Guo et al. studied the effect of a radiation-induced bystander in cells not directly irradiated in C. elegans [66]. Worms were grown in room conditions and were fed with standard E. coli OP50. Proton radiation was used in these experiments, and the dose delivered by each proton hitting the C. elegans was calculated as 0.033 Gy. The L₄ stage worms were irradiated by proton beams with 1.65, 6.6, 16.5, 33, and 66 Gy and were compared with the control group. The number of dead germ cells was counted 24 h after the irradiation. The results clearly showed that bystander effects happen in C. elegans after ionizing irradiation, including effects on germ cells [66]. In a similar study, Tang et al. used a combination of irradiation with a proton beam and gamma rays to investigate the interaction between the radio-adaptive response and the radiation-induced bystander effect (RIBE) in C. elegans [67]. In this experiment, they performed whole-body exposure of a 14-h-old C. elegans to a microbeam of 1000 and 2000 protons, respectively. After two hours, the worms were irradiated with gamma radiation with 75 and 100 Gy, respectively. Their study suggested the phenomenon of RIBE, where defects such as the vulva abnormalities (non-irradiated cell) can be significantly decreased because of a signal received from the irradiated rectal valves (irradiated cell) of C. elegans; therefore, the radio-adaptive response of the C. elegans can be improved by a radiation-induced bystander effect. Xu et al. exposed C. elegans to X-rays to study the response of C. elegans to ionizing radiation [68]. In this study, the following three C. elegans cohorts were included: a control group and two groups exposed to X-ray doses of 200 and 400 Gy. After irradiation, total RNA was extracted from each group, also RNA was sequenced to compare the transcriptomes between these groups. They found that many genes expressed differentially and were enriched significantly in ionizing radiation-related biological pathways and processes [68]. This demonstrated that C. elegans genes related to various biological processes, such as behavior, regulation of growth and locomotion, positive regulation of growth, calcium ion transport, and di- and trivalent inorganic cation transport, were modulated by different doses of X-ray radiation [68]. Ionizing radiation affected the locomotion of C. elegans and influences the reaction of C. elegans to the environment. Sakashita et al. studied the effects of dietary NaCl on the learning (learning function of nervous system) of radiation-induced C. elegans [69]. The C. elegans were grown at room condition and fed with E coli until the adult stage. The C. elegans were exposed to both acute and chronic irradiation in two separate groups. The animals were irradiated with a 100 Gy dose of gamma-ray at the rate of 32 Gy/min as acute irradiation. In chronic irradiation, the animals were also irradiated with a 100 Gy dose of gamma-ray but at a rate of 0.42 Gy/min for four hours. The animals were then treated in the following three sets of NGM plates: (1) without NaCl, (2) 10 Mg NaCl, and (3) 50 mM NaCl to measure the chemotaxis toward NaCl. Their experiment showed that both acute and chronic irradiation decreases the chemotaxis of C. elegans to NaCl. They suggested that irradiation in C. elegans may have a modulatory effect on diet NaCl associated with animals learning.

3.2. Effect on Aging

The effects of exposing C. elegans to ionizing radiation are a subject of interest in radiation biology research investigating the process of aging in this organism as the life span of C. elegans is affected by the radiation exposure. This was investigated in a study by Kuzmic et al., where they show an acceleration of the aging process by exposing C. elegans to chronic irradiation. Irrespective of the dose rate and dose delivered, chronic exposure reduced the level of oxidative protein damage (carbonylation), which is a biomarker of the aging process [70]; Dubois et al. also experimented with the response of C. elegans to acute and chronic exposure [71]. After chronic exposure, 168 proteins were significantly changed in the experimental group, while 369 proteins were significantly changed following acute radiation exposure. Analysis of global protein expression in this study showed the modulation of proteins involved in regulating biological processes such as lipid transport, DNA replication, development of germ cells, apoptosis, transport of ions, and the development of cuticles. These results confirmed that chronic irradiation induced different molecular mechanisms than acute irradiation [71].

Besides the whole-body irradiation of C. elegans, partial irradiation is becoming more important in recent studies. Suzuki et al. irradiated specific regions on the C. elegans to compare animal movements with whole-body irradiation [72]. C. elegans were grown at 20 °C and were fed with E coli. Then, the three-day-old animals were irradiated with a 500 Gy dose of heavy carbon ions. The worms were irradiated within four groups. The groups were as follows: whole-body irradiation, central nervous system, CNS, irradiation or head, middle of the body irradiation, and tail irradiation. They also used active animals in their experiment and studied the motion of the animals immediately after the irradiation, which was the novelty of their experiment method. The body bend rate was calculated for five animals in each group. The results showed that the whole body irradiation reduces the mobility of the worms, while region-specific irradiation did not significantly affect the motion of the C. elegans [72]. Another study by Suzuki et al. presents evidence of the CNS’s activity in reducing the mortality in adult hermaphrodite C. elegans due to acute irradiation. Research shows that decreased mortality after irradiation of the CNS depends on the dose, and the body muscle around the CNS is also partly involved in reducing the mortality [73]. A device that makes the researcher able to partially irradiate C. elegans has recently been developed by Funayama et al. [74]. They have designed and built a device to irradiate animal models such as C. elegans with an ionized microbeam. They obtained a beam size smaller than 10 μm in their device to target specific cells in their animals under the microscope. They irradiated intracellular objects with the newly developed device. They grew C. elegans in a room temperature environment at 20 °C for three days, and the animals were cultured on nematode growth medium [74]. The CNS of the C. elegans was irradiated with 500 Gy of carbon ions. The microbeam’s size was 8 μm, which helped to irradiate the worms’ nervous system spot. Following irradiation, samples were collected, and the animals were placed on an NGM plate where they were not fed anymore. Then, they evaluated the effects of the irradiation on the locomotion of the C. elegans. The results showed that the irradiation did not change the animals’ locomotory rate, and thus, the 500 Gy had no effect on the movement of the C. elegans. They also designed a frame in which they can irradiate the C. elegans with micrometer precision, which can be used for further investigations on the heavy ion irradiation on C. elegans and cultured cells [74].

3.3. Effect on Reproduction

Radiation exposure affects C. elegans in its reproduction. Min et al. showed that C. elegans irradiated using a proton beam at the L₄ larval stage grew almost generally in the adult stage, but later experienced reproductive deficiencies demonstrated by the decreased size of brood as measured by the total number of eggs that are fertilized by hermaphrodite animals; moreover, an increase in germline apoptosis caused reduced fertility [75]. The effects of a heavy-ionized beam on C. elegans germline cells were studied by Sugimoto et al. [76]. The nematodes were grown in room conditions at 20 °C for 24 h on NGM plates. When the animals entered the L₄ larval stage, they were exposed to 20, 40, 60, and 100 Gy doses of carbon ion microbeams for both whole-body irradiation and germline cells. Then, the nematodes’ hatching rate was calculated at 0 to 4 h and 11 to 15 h after the irradiation. The hatching rate for the delivered dose before 4 h was 65, 20, 2, and 0 percent, respectively for the surviving eggs. The calculated rate for 11 to 15 h was about 100, 90, 60, and 0 percentages for the surviving eggs. The experiment results showed that both whole-body irradiation and germline spot irradiation decrease the hatching rate of the larva [76]. Histone-modifying enzymes have been reported to affect longevity through several generations [77,78]. Their progeny may gain trans-generationally inheritable survival benefits [79].

Min H et al. reported major increases in the number of proteins named RAD-51 and HUS-1 (RAD-51 and HUS-1 are both critical components in the DNA damage response of C. elegans) and in the number of chromosomal aberrations in gonads and oocytes, respectively, following proton beam irradiation in hermaphrodites P0. However, these defects were recovered mainly in progeny F1 and F2 [75]. Homologous recombination resects the double-strand breaks (DSBs) and produces single-stranded DNA tails. Since RAD-51 binds directly to these single-stranded DNA tails, a good estimate of the number of DSBs in a cell is given by the number of these proteins [80]. An increase in the number of RAD-51 foci (6.0 ± 1.75 per nucleus) in hermaphrodite gonad germ cells relative to those of non-irradiated hermaphrodite gonad germ cells (0.9 ± 0.61 per nucleus) was observed following irradiation at a dose of 10 Gy with a proton beam [80]. A critical component of the response to DNA damage in C. elegans is HUS-1; compared to non-irradiated gonads, the number of HUS-1 proteins was found to have increased in the irradiated hermaphrodite gonads [81]. Ryu et al. studied the roles of a homolog of p53 binding protein 1, HSR-9, in the DNA repair and the ionized irradiation response of C. elegans [82]. Standard C. elegans strains were cultured in room conditions and fed with E. coli. Furthermore, the worms were grown until the L₁ stage, and then the worms were used for different experiments such as survival gem cells rate and abnormality developments in the eggs of the next generation of worms. Twenty different RNA-types of L₄ stage animals were irradiated with 60 and 120 Gy doses of gamma-ray to find the germ cell survival of C. elegans. The hatching rate for the eggs was calculated at 24 and 48 h after the irradiation. Additionally, the survival rate for L₁ stage worms was calculated in a separate experiment [82]. The nematodes were exposed to 25 and 50 Gy doses of acute gamma irradiation, and the hatching rate was calculated after 48 h. Furthermore, the 4-h-old eggs of C. elegans were irradiated with a 90 Gy dose of Gamma-ray searching for any abnormalities in the hatched worms after the irradiation. Both the irradiated and the control group of worms were examined until the L4 stage and abnormalities were recorded in the irradiated worms after three days. The results of these experiments showed that although p53 binding protein one homolog increases apoptosis, it does not directly influence the C. elegans response to DNA damage [82].

3.4. Effect on DNA Response

The DNA response to ionizing irradiation in C. elegans has gained much interest recently. Bertolini et al. studied DNA repair mechanism in irradiated C. elegans [83]. They used wild-type C. elegans and compared their DNA response to other mutant C. elegans strains, such as brc-1, bub-3, and san-1. The C. elegans were grown in room temperature conditions and fed E coli OP-50 and irradiated using a 137Cs gamma source. The L₁ stage worms were exposed to a 60 Gy dose of ionizing irradiation, which resulted in vast DNA damage in C. elegans, but it did not decrease the worms’ reproduction. Other C. elegans were irradiated with 90 and 120 Gy doses, and results were compared to a control non irradiated group [83]. They found that the lethality increased in the bub-3 mutant compared to wild-type C. elegans. Additionally, ionized sensitivity in both bub-3 and san-1 was increased. These studies suggested that the involvement of bub-3 in response to the DNA damage in C. elegans occurs in cell cycle timing [83]. In another genetic study, Vujin et al. exposed different mutants of C. elegans, as well as wild-type N2 and wild-type NHJ1 strains to ionizing radiation [84]. The C. elegans were irradiated by 0, 25, 37.5, 50, and 75 Gy doses of X-ray in different experiments and different factors, such as ionizing radiation sensitivity or resistance, were determined. They suggested that damage in the nhj-1 sequence caused sensitivity to ionized radiation in wild-type N2 C. elegans [84]. Yang et al. showed ceramide regulated DNA damage response to mediate radiation induced germ cell apoptosis [85]. Buisset-Goussen et al. described a decrease in reproductive capacity in future generations after chronic exposure of C. elegans mother hermaphrodites to gamma radiation [86]. Barber et al. reported that the genomic instability in the progeny, which was not irradiated, was caused by irradiation of the mother C. elegans and concluded that the damage due to radiation is a long-term risk that lasts for generations [87]. Proton beam irradiation was also reported to cause transgenerational effects in three successive generations by Min et al. [75].

In another experiment, C. elegans were exposed to both chronic and acute γ-ray radiation in two different experiments by Dubois et al. [88]. They showed that acute irradiation influences the hatching of the worms, but chronic irradiation does not. C. elegans were exposed to six different doses and four doses in acute and chronic irradiation experiments. C. elegans were irradiated for 65 h during a whole life cycle from the embryo stage to the L4-YA adult stage for chronic exposure. The study’s radiation source was 137Cs, which provided a homogeneous dose rate on the plate [88]. A similar study by Maremonti et al. about the acute and chronic exposure of C. elegans to γ-ray radiation demonstrated that toxicity of chronic exposure in reproduction compared to acute exposure [89]. While acute irradiation did not significantly affect reproduction, chronic irradiation of 62 h from egg to young adult stage decreased the number of larvae hatched per adult hermaphrodite C. elegans up to 61% with a total dose of 14 Gy. These effects were mainly due to increased germ cell apoptosis, impaired sperm meiosis, with adverse effects on sperm production [89]. Later studies by Maremonti et al. showed an increase in the copy of mitochondrial DNA (mtDNA), which plays an important role in reproduction influencing germ cell development, quality, and embryonic development after chronic exposure [90]. Moreover, in their latest study, Meremonti et al. investigated effects of chronic exposure of gamma radiation on accumulation of free radical and the subsequent antioxidant responses to the apical reproductive and developmental processes in C. elegans. This research showed a reduction in the number of offspring by 20 and 40% after 96 h of gamma exposure of rate 40 100 mGy h⁻¹ (total dose ~3.9) and 100 mGy h⁻¹ (total doses ~9.6 Gy), respectively [91].

Table 1. Summary of Acute Irradiation of C. elegans.

Type of Radiation and Exposure Time	Radiation Dose/Dose Rate	Findings	Year/ Studies
Gamma Radiation for about an hour	0.027 Gy/min	≥0.1 Gy is needed to reduce the mean life of C. elegans. Dauers are the most sensitive and 8-day-old adults are the most resistant to ionizing radiation.	Johnson et al., 1984 [36]
Targeted micro bream (12C ion particles)	20, 40, 60, and 100 Gy	Reproduction in C. elegans eggs arrested for both whole body and tip irradiation. In spot irradiation, the neighboring cells around the targeted point did not arrest the reproduction of germ cells nor did apoptosis happen.	Sugimoto et al., 2006 [76]
Gamma radiation	100 Gy (32 Gy/min)	Chemotaxis reaction of C. elegans toward NaCl decreases. The role of ionizing irradiation in associative learning of C. elegans toward NaCl is elaborated.	Sakashita et al., 2008 [69]
Funayama et al., 2020 [
74
]
Gamma Radiation
0, 5, 10, 25, and 50 Gy (3.37 Gy/min)
Germ cell apoptosis decreases when
C. elegans
Vujin et al., 2020 [
84
]

Table 2. Summary of Chronic Irradiation of C. elegans.

Type of Radiation & Exposure Time	Radiation Dose	Findings	Year/Studies
Low dose gamma-ray radiation for 219.5 h	61	]
Low dose gamma-ray radiation for 11 days	Not specified	No significant increase in the mutation rate Introduction of eT1 balancer system for longer-term measurement of biological damage in space.
are treated with Ceramide.
Ceramide influences
C. elegans
’s response to DNA damage.
Ceramide involves in the functioning of mitochondria in
C. elegans
under ionizing irradiation.
Yang et al., 2020 [
85
]
0.268 to 0.306 cGy	The mutation frequency increased significantly due to exposure to space radiation.	The charged iron particles are the major mutagenic component and the increased mutation frequencies caused significant cancer risk inherent in extended space travel.	Hartman et al., 2001 [Zhao et al., 2006 [62]
Gamma radiation for 4 h	100 Gy (0.42 Gy/min)	The avoidance response of C. elegans toward NaCl decreased significantly.	Sakashita et al., 2008 [69]	Gamma radiation for 18 s	6 × 10−3 to 2.8 × 10−2 Gy (for 1 month) 36 × 10–3 to 16.8 × 10–2 Gy (for 6 months) 144 × 10–3 to 67.2 × 10–2 Gy (for 2 years)
Accelerated proton for 18 s	33.6 × 10–3 to 16.8 × 10–2 Gy (for 6 months) 144 × 10–3 Gy (for 2 years)	C. elegans would experience some damage from irradiation during long-term space flight, there are changes in genes related to DNA damage response, oxidative stress, and cell death, and the gamma rays induce apoptosis.	DNA repair mechanism was reduced due to proton exposure. Accelerated protons induce the expression of genes that are related to the DNA damage response and anti-apoptosis.S. Yi et al., 2013 [64]
Yi et al., 2013 [	64	]	Accelerated proton for 18 s	33.6 × 10–3 to 16.8 × 10–2
Gamma Radiation (137Cs source) for 64 h	Gy (for 6 months) 144 × 10–3 Gy (for 2 years)	DNA repair mechanism was reduced due to proton exposure. Accelerated protons induce the expression of genes that are related to the DNA damage response and anti-apoptosis.	S. Yi et al., 2013 [64]
Dose rate 7 and 52 mGy/h	Dose: 0.5 and 3.3 Gy	Life span is significantly shortened in irradiated C. elegans. There was a significant difference between different absorbed doses for the same dose rate.	Kuzmic et al., 2019 [70]	Gamma radiation	25, 50, 60, 90, 120 Gy	53BP1 homolog, HSR-9 increases the cell death in muted
Gamma Radiation (137Cs source) for 19 days	Dose rate 7 and 52 mGy/h Dose: 3.3 and 24 Gy	C. elegans exposed to acute irradiation. HSR-9 does not involve in C. elegans cells’ response to DNA damage due to ionizing irradiation.	Life span is significantly shortened in irradiated C. elegans. Ryu et al., 2013 [82]
There was a significant difference in absorbed doses in the treatments between 3.3 Gy cumulative irradiation (with 7 mGy/h) and 24 Gy cumulative irradiation (with 52 mGy/h)	Kuzmic et al., 2019 [	70	]	Proton particles microbeam	Ionized irradiation causes bystander effects in C. elegans cells
Gamma Radiation (137Cs source) for 65 h	1.65, 6.6, 16.5, 33 and 66 Gy (0.033 Gy per proton particle)	Six dose rates 7, 14, 45, 50, 75, and 100 mGy/h Dose: 0.5, 1, 3.3 GyProton irradiation increases the germ cell apoptosis in neighboring cells around the radiation spot.	Guo et al., 2013 [66]
There are no effects from irradiation on the percentage of the hatch after chronic irradiation compared to control	C. elegans	.	Dubois et al., 2018 [88]	Proton microbeam and gamma rays (137Cs) (Time not specified)	Proton bream: 3.2 MeV with linear energy transfer rate Gamma rays: 75 and 100 Gy
Gamma radiation (137Cs source) for 65 h	DNA damage in worms was unique and in somatic cells, which include vulval cells the DNA damage checkpoint was not active.	Radio-adaptive responses of the whole C. elegans organisms were improved by bystander effect, which was induced by radiation.	Tang et al., 2016 [	Dose rate: 7, 14, 50 mGy·h−1 corresponding to cumulated doses (0.5, 1, and 3.3 Gy)67]
168 proteins were found with significant differences.		The molecular mechanisms induced by chronic irradiation differ from those that were induced by acute irradiation.	Dubois et al., 2019 [71]	Targeted micro bream (12C ion particles)	500 Gy	Development of a method to irradiate active C. elegans. Whole-body irradiation decreased the movement rate of C. elegans significantly. Regional irradiation on the head, middle, and tail of C. elegans did not have a significant effect on the movement rate.	Suzuki et al., 2017 [72]
Gamma Radiation for 62 h	Dose rate: 0.9 to 227 mGy·h−1 Total dose up to 228 Gy	The number of larvae hatched was significantly decreased (by 43 and 61%, when chronically exposed from egg to young adult stage to a total dose of 6.7 Gy and 14 Gy, respectively) with increased germ cell apoptosis, impaired sperm meiosis, and adverse effects on sperm production.	Maremonti et al., 2019 [90]	Gamma radiation (137Cs)	0, 60, 90, and 120 Gy	Ionized irradiation caused significant damage in C. elegans DNA but did not reduce the reproduction cells. Sensitivity to ionized irradiation increased in C. elegans mutants compared to the wild-type strain. BUB has a role in the response of C. elegans to DNA damage.	Bertolini et al., 2017 [83]
Gamma Radiation for 72 h	Dose rate: 0.4 to 100 mGy Dose: 0.03 to 72	Significant increase in mtDNA copy number (approx. 1.6-fold).	Maremonti et al., 2019 [90]	Gamma radiation (137Cs)	2.5, 6.5, 14.4, 50, 100, and 200 Gy	Dependency of hatchability on irradiation dose was shown by the result of the decrease in significant number of progenies per individual after irradiation from and above 50 Gy, until 200 Gy.	Dubois et al., 2018 [88]
Gamma Radiation for 96 h	Dose rate: 40 and 100 mGy·h−1 Total dose: ~3.9 and 9.6 Gy	Toxic effect in reproduction. No of offspring reduced by 20 and 40%.	Maremonti et al., 2020 [91]	Gamma radiation (137Cs)	1 Gy·min−1 0.5 Gy, 1 Gy, and 3.3 Gy	369 proteins were found with significant differences. The molecular mechanisms induced by chronic irradiation differ from those induced by acute irradiation.	Dubois et al., 2019 [71]
X-ray (600C/D linear accelerator)	0 gray [64], 200 Gy, and 400 Gy (not specified)	Genes related to several biological processes, such as behavior, regulation growth and locomotion, positive regulation of growth, calcium ion transport, and di- and trivalent inorganic cation transport, are differentially expressed	Xu et al., 2019 [68]
Gamma Radiation	Dose Rate: 1445 mGy·h−1 Total dose up to 6 Gy	Acute irradiation does not induce a significant change in reproduction.	Maremonti et al., 2019 [89]
Targeted micro bream (12C ion particles)	0, 500, 100, and 1500 Gy	The decrease in mortality depends on the dose due to central nervous system (CNS) targeted irradiation and may partly be due to body-wall muscle cells around the CNS.	Suzuki et al., 2020 [73]
Targeted micro bream (12C ion particles)	500 Gy	Targeted heavy ion microbeam smaller than 10 µm. The preparation and irradiation method for the device is provided. Targeted irradiation on the specific spots did not have an impact on the movement of C. elegans.	X-ray	0, 25, 37.5, 50, and 75 Gy	NHEJ factor in C. elegans is reported. NHJ-1 causes ionized radiation sensitivity in N2 wild-type C. elegans.

Besides these effects on the various processes of the life cycle of C. elegans, there are several other important radiation studies that have been conducted. Weidhaas et al. developed a tissue model of reproductive cell death induced by radiation in C. elegans [92]. Huangqui et al. demonstrated the radio-adaptive response for reproductive cell death in the valval tissue in C. elegans for the first time and showed that the C. elegans is an excellent in vivo model for radiation-induced reproductive cell death [93]. Similarly, Liangwen et al. demonstrated reproductive cell death across the entire range of carbon-ion irradiation [94] These studies opened new horizons on radiation-induced reproductive cell death in C. elegans and further confirmed that this nematode is an excellent model to study human reproductive cell death from the radiation therapy that is used in cancer therapy.

It can be inferred from the study of Table 1 and Table 2 that there is a variation of biological responses by the C. elegans irradiated with different radiation types and dose rates. For a similar type of radiation, it is obvious that the higher dose of radiation affected biological responses more than that the lower dose did. Moreover, there is distinctive variation of biological responses by irradiation from low-LET and high-LET (LET- linear energy transfer). High-LETs are highly mutagenic compared to low-LETs to diploid animal cells in an in vivo based system such as the C. elegans model organism. On the other hand, biological response are also affected by the treatment of C. elegans and the environment where they were grown, before and during the irradiation. Despite all these conditions, C. elegans has been regarded as an excellent model organism for radiation biology research. However, there are also some disadvantages or limitation of the C. elegans model system. First, C. elegans, having a simple body plan, lack many defined organs or tissue such as brain, blood, defined fat tissue or cells, and internal organs found in humans. Second, C. elegans has a very short length of about 1mm, which make it difficult to conduct some biochemical experiments such as understanding tissue-specific signaling [95].