Detection of immune system activation in hemolymph of Drosophila larvae exposed to chitosan-coated magnetite nanoparticles

Drosophila melanogaster hemolymph cells are confirmed as a model to study the activation of immune system due to foreign stimuli like iron nanoparticles. The toxicity of nanoparticles is a cause for concern due to their effect on human health and the environment. The aim of this study was to detect the activation of cellular immune response in Drosophila larvae through the observation of hemolymph composition, DNA damage and larval viability, after the exposure to 500 ppm and 1000 ppm chitosan-coated magnetite nanoparticles for 24 hours. Our results showed activation of cellular immune response after exposure to the nanoparticles owing to the increment of hemocytes, the emergence of lamellocytes and the presence of apoptotic hemocytes. In addition, chitosan-coated magnetite nanoparticles produce DNA damage detected by comet assay as well as low viability of larvae. No DNA damage is showed at 500 ppm. The cellular toxicity is directly associated with 1000 ppm.


Introduction
Drosophila melanogaster has proved to be a suitable organism to test toxic effects of 17 different chemical elements due to its short life cycle and abundant offspring. In Drosophila 18 there are two main components of the innate immune response: the humoral and cellular 19 systems, both of which are activated upon immune challenge. The cellular response refers to 20 processes such as phagocytosis, encapsulation, and clotting that are directly mediated by 21 hemocytes [1][2][3][4]. The hemolymph of Drosophila is composed of three types of hemocytes: 22 plasmatocytes (95%) (macrophages) have the capacity to remove foreign material by 23 phagocytosis; crystal cells (5%) are involved in melanin synthesis during pathogen 3 24 encapsulation [5] and lamellocyte, which are large flattened cells whose differentiation is 25 induced in response to the immune system activation, i.e. the presence of foreign particles 26 in the hemocoel.

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The hemocytes of Drosophila are widely regarded as an excellent model for deciphering 29 general innate immune mechanisms and DNA damage in animals [2,6-8]. In vitro and in 30 vivo studies, no obvious toxicity of magnetic nanoparticles has been detected, but potential 31 toxicity has been observed in blood and also activation of the immune systems [9].  It is a non-toxic, biodegradable and biocompatible polysaccharide obtained from the 44 deacetylation of chitin [12]. Chitosan provides nanoparticles with free amino and hydroxyl 45 groups that enable the possibility to bind to a diversity of chemical groups and ions, leading 46 to a number of applications such as protein and metal adsorption, guided drug and gene 47 delivery, magnetic resonance imaging, tissue engineering and enzyme immobilization. 48 Furthermore, this type of nanoparticle could be used in hyperthermia treatment for destroying 49 malignant cells [13]. 50 Chitosan in Drosophila has been well studied. Drosophila has been utilized for both 51 production of chitosan [14] and as an in vivo model to investigate the transport and uptake of 52 nanoparticles covered with chitosan in the larval digestive tract after oral administration [15].

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In this study, we have observed in vivo, the cellular immune system activation in the 54 hemolymph of Drosophila larvae by the effect of Ch-Fe 3 O 4 NPs exposure. To achieve these 55 objectives, third instar larvae were exposed to two concentrations of Ch-Fe 3 O 4 NPs (500 and 56 1000 ppm) for 24 hours. Immune system activation was evaluated through hemolymph in 57 terms of total number of hemocytes, apoptotic plasmatocytes, lamellocytes and DNA damage 58 (comet assay). Additionally, the viability of larvae after the exposure to Ch-Fe 3 O 4 NPs was 59 estimated.   were identified. The hemocytes were counted using a Neubauer chamber in a microscope 90 ZEISS Imager A2 (40x/0.75).

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The number of hemocytes (normal hemocytes, apoptotic plasmatocytes and lamellocytes) in 92 larvae exposed to 500 ppm, 1000 ppm Ch-Fe 3 O 4 NPs and non-exposed was established.

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Statistical differences between treatments were analyzed through a one way analysis of   The data was compared with an analysis of variance (ANOVA) test with the SPSS 109 statistical software 23.0v. A probability of less than 0.05 (p < 0.05) was considered 7 110 statistically significant. The Bonferroni post-hoc test was performed to compare the control 111 versus the treatments exposed to Ch-Fe 3 O 4 NPs.

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Viability 113 A hundred of third instar larvae were exposed to each treatment (500 ppm and 1000 ppm,   inhomogeneity, we have averaged the spectra obtained from 25 points grid was averaged.

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The normalized weight average of each element and the standard deviation obtained by EDS 132 analysis are listed in Table 1. We found the organic elements that comes from chitosan, C,    Samples of XRD were dried on a microscope slide at 40ºC to avoid any organic degradation.

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Analysis XRD of the obtained average is the result of 6 different measurements from 5º to 90º (θ-142 2θ) angle. The Fe 3 O 4 NPs crystalline nature is confirmed from the XRD analysis (Fig 3). It is as large and irregular cells (Fig 4).  The total number hemocytes increased in larvae exposed to 1000 ppm (mean: 411.33) but 166 decreased in the larvae exposed to 500 ppm (mean: 201.67) compared with the control group 10 167 (235.67). In the case of apoptotic plasmatocytes, the larvae exposed to 1000 ppm also showed 168 an increase in the number of apoptotic plasmatocytes (mean: 54.33) compared with the 500 169 ppm (mean: 8.6) and control group (0.33). Lamellocytes were not present in the control 170 larvae, but this type of cell was observed in the larvae exposed to 500 ppm (1.3) and 1000 171 ppm (13.3) (Fig 5).  The comet assay was used to observe potential DNA damage in the hemocytes of larvae were identified (Fig 6).

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The level of DNA damage produced for each treatment was estimated in function of the % 196 of DNA in the comet tail ( Fig 7A) and the comet tail length (µm) (Fig 7B). The highest level 197 of DNA damage was observed in the larvae exposed to 1000 ppm, followed by the larvae 198 exposed to 500 ppm and finally the control larvae. However non-statistical differences were 199 observed between the larvae exposed to 500 ppm and 1000 ppm (p<0.05). Therefore, both  (Table 3). viability was observed in the larvae exposed to 1000 ppm Ch-Fe 3 O 4 NPs (51.3%). A higher 214 percentage was observed in the larvae exposed to 500 ppm (61.0%) and the highest 215 percentage of viability was observed in the control test (84.0%). It is evident that mortality 216 is directly associated to Ch-Fe 3 O 4 NPs concentration, therefore, exposure to high-dose 217 concentration of Ch-Fe 3 O 4 NPs produce high mortality of larvae (Fig 8).

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Significant statistical differences were observed in the viability between 1000 ppm and 219 control treatment. eclosioned adults (Fig 9).

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A) larva not exposed (control), B) larvae exposed to 1000 ppm, C) adult exposed to 1000  strand breaks [25,26]; this will allow identification of the possible mode of action of 286 nanoparticles at the molecular level.

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In addition, the effect of Ch-Fe 3 O 4 NPs on the viability of larvae was evidently toxic, 288 producing up to 50% of mortality in larvae exposed to 1000 ppm Ch-Fe 3 O 4 NPs, while the 289 non-exposed larvae presented only up to 16% of mortality. The low viability is associated to associated to the dose concentration Ch-Fe 3 O 4 NPs. The toxic effect of nanoparticles is higher 309 in larvae exposed to 1000 ppm concentration, while 500 ppm could have toxic risks but have 310 not been detected in this study.