the control [60]. Still, through the use of an in vitro culture system, the gut microbiota of a marine benthic polychaete, ‘Nereis succinea’, showed fundamental metabolizing activity towards two representative organophosphates, CPF and malathion [61]. The impact of CPF on the gut microbiota has also been studied in vitro using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME^®) and Caco-2/TC7 cells of the intestinal mucosa. After 3.5 mg/day CPF exposure, Lactobacillus (Firmicutes) and Bifidobacterium (Actinobacteria) concentrations resulted as being strongly decreased [62]. A low-dose exposure (1 mg/day) after 15 and 30 days increased the Enterobacteria spp. (Proteobacteria), Bacteroides spp. (Bacteroidetes) and Clostridia (Firmicutes) counts but decreased the bifidobacterial (Actinobacteria) counts, reducing fermentative efficacy [63]. Similar results were obtained from other authors who aimed to assess the effect of CPF chronic low-dose exposure (1 mg for 30 days), both on SHIME and rats. They observed an increment in the entire bacterial flora, primarily caused by Enterococcus spp. (Firmicutes) and Bacteroides spp. (Bacteroidetes), but a reduction in bifidobacterial (Actinobacteria) and lactobacilli (Firmicutes) [64].

The effects of trichlorfon administered to common carp at different concentrations (0, 0.1, 0.5 and 1.0 mg/L) were evaluated by Chang et al. Gut histological changes resulted in a reduced size of intestinal villus and an altered expression of tight junction proteins, such as claudin-2, occludin and ZO-1. In addition, many antioxidant enzymes were decreased and proinflammatory cytokines upregulated after treatment. The most represented phyla were Fusobacteria, Proteobacteria and Bacteroidetes. Trichlorfon administration led to a reduction in Fusobacteria and an increment in Bacteroidetes [65]. Inversely, diethyl phosphate exposure did not show proinflammatory effects in exposed rats. Opportunistic pathogens were found significantly enriched, but IL-6 was reduced [66]. The effects of another organophosphorus pesticide, diazinon, were evaluated in crucian carp. Diazinon exposure increased the counts of Bacteroidetes, Fusobacteria and Firmicutes, but not the abundance of Proteobacteria. After 14 days of exposure, also Patescibacteria were found to be incremented [67]. Low monocrotophos exposure led to a depletion of the bacterial and fungal populations in the earthworm gut (Lampito mauritii). After 15 days of exposure, the bacteria and fungi showed a minor increment, but on day 30, the total counts were lower than those of the control earthworms [68]. It has been hypothesized that gut microbiota could modulate resistance to dichlorvos in a species of beetles, Callosobruchus maculatus [69]; a similar mechanism has been suggested to explain the resistance of some strains of Anopheles mosquitoes to organophosphate fenitrothion [70]. Phoxim exposure in silkworms showed immunosuppressive effects and increased bacterial abundance, particularly in two genera, such as Methylobacterium and Aurantimonadaceae (Proteobacteria) [71]. See Table A2.

Dichlorodiphenyldichloroethylene (DDE) is the most prevalent metabolite of dichlorodiphenyltrichloroethane (DDT), a common organochloride pesticide banned several decades ago. Among all the adverse effects of organochlorides, the GM is also a target of these chemicals. In mice exposed for 8 weeks to DDE, alterations of gut microbiota composition resulted in an increment in Bacteroidetes, and a reduction in Proteobacteria, Deferribacteres and Cyanobacteria [72]. Contrariwise, in rats exposed to DDE, gut dysbiosis resulted in a reduction in Bacteroidetes and Proteobacteria accompanied by increased Firmicutes and Tenericutes, with a substantial rise in the Firmicutes-to-Bacteroidetes ratio [73]. Other authors investigated the effects of both DDE and β-hexachlorocyclohexane (β-HCH), an active metabolite of hexachlorocyclohexane (HCH), on mice, observing changes in hepatic and bile acid metabolism besides the aforementioned effects on the microbiota composition [74]. HCH, being a persistent organic pollutant, can alter the microbial composition in the human colostrum and, consequently, the colonization of the infant gut [75]. These results were confirmed by another study that investigated whether environmental pollutants, including organochlorine, in breastmilk could impact the homeostasis of the infant gut microbiota at one month, revealing differences in the abundance of some strains of Firmicutes [76]. Zebrafish fed with dieldrin did not show histological modifications of the gastrointestinal tract or body weight changes after four months of exposure. However, Firmicutes resulted as less abundant [77].

Malachite green (MG) (an organic chloride salt used as a fungicide in the aquaculture industry) has been shown to influence not only gut microbiota but also immune system homeostasis in goldfish. The altered gut microbiota was linked to opportunistic bacteria overgrowth and thus to potential infections [78]. Other authors observed a diet-specific effect of endosulfan sulphate, a major metabolite of the organochlorine insecticide endosulfan, in mice. The abundance of most the represented phyla (such as Actinobacteria, Proteobacteria, Bacteroidetes and Firmicutes) was different in the mice fed with a high-fat diet from low-fat diet fed mice [79]. The GM’s role against insecticide resistance has been demonstrated in the German cockroach. Indeed, using antibiotic treatments, the susceptibility to indoxacarb insecticide was incremented. The main phyla composing microbiota were Proteobacteria, Bacteroidetes, Firmicutes and Fusobacteria, and via faecal transplant, indoxacarb-resistant could be transferred to susceptible strains [80]. See Table A3.

The toxicity of imidacloprid (IMI), a neonicotinoid insecticide, has received growing attention in the last years. In a Drosophila melanogaster model, IMI led to microbial dysregulation consisting of an increment in Lactobacillus spp. (Firmicutes) and Acetobacter spp. (Proteobacteria) [81]. Bees can be an indirect target of neonicotinoid insecticides such as IMI, thiacloprid, nitenpyram, amitraz and dimethoate. Several studies suggested that long-term exposure can lead to impairment of the gut microbial in bees with a reduced abundance of total bacterial flora, mainly Firmicutes [82,83,84,85,86]. In pregnant mice, nitenpyram also altered lipid metabolism and decreased the abundance of Proteobacteria, specifically of the Desulfovibrionaceae family [87], and IMI was shown to reduce liver weights and alter the histological structure of hepatic tissue, disrupting the gut microbial composition with consequently impaired barrier function [88]. Hong et al. evaluated the effects of IMI in crab, ‘Erocheir sinensis’. On the one hand, Bacteroidetes abundance was increased in a dose-dependent manner and reached almost three times as much as the control. On the other hand, the relative abundance of Proteobacteria diminished [89]. Onaru et al. described the effects of clothianidin on rats. The animals showed a loss of body weight and significant alterations in the gut microbiota, principally Firmicutes was strongly decreased, inversely Bacteroidetes was augmented and the dominance of Proteobacteria and Actinobacteria was inverted [90].

Among all pyrethroids, permethrin (PEM) is one of the most commonly used for pest control. In low-dose exposed rats, PEM decreased the amount of three microbial species belonging to the Bacteroidetes phylum (Bacteroides, Prevotella and Porphyromonas). Moreover, it showed antibacterial properties against beneficial bacteria such as Bifidobacterium (Actinobacteria) and Lactobacillus (Firmicutes) [91]. The widespread use of pyrethroids, such as alpha-cypermethrin, deltamethrin and PEM, in the ecosystem could modify the bacterial composition in mosquitoes, promoting insecticide-metabolizing microbiota, thus supporting insecticide resistance [92]. Further, methoprene, a biochemical insecticide, was capable of affecting the microbiota in larval mosquitoes, arising the abundances of Proteobacteria, and precisely the Gammaproteobacteria class and Enterobacteriaceae family [93]. In vitro analysis of intestinal epithelial cells confirms that gut microbiota activity can be disrupted by deltamethrin [18]. The gut microbial community in wasps has been suggested to determine animals’ susceptibility to insecticides such as abamectin [94]. Rouzé et al. investigated the effect of four pesticides (IMI, coumaphos, fipronil and thiamethoxam) on the GM of honeybees, highlighting how the microbial community’s composition differed in summer and winter bees. Sublethal doses of the compounds mentioned above showed similar effects and these were more substantial in the winter bees [95]. Other authors focused on the toxicity of aldicarb, a carbamate insecticide, on mice models, demonstrating through a metabolomic approach how this chemical can disrupt the gut microbiota leading to alteration in lipid profile, oxidative stress induction, DNA damage and susceptibility to bacterial pathogenicity [96]. See Table A4.