An Overview of Antimicrobial Compounds from African Edible Insects and Their Associated Microbiota

The need for easily biodegradable and less toxic chemicals in drug development and pest control continues to fuel the exploration and discovery of new natural molecules. Like certain plants, some insects can also respond rapidly to microbial infections by producing a plethora of immune-induced molecules that include antibacterial and antifungal peptides/polypeptides (AMPs), among other structurally diverse small molecules. The recent recognition that new natural product-derived scaffolds are urgently needed to tackle life-threatening pathogenic infections has been prompted by the health threats posed by multidrug resistance. Although many researchers have concentrated on the discovery of AMPs, surprisingly, edible insect-produced AMPs/small molecules have received little attention. This review will discuss the recent advances in the identification and bioactivity analysis of insect AMPs, with a focus on small molecules associated with the microbiota of selected African edible insects. These molecules could be used as templates for developing next-generation drugs to combat multidrug-resistant pathogens.


Introduction
Plants are the primary sources of natural products, having contributed immensely to drug discovery for the treatment of a variety of human diseases. However, the arsenal of secondary metabolites has recently expanded to include those of microbial origin associated with insects [1]. The early regime of antibiotics was boosted by the "accidental" discovery of penicillin (β-lactam), isolated from the fungus Penicillium sp., whose mode of action entailed targeting bacterial cell wall synthesis. Subsequent investigations in the field of antibiotic metabolites led to the discovery of many other classes of antimicrobial compounds through conventional fermentation procedures. However, the systematic emergence of resistance towards the available classes of antibiotics has increasingly posed a major challenge [2]. This has continued to justify the search for new antimicrobial metabolites, with potentially new modes of action, towards countering the problem of multi-drug resistance.
Bacteria and fungi, like many other organisms, must compete for resources by using a range of strategies, including those inflicted directly by toxins and indirectly through the activity of host immune responses, which results in changes in pathogenicity [3]. Bacterial and fungal interactions with their hosts including insects are crucial for many biological processes in agriculture, waste management, food production, and medicine [4,5].
Reviews on the nutritional value of certain insects, such as black soldier fly larvae, housefly maggot and pupae, mealworms, silkworm pupae, locusts, beetles, grasshoppers, and crickets, show that they have the potential to be used as an alternative protein source in various livestock feeds [6,7]. Additionally, insect meals can be used effectively in rearing of animals, such as poultry, to enhance animal health, intestinal morphology, and the composition of the intestinal microbiota [8], with an effect close to that of plant bioactive substances [9]. These insects' gut microbiota comprises a diverse range of microorganisms that produce bioactive compounds that, among other things, protect their host from  [15] and CLP1 (46_amino acid sequence, 4840 Da) [16]. (B) Structural representation and molecular modeling of DLP4 (pdb code: 2nz3) and CLP1 (pdb code: 2npi). Molecular models were generated with PyMOL 2.4.1.
Infections caused by resistant bacteria, on the other hand, are more difficult to treat, jeopardizing the efficacy of first-line antibiotics. As such, drugs that are more effective, more widely available, and have less toxic side effects are in high demand to treat these infections [17,18]. Thus, this review will discuss the recent advances in the identification and bioactivity of antimicrobial compounds derived from selected African edible insects and opportunities for their use as templates for developing next-generation drugs to combat multidrug-resistant pathogens. Although this review targets edible insects from Africa, in some sections, references are drawn from other parts of the world to buttress the growing demand for antibiotics.

Edible Insects in Africa and Their Microbiota
The consumption of insects (entomophagy) is an ancient and traditional practice, which has been recognized as one of the ways to alleviate hunger around the world, particularly in Latin America, Africa, and Asia. In sub-Saharan Africa, edible insects have been used as food because they are a good source of protein and essential fatty acids [19][20][21]. They are also high in micronutrients such as copper, iron, magnesium, manganese, phosphorus, selenium, and zinc, as well as vitamins such as riboflavin, pantothenic acid, biotin, folic acid, vitamin A, B complex, and C [22,23].
Spore-forming bacteria and Enterobacteriaceae, for example, have previously been detected in crushed mealworms and crickets and were believed to be released from the gut [25]. Furthermore, research on cricket (Gryllotalpa Africana), weevil (Rhynchophorus phoenicis), and butterfly (Bematistes alcinoe) species showed that the majority of microorganisms belonged to the two bacterial genera Bacillus and Staphylococcus, with saprophytes accounting for the remainder [26].
Additionally, a recent study profiled the black soldier fly larval gut microbiota, which included bacterial and fungal communities from four different substrates (brewers' spent grain, kitchen food waste, poultry manure, and rabbit manure). Metagenomic analysis revealed 21 bacterial and 20 fungal genera. These findings further indicated that the composition and abundance of the identified microbes differed depending upon the substrate [27]. The highly represented bacterial population in BSF larvae reared on all substrates except rabbit waste, for example, belonged to the Dysgonomonas genus. In contrast, the Campylobacter genus was more abundant in BSF larvae reared on rabbit waste than in larvae raised on the other three substrates [27]. Pichia, Cyberlindnera, and Saccharomycecodes were found in high concentrations in brewers' spent grain, kitchen food waste, and rabbit manure, respectively, in the fungal community. These findings suggest that there is a large population of microorganisms that is yet to be identified, including those found in edible insects, from which new scaffolds of drugs with potentially new modes of action could be developed to target multidrug-resistant bacteria.

Multi-Drug Resistance (MDR)
The development of antibiotic resistance has continued to warrant the search for novel bioactive metabolites in the field of natural products. A contributing factor to the problem of multi-drug resistance is the widespread and uncontrolled usage of antibiotics to treat bacterial infections. Scientific research has shown that some disease-causing microbes have developed resistance against certain available classes of antibiotics. These include the "ESKAPE" organisms, i.e., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp. [28]. Methicillinresistant Staphylococcus aureus (MRSA) is a classic example of a notorious case of multi-drug resistance development. It has developed resistance against the major classes of antibiotics such as aminoglycosides, macrolides, tetracycline, chloramphenicol, and lincosamides [29].
Multi-drug resistance (MDR) is therefore linked to several disease treatment challenges, including prolonged time of infection in patients due to the increased spread of resistant pathogens as a result of first-line drug efficacy, high treatment costs that may lead to high morbidity and mortality rates, and the exposure of immune-compromised patients as an easy target due to decreased drug efficacy [30]. These reports encourage the exploration of alternative sources of antibiotics to overcome MDR. That begs the question of whether insects could serve as a potential source for identifying novel antibiotics.

Insects as Potential Antibiotic Producers
Insects not only perform a variety of roles in the environment, but also host a diverse community of microorganisms. The multifaceted cellular and humoral mechanisms comprise the innate immune system of an insect [31,32]. The cellular mechanism is based on phagocytosis being activated by enzymes and invading microorganisms being encapsulated by the hemolymph. The humoral response, on the other hand, is involved in the production of broad-spectrum antimicrobial peptides (AMPs), reactive oxygen or nitrogen intermediates, and complex enzymatic cascades that help to regulate hemolymph coagulation or melanization [33,34]. The presence of microorganisms invading insects causes the fat body to rapidly synthesize AMPs, which are then secreted into the hemolymph [35,36].
Previous research indicates that each insect species produces a distinct antimicrobial peptide that acts against specific microorganisms, as shown in the review by Yi et al. [11]. However, in order to boost the insect's defense system against other pathogens, some of the peptides are expressed concurrently, encouraging synergism [36][37][38]. For example, when a cecropin (LSer-Cec6) and a defensin (LSer-Def4) from the wound maggot Lucilia sericata were examined together, they showed significantly increased antibacterial activity [39]. Furthermore, the amount of AMPs produced by insects varies greatly depending on the species. As such, AMPs have distinct modes of action, such as altering the electrochemical gradient at the membrane, producing reactive oxygen/nitrogen species (ROS/RNS) that cause cell death, inhibiting protein synthesis, and permeabilizing the cell membrane [38,40]. Antimicrobial peptides (AMPs) have pharmacological properties such as low molecular weight, high water solubility, broad-spectrum antimicrobial activity, and low levels of cytotoxicity [41].

The Chemistry of Microorganisms from Selected Edible Insects
In this section, chemical compounds identified in microbes found in the six selected edible insects (black soldier fly, termites, beetles, locusts, caterpillars, and crickets) are highlighted. Some of these chemicals have been reported to have antibacterial, antifungal, antimalarial, anti-inflammatory, and cytotoxic activities.

Black Soldier Fly Hermetia Illucens (Diptera: Stratiomyidae)
The gut of the black soldier fly (BSF) larvae has been shown to harbor beneficial microbes and fungi that also control pathogens [42]. Thus, the microbes linked to it are a good target for the discovery of new antimicrobial compounds of pharmaceutical relevance. In some studies, the Pichia genus was found to be related to the larvae fed on vegetable waste, whereas Trichosporon, Rhodotorula, and Geotrichum were the most abundant genera in the larvae fed on chicken feed only [42]. Regarding the production of AMPs, it is highly conserved between insects and may only vary between species depending on their respective habitats [43]. In BSF larvae, AMPs have been classified as defensins (cysteine-rich peptides), cecropins (α-helical peptides), attacins (glycine-rich peptides/proteins), and diptericins (a family of related glycine-rich antibacterial peptides), as well as lipids (hexanedioic acid) [43,44]. In a previous analysis, scores that predicted the biological activity of unknown peptides in the transcriptomes of BSF larvae and adults were determined using different algorithm in in silico tests. Biological activities that were predicted included antimicrobial, anticancer, antiviral, and antifungal properties [13]. Follow-up in vitro studies are yet to be carried out to validate these findings.
The general mechanisms of action of AMPs have been investigated in a few studies [45,46]. For example, Park et al. [47], demonstrated that H. illucens larvae extracts have antibacterial properties against Gram-positive S. aureus, methicillin-resistant S. aureus (MRSA), and Gram-negative Pseudomonas aeruginosa [47]. The methanol extract of BSF was found to inhibit the growth of K. pneumoniae, Neisseria gonorrhoeae, and Shigella sonnei bacteria, whereas the extract elicited no antibacterial effects against the bacteria B. subtilis, Streptococcus mutans, and Sarcina lutea [48].
The expression and characterization of stomoxynZH1 (encoded by a 189-basepair gene) with antimicrobial activity against Escherichia coli and S. aureus is one of two recent promising results involving AMPs from H. illucens [49]. In addition, DLP4, a novel AMP isolated and characterized from H. illucens hemolymph larvae, showed potent activity against MRSA and methicillin-susceptible S. aureus (MSSA) [15].
Furthermore, secondary metabolites from Chrysosporium multifidum broth extract, a fungus isolated from the midgut of BSF larvae fed on fresh unsterilized chicken guano, have been described in the literature. As shown below, these molecules include six pyrone derivatives (1-6) and one diketopiperazine (7) (Figure 2). The epoxy moiety containing α-pyrone (5) was found to have moderate antibacterial activity against 43,300 ATCC strains of methicillin-resistant Staphylococcus aureus (MRSA) [10]. Thus, AMPs and small molecules from H. illucens are still understudied, despite the fact that they have strong antibacterial properties. So far, only one fungal strain has been targeted within the rich microbiota of H. illucens, with no reports on the antifungal and cytotoxic effects of the identified molecules.

Termites (Isoptera)
Termites are recognized as beneficial insects in agriculture, entomotherapy, and the environment. Their ability to act as mediators of the process of decomposing plant organic matter and as influential agents in soil formation, particularly in tropical forests, is one of their important ecological roles [50]. The diversity of termite gut communities is astounding, but the function of each group of symbionts is poorly understood. Hemicellulosedegrading bacteria, lignolytic bacteria, cellulolytic bacteria, aromatic compound-degrading bacteria, and nitrogen-fixing bacteria are among the primary termite intestinal microorganisms [51][52][53][54][55][56][57][58][59]. According to research, the most abundant bacteria found in both higher and lower termites are strict aerobes or facultative anaerobes. There is also evidence that the most abundant bacteria in termite guts belong to the Staphylococcus and Bacillus genera [60,61]. Moreover, research has previously discovered a link between the major gut bacteria and the termite family (see Table 1) [62]. Enterobacter sp. Gut [62] Termitidae species (Nasutitermes exitiosus, N. graveolus, N. walkeri) Staphylococcus sp. Gut [62] Additional studies have shown that Streptomyces strains isolated from termites had significantly higher inhibition activity against Gram-negative bacteria than soil isolates [64]. Chemical analysis of termite-associating microbes, in particular fungi from O. formosanus, revealed three small molecules named 5-hydroxyramulosin (8), biatriosporin M (9) from the Pleosporales sp. BYCDW4, and 1-(2,5-dihydroxyphenyl)-3-hydroxybutan-1-one (10) from the Microdiplodia sp. BYCDW8. Investigation of their biological activity indicated that compound (10) had moderate inhibitory activity against B. subtilis and S. aureus [65]. Streptomyces davaonensis YH01, isolated from the body surface of the queen of the termite O. formosanus, demonstrated antibacterial activity. Roseoflavin (11) and 8-methylamino-8demethyl-D-riboflavin (12), which exhibited antibacterial activities, were discovered in a subsequent study on the same strain [66]. Their absolute stereochemistry, however, is yet to be assigned ( Figure 3).
In silico analysis of the genome of Actinomadura sp. RB29 from Macrotermes natalensis, using antiSMASH and tandem MS 2 data submitted to Global Natural Product Social Molecular Networking (GNPS), and subsequent RiPPquest processing revealed the presence of two lanthipeptides with proposed structures of rubrominin A (55) and B (56) [70]. Their gene cluster resembles that of cinnamycin (Figure 7) [100][101][102]. Except for the Actinobacteria microorganisms, the termite family has a large range of microbiota that has not been thoroughly investigated in terms of their chemical capacity as presented here. The bulk of their chemical structures consists of peptides and isoflavonoids, which makes them peculiar.

Beetles (Coleoptera)
Beetles are generally known to benefit the environment (nutrient recyclers, pollinators), but a significant portion of them are pests of economically important crops and storage products [103]. Bark beetles, in particular the southern pine beetle (Dendroctonus frontalis), are damaging to trees [104]. They have a symbiotic relationship with the fungus Entomocorticium sp. A, which serves as food for the beetle larvae [105].
Recent research has revealed a link between various beetle species and four bacterial phyla, Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes, as well as three fungal phyla, Ascomycota, Zygomycota, and Basidiomycota. These microbial communities were discovered to differ depending on the beetle host, individual organism, and environment [106].
Actinobacteria, particularly Streptomyces, have been described as having a chemical defense mechanism that produces antimicrobial compounds that aid in the fight against infectious disease. Mycangimycin (57), frontalamide A (58), and frontalamide B (59), for example, were isolated from a Streptomyces strain that was symbiotically associated with the southern pine beetle (Dendroctonus frontalis). Mycangimycin inhibits the beetles' antagonistic fungus Ophiostoma minus and has potent antimalarial activity. Frontalamide A and frontalamide B have antifungal properties [87,88].
Nakashima et al. studied the fungal strain Fusarium sp. from the ambrosia beetle Euwalecea validus in the early 1980s. Its culture extract contained two antifungal compounds, cerulenin (60) and the nortriterpenoid helvolic acid (61), which inhibited the growth of mold fungi and are thought to suppress bacterial contaminations [89]. Chemical analysis of rove beetles (Paederus sp.) yielded a complex polyketide pederin (62) molecule from an endosymbiotic Pseudomonas sp. [49][50][51][52] that showed toxicity against predators such as wolf spiders in a study by Kellner and Dettner [90].

Locusts (Orthoptera: Acrididae)
Locusts, like other insects, have bacterial cells both inside and outside their bodies, such as on their cuticles. With respect to other locust species, the bacterial composition of the desert locust (Schistocerca gregaria) gut has been extensively described [107]. For example, the bacteria in the hindgut of the desert locust were discovered to be relatively simple, consisting of members of the families Enterobacteriaceae (including Enterobacter and Klebsiella) and Enterocococeaea [108]. This study followed previous research that found gut bacterial flora in S. gregaria that included Escherichia coli, Enterobacter liquefaciens, Klebsiella, Pneumoniae, and Enterobacter cloacae [109,110]. The lack of complexity in the desert locust's gut microbiota could be attributed to its simple structure and short throughput time.

Caterpillars (Lepidoptera)
Caterpillars are among the most popular, consumed, and economically valuable edible insects in the tropics, because they have high protein and fat content [21]. They are mostly found in tropical rainforests. There are over 130 edible caterpillar species consumed in Africa alone, compared to nearly 400 species worldwide [112]. Despite their enormous contribution to global food security, little is known about their ecological relevance [113], associated microbiota, and antimicrobial potential. Caterpillars, unlike other living organisms, do not have a gut microbiota, demonstrating their freedom from symbionts [114].
So far, volicitin (80), N-(17-hydroxy-linoleoyl)-L-glutamine (81), N-linolenoyl-L-glutamine (82), N-linoleoyl-L-glutamine (83) and volicitin-related compounds (84), linolenic acid (85), and the 17-hydroxylinolenic acid (86) (Figure 10) have been discovered in the oral secretions of three Noctuidae species, i.e., Helicoverpa armigera, Mythimna separata, and Spodoptera litura, as well as one Sphingidae species, Agrius convolvuli [98]. The structure-activity relationships of volicitin-related compounds were investigated to determine the elicitor activity of volatiles from corn seedlings, revealing that chirality at C-17 in the linolenic acid chain had no effect on bioactivity. However, it was discovered that the L-glutamine moiety was more important than the hydroxyl moiety [115]. Derivatizing these molecules can lead to a wide variety of agricultural applications, such as identifying volatile components that can be used in pest control. Interestingly, there is no evidence of these compounds being associated with the gut microbiota of caterpillars.

Crickets (Orthoptera)
Crickets have traditionally been used as a source of medicine for a variety of ailments. In Korea, for example, the mole cricket Gryllotalpa Africana (Gryllotalpidae) is used to treat urine retention, urolithiasis, edema, lymphangitis, and furuncles [116]. In Latin America, the house cricket Acheta domesticus (Gryllidae) is used to treat scabies, asthma, eczema, lithiasis, earache, oliguresis, rheumatism, urine retention, urinary incontinence, and ophthalmological problems [117]. The crickets Paragryllus temulentus (Gryllidae) and Gryllus assimilis (Gryllidae) are both useful in the treatment of rheumatism and warts. Interestingly, bacteria from the Citrobacter, Klebsiella, Yersinia, Bacteroides, and Fusobacterium genera were found in the gastrointestinal tract of the house cricket Acheta domestica [118]. The Photorhabdus asymbiotica bacterium is known to have a two-part life cycle that colonizes the intestines of entomophagous nematodes and necessitates adaptation to both symbiotic and pathogenic phases [119]. The infection of live crickets with the Photorhabdus asymbiotica bacterium resulted in the isolation and characterization of glidobactin A (87), luminmycin A (88), and luminmycin D (89). Only compound (89) was found to be cytotoxic to human pancreatic cells and inhibited proteasome activity ( Figure 11) [99]. The macrolactam center of these molecules consists of nonribosomal peptide synthetase modules catalyzing the condensation of 4-hydroxylysine, L-alanine, and malonyl-CoA [120]. Additionally, there is a threonine moiety connecting it to the fatty acid side chain. In this regard, from the perspective of structure-activity relationships, the hydroxyl group on the threonine side chain and the R 1 positions can be targeted as accessible sites for modifying the molecule to potentially enhance its biological activities.

Future Perspectives
Insects thrive in Africa's tropical climate. However, this review reveals that very little research has been done on the chemistry of edible insects and their microbiota in Africa. With the current knowledge, it is imperative to advance studies to a higher level, by developing various methods to isolate and characterize new AMPs and small molecules. This may involve varying the habitats in which the insects are reared to stimulate different microbiota, which may increase the likelihood of discovering new pharmacologically relevant metabolites. It is also possible to screen different life stages because insects may live in different habitats, aquatic and/or terrestrial; hence, their potential association with different microorganisms. Moreover, derivatization can be facilitated by using known chemical structures as a template. Aside from that, the identified molecules can be tested in binary or ternary mixtures to increase their synergistic/additive effectiveness against multidrug-resistant species. Bioengineering and biotransformation of species and scaffolds, respectively, may also enhance the observed biological activity. In addition, insights into how these molecules are synthesized by various organisms would be useful to identify the enzymes involved. These studies should therefore help to clarify what function these microorganisms play in their hosts and pave the way for the production of antimicrobial agents.

Concluding Remarks
This review has shown that edible insects are a good source of novel antimicrobial peptides and compounds that can be screened against multidrug-resistant pathogens. StomoxynZH1 and DLP4 are among the AMPs found in the BSF with potent antibacterial activity. The majority of isolated microorganisms from which small molecules have been identified were sourced from the insect's gut. Overall, termite-related microorganisms are the most researched in terms of chemical diversity. In comparison to fungal strains, bacterial strains, especially Actinobacteria, have been extensively studied. As it is evident, Streptomyces yielded 25 (52%) of the 48 molecules isolated from termites, while 13 (27%) were isolated from Actinomadura species. In terms of the chemistry of BSF-associated microorganisms, only one fungal strain has been investigated so far and identified to contain primarily antibacterial α-pyrone molecules. In addition, about 29% of the small molecules characterized herein were obtained from the gut microbiota of beetles. Apart from sterols that have been identified in locusts, there is little knowledge on antimicrobial compounds that they may produce. Caterpillars, on the other hand, contain mainly linolenic acid-derived molecules in their oral secretions. Although the number of studies documenting antimicrobial peptides and small molecules is on the rise, the microbiota of edible insects still remains an understudied topic.  The views expressed herein do not necessarily reflect the official opinion of the donors. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Therefore, the views expressed herein do not necessarily reflect the official opinion of the donors.