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Macpherson et al. Microbiome Res Rep 2024;3:20 https://dx.doi.org/10.20517/mrr.2023.66 Page 9 of 15

Entomopathogens
Certain pathogens incite tumorigenesis or reduce apoptosis in the gut epithelial tissue of insects. In one
study, the European spruce sawfly (Gilpinia hercyniae) developed multiple midgut tumors after exposure to
a polyhedral virus . Additionally, Nosema cerenae, a fungal pathogen of A. mellifera, reduces apoptosis of
[75]
[76]
midgut epithelial cells by increasing the transcription of the inhibitor of apoptosis-2 gene . The direct
application of entomopathogens in insect cell line production has yet to be explored; however, this is not to
suggest that pathogens may not have already played a critical role. Both existing A. mellifera cell lines
[77]
(AmE-711 and MYN9) are known to be infected with deformed wing virus , which could have contributed
to their immortalization.

CRISPR-Cas9
CRISPR-Cas9 allows for the targeted insertion and deletion of genes from a host genome. CRISPR-Cas9
technology has recently been used to immortalize mature human prostate epithelial cells of the PrEC cell
line . In this cell line, the inactivation of CDKN2A at two loci induced immortalization, but also
[56]
[56]
maintained the characteristics of normal cells, including a normal p53 response . CRISPR-Cas9 has also
been used to edit several existing insect cell lines, including insect cell lines Sf-21 and S2R+ [78,79] . Despite
great potential, CRISPR-Cas9 has not yet been employed to produce a novel insect cell line. Future research
endeavors may benefit from editing genes that have been empirically linked to midgut tumorigenesis in
D. melanogaster, a commonly used insect model of colon cancer [66,69] . Two of these genes are APC and ras,
which control the activation of the Wnt signaling pathway and drive the proliferation and migration of cells,
respectively . These two genes could be key targets in future studies.
[66]

Radiation
Ionizing radiation (X-rays and Gamma-rays) induces direct DNA damage in the form of single- and
double-stranded breaks and indirect DNA damage through the production of reactive oxygen species .
[80]
Following the theory of radiation hormesis, low-dose radiation is purported to improve cellular health, but
high-dose radiation is extremely damaging and results in DNA breaks . Radiation causes apoptosis in the
[81]
majority of exposed cells, but has the potential to produce providential mutations, leading to
immortalization in a subset of these cells. The KMST-6 human fibroblast cell line was produced in this way
[53]
using Gamma radiation , illustrating the promise of this technique. Furthermore, literature from as early
as 1928 has associated X-ray exposure with genetic mutation in insects, indicating promise for future work
[69]
using radiation to deliberately transform insect cells to immortalize them .

INSECT GUT CELL LINES HAVE THE POTENTIAL TO ADDRESS MICROBIOME-RELATED
KNOWLEDGE GAPS
Defining a “healthy” insect microbiome
A substantial knowledge gap that could be addressed by insect gut cell lines is the balance of microbes that
[82]
constitutes a healthy, or “eubiotic,” gut microbiome . Eubiosis in insects is poorly defined, as is the
opposite state, dysbiosis. This, in large part, is due to the context-dependent nature and inherent subjectivity
of the gut microbiome . Additionally, very few insect species have well-studied microbiomes, furthering
[83]
this lack of understanding.

One theory that attempts to define microbiome health in insects follows the Anna Karenina principle of
“Happy families are all alike; every unhappy family is unhappy in its own way” [84,85] . According to this
framework, once a eubiotic species balance is achieved, any deviation from this species balance will
destabilize the system, leading to dysbiosis. This principle applies well to social insect species, which possess
highly conserved and vertically transmitted microbiome compositions [86,87] . However, the theory fails to
consider solitary insect microbiomes, which more often mimic the composition of their environment [6,88] .