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Page 10 of 15 Macpherson et al. Microbiome Res Rep 2024;3:20 https://dx.doi.org/10.20517/mrr.2023.66

Other researchers have argued that the level of connectedness determines the health of the microbiome,
[84]
rather than the balance of species . Just as for the human microbiome, it is unknown what truly constitutes
a “healthy” insect microbiome, especially when the correct balance differs from species to species. Insect gut
cell lines hold the potential to elucidate the roles of microbes and their contributions towards eubiosis or
dysbiosis within the insect gut, and the animal as a whole.

Culturing the uncultured
For most animals, the gut houses the greatest number of microorganisms in the body, many of which
remain uncultured [72,89] , meaning culture cannot be achieved using traditional microbiological methods,
likely because agars and broths utilized in laboratory settings do not replicate the complex physiological and
[29]
chemical conditions of the gut environment . One option to improve culture conditions is the
employment of bioreactor models, which show great promise in cultivating entire gut-derived communities,
[90]
but still lack the host cell component . In future studies, bioreactors could be linked to cell culture through
microbial metabolites. Cell-free supernatant from bioreactors is a source of microbial metabolites, which
can be filter-sterilized and applied to insect cell lines to replicate host-microbial metabolite interactions.
Since much of the interaction between a microbe and an animal cell is mediated through metabolite
signaling, the use of metabolites alone helps preserve cell line integrity while understanding host-microbe
interactions. This may be achieved by assessing cytokine release, localization within a host cell, and the up-
or downregulation of host gene expression.

In addition, since cell culture replicates the gut tissue microenvironment, it allows for the cultivation and
isolation of sensitive microbes that are strictly dependent on the presence of host cells . For example,
[29]
obligate intracellular microbes, which rely entirely on eukaryotic host cells, must be cultured in this
[29]
manner , including the endosymbiont W. pipientis, which was first cultured and isolated utilizing the Aa23
cell line, derived from the Asian tiger mosquito (Aedes albopictus) . Through the use of insect gut cell
[28]
lines, previously uncultured bacteria, archaea, and fungi can be isolated, enhancing our comprehension of
insect pathogens, commensals, and symbionts. Further, undiscovered constituents of the insect microbiome
may possess characteristics valuable to human industry and pharmaceuticals.

In highlighting the efficacy of cell lines for investigating facultative symbionts, the 2018 study by Chevignon
et al. stands out as a notable example . To explore the “unculturable” aphid symbiont Hamiltonella
[91]
defensa, crucial for safeguarding its host against parasitoid wasps, Chevignon et al. leveraged the
immortalized TN5 insect cell line . The utilization of cell lines in cultivating H. defensa yielded a higher
[91]
abundance of the symbiont compared to in vivo cultures . This enhancement enabled the researchers to
[91]
successfully generate the complete genomes for various H. defensa strains, thereby unraveling the genetic
[91]
variance of strains and genome elements responsible for the protective abilities exhibited by H. defensa .

Understanding the microbiome’s toxin defense systems
The insect microbiome serves as a critical barrier between toxins and host tissue. The scientific literature has
documented the ability of insect gut microbes to mitigate toxin exposure to the host through the breakdown
[6]
of external compounds, such as insecticides, as well as host-secreted wastes . The insect microbiome also
exhibits a capacity to adapt to toxin exposure. Namely, microbes of the brown plant hopper (Nilaparvata
lugens), an agricultural pest, displayed heightened expression of detoxifying genes, including NlCYP6ER1,
after repeated exposure to insecticides . Additionally, a commensal bacterial species of D. melanogaster,
[92]
Lacticaseibacillus (formerly Lactobacillus) rhamnosus, reduced the absorption of organophosphate
[93]
insecticides by the host . Furthermore, several lactic acid bacteria strains, isolated from the gut of managed
A. mellifera, had the ability to detoxify representative insecticides in cell line models . While encouraging,
[94]
it is important to note that none of the cell lines used (ovarian insect Sf-9, rat intestinal IEC-6, and human