The effect of UV radiation on the frequency of crossing over in Drosophila melanogaster

The increasing ozone layer depletion due to exposure to high environmental pollutants may contribute to the high probability of cells being exposed to UV radiation, causing various cellular responses, such as genomic instability. This study aimed to determine the effect of UV on the frequency of crossing over in a cross between the wild-type and the black vestigial strain of Drosophila melanogaster, using four variations of duration UV exposure as follows: 0’, 3’, 6’, and 9’. Recombinant progeny were calculated and analyzed using single-factor ANOVA. The results showed that: (1) The longer the duration of the UV exposure, the higher the percentage of recombinant progeny. (2) The body size of the D. melanogaster progeny exposed to UV light was more petite, and their movements were slower. (3) The single factor ANOVA showed that the length of UV had not affected the frequency of crossing over; but there was a phenomenon of crossing over, which has been showed that UV exposure made higher the percentage of recombinant progeny and affected to the body size of D. melanogaster. Therefore, further research is needed to examine the effect of UV radiation with longer periods of crossing over frequency and molecular analysis needs to be considered.


Introduction
Excessive exposure to ultraviolet radiation (UV-R) from sunlight and artificial sources impacts various sectors [1].In the environmental sector, the increased UV-R can contribute to climate change [2].In the context of broader societal influences, the increase in UV-R greatly impacts the health sector [3].UV-R type A (315-400 nm) is highlighted as low-energy radiation that is not filtered and can penetrate both the ozone layer and the atmosphere.The increase in UV-R intensity triggered by ozone can enormously affect living organisms [4].
Increased UV-R can impact the cellular level, especially DNA [3].If a fundamental part of cell DNA is exposed to UV light, it will affect genome instability.Genome instability due to exposure to UV light can cause double-strand breaks, which play an essential role in homologous recombination in crossing over [5].Crossing over is critical in contributing to genetic and phenotypic variations in living things, resulting in offspring with different types from their parents, namely the recombinant type [4].
Fan et al. [5] showed that crossing over is influenced by several factors, one of which is radiation.Radiation can increase crossing-over in fruit flies and typically reduce part of DNA [3], [6].Ultraviolet radiation is known to be one of the agents that cause physical mutations.Ultraviolet radiation has different wavelengths, does not cause ionization and has low penetrating power [7].Ultraviolet radiation can only penetrate the surface layer of cells and does not reach the gonads.Embryonic cells actively growing and dividing have a higher sensitivity to radiation.However, ultraviolet radiation can act as a mutagen and can kill cells at high doses [8], [9].This research used Drosophila melanogaster as an organism model.D. melanogaster has a genetic constitution relatively similar to humans.On the other hand, D. melanogaster is easy to obtain and maintain, lays lots of eggs, reproduces quickly and matures quickly (10-14 days old) [10]- [13].D. melanogaster is often used as a research object as a model organism for multicellular organisms because it is small and cheap, and most of the genes that cause disease in humans are also found in D. melanogaster [14].
Furthermore, the increase in UV-R impacting various sectors suggested the importance of revealing the extent of UV-R's impact on our genome.However, research on the effects of UV-R on D. melanogaster, is still scarce.Therefore, this study aimed to evaluate the impact of UV-R on crossing over in D. melanogaster as a model organism.In addition, this research needs to be conducted to reinforce the importance of protecting from UV-R.

Tool and material
The tools used during the research were a binocular microscope, culture bottles, a UV-R tool, and a laptop.The materials used were D. melanogaster, culture media, water, and banana varieties Rajamala.

Preparation
D. melanogaster used in this research was obtained from the Genetics Laboratory, State University of Malang.Two strains were chosen as research objects: wild type (N) and black body vestigial wing (bvg).The composition of the media consists of ± 2500 mL water, 700 g banana varieties Rajamala, 200 g fermented cassava, and 100 g palm sugar.All media was boiled for 45 minutes.The quantity of food was enough for 35 culture bottles.D. melanogaster was cultured in glass bottles measuring 200 ml (diameter = 7 cm, height = 9 cm), and each glass bottle was filled with 30 ml of standard media.

Treatment UV-R
Crossing and UV-R treatment were carried out in several stages.First, the ♀N >< ♂bvg strain was crossed into glass bottles with solid medium (slices of banana varieties Rajamala with a thickness of ± 1.5 cm).Second, the ♂bvg strain was released after two days of crossing.Third, the ♀N strain was released when there were eggs (F1).Fourth, the eggs in the solid medium were exposed to UV light for different durations: 0, 3, 6 and 9 minutes.Fifth, tweezers took the solid medium from the bottle and then transferred it to a sterile petri dish.Sixth, the solid medium was transferred to a liquid medium after UV treatment.Seventh, after the pupa formed, the pupa was collected.Eighth, ♀N progeny F1 >< ♂bvg were crossed in a liquid medium.Ninth, the progenies of F2 were calculated and recorded for further analysis.

Statistic analysis
Data analyzed were progenies of F2.The frequency of recombinant progeny can be calculated by: The frequency of recombinant progeny = Data on the number of phenotypes and sex of the F2 progeny were analyzed using single-factor ANOVA using SPSS.The analysis was conducted to determine whether there were differences in the duration of UV exposure to crossing-over.Data on recombinant progenies were also presented in diagram form.

Results and Discussion
This study showed three main results.The first was the results of the frequency of recombinant progeny F2.Based on the results of calculating the frequency of recombinant progeny F2, it showed that the 3 longer the duration of UV-R, the greater the frequency of recombinant progeny.Figure 1 shows the lowest recombinant progeny frequency when not given UV-R but the highest recombinant progeny frequency when given UV-R for 9 minutes.

Figure 1. Difference in recombinant frequency over varying time durations
The frequency of recombinant progeny in each treatment increased because UV-R initiated the formation of strand breaks and triggers crossing over.In line with research conducted on the budding yeast group Saccharomyces cerevisiae.UV can induce recombination between sister chromatids and homologous or heterologous chromosomes [14].Crossing over is influenced by UV-R and can initiate strand breaks in DNA structure [5].
The second was the different morphology.The morphological view showed the differences in the morphology of D. melanogaster that was given UV-R and was not given UV-R.D. melanogaster given UV-R became smaller and slower than not given UV-R.Susmita et al. [21] showed that UV-R can change the morphology of fruit flies, especially in their wings.These results indicated that UV-R is more likely to influence longevity, lifespan, fertility, and the morphological appearance of D. melanogaster than its direct influence on crossing-over [10].
The third was the results of single-factor ANOVA.Based on the results of single-factor ANOVA, the p-value (0.456) was more than α (0.05), meaning Ho was accepted and Ha was rejected.The effects of the single factor ANOVA pointed out that the length of UV-R had not affected the frequency of crossing between the two strains♀N><♂bvg.Table 1 summarises the results of the single-factor ANOVA analysis.UV-R can act as a mutagen, and high doses of UV-R can kill cells [9].Embryonic cells (D. melanogaster eggs) that are actively growing and dividing have a higher level of sensitivity to radiation.Wu  UV-R to penetrate [15].However, based on the data analysis, the results showed a significant value greater than alpha, indicating that the length of UV-R does not affect crossing-over.This phenomenon can happen because the duration of UV exposure is too short and needs to be increased to 0-24 hours.Cui et al. [16] found that the length of exposure to UV light required to affect increasing mortality and reduce the antioxidant effect was 0.5 -3 hours per day.
UV wavelengths also play a significant role.In line with Masuma regarding various UV wavelengths on the viability of D. melanogaster.The UV wavelength of 320 nm has a more harmful impact [17].Research on exposure to UV-R with certain wavelengths disrupts and reduces the longevity, lifespan and fertility of D. melanogaster [6], [10].Physical stressors such as radiation can reduce fruit fly survival and change the shape of their reproductive organs [18].Research from Liu et al. [19] showed that exposure to UV light has more impact on the susceptibility of D. melanogaster to parasite infection than its effect on crossing-over.
All in all, this research showed that there are three main results.(1) The longer the duration of the exposure to UV light, the higher the percentage of recombinant progeny.(2) The body size of the D. melanogaster progeny exposed to UV light was more petite, and their movements were slower.(3) The statistical analysis of single factor ANOVA revealed that the p-value (0.456) was more than α (0.05), which means the length of UV light had not affected the frequency of crossing over between the two strains.The limitations of this research are: this research used four durations of time of UV-R, conducted in D melanogaster, and viewed from a morphological view.The suggestions for further research related to molecular analysis of the effect of UV-R with the right wavelength and exposure time on the formation of strand breaks to initiate crossing over are very important

Conclusions
Based on the results showed that: (1) The longer the duration of the exposure to UV light, the higher the percentage of recombinant progeny.(2) The body size of the D. melanogaster progeny exposed to UV light was more petite, and their movements were slower.(3) The statistical analysis of single factor ANOVA revealed that the p-value (0.456) was more than α (0.05), which means the length of UV light had not affected the frequency of crossing over between the two strains.Several studies have shown that UV-R with a certain wavelength and exposure time of 0.5-3 hours per day has a more significant impact on the survival and mortality susceptibility of D. melanogaster than the direct effect on crossing-over events.Studies related to longer periods of exposure to UV radiation on crossing over frequency and molecular analysis need to be considered.This research can provide information regarding whether UV-R exposure can induce or inhibit recombination directly or indirectly.Further research can reveal the effect of UV-R from molecular analysis.

Table 1 .
The summary of ANOVA results on the effect of UV-R on crossing over.
et al. added that Drosophila eggs have two thin layers, namely vitelline and chorion, which allow