1. Introduction
The fruit fly
Drosophila melanogaster (Meigen) (Diptera: Drosophilidae) is a commonly used model for a wide range of studies, because of its short life cycle, adaptation to different ecological conditions, and ease of rearing in the laboratory. Previous experiments have demonstrated that different concentrations of some organophosphorus insecticides, plant secondary metabolites, and food dyes have toxic effects on survivorship of
D. melanogaster [
1,
2,
3,
4]. Antibiotics that possess antibacterial, antifungal, and anthelmintic properties have been incorporated into insect-rearing media for decades. Their influence on life parameters and their effective concentrations for control of microbial contamination in diets have been studied for many species [
5,
6,
7,
8,
9,
10]. It has been shown, for example, that clinically significant doses of some antibiotics lead to mitochondrial dysfunction and oxidative damage in patients caused by reactive oxygen species (ROS) [
11]. This raises the possibility that antibiotics incorporated into diets may also exert damaging effects on insects or decrease quality of the products of their activity, like honey [
12].
Numerous studies have indicated that the use of antimicrobial substances that prevent mold and bacteria from contaminating insect cultures can lead to developmental retardation, decreased survival, and short longevity of certain insects [
6,
7,
9,
13,
14,
15,
16,
17]. Many insect species are routinely maintained in very large colonies for research purposes and for large-scale insect pest management. The potential influence of dietary antibiotics on insect biology is of importance because their use may increase the costs of maintaining insect cultures, affect insect physiology, and potentially reduce the biological performance of insects reared for research and biological control programs. In the case of
D. melanogaster, it may also lead to misinterpretation of the results of experiments carried out using this model organism.
One of the antibiotics used in insect breeding is neomycin aminoglycoside—a broad-spectrum substance, naturally produced by
Streptomyces fradiae, often found in the environment [
18]. This antibiotic inhibits protein synthesis at initiation and elongation stages by binding 30S subunits, and sometimes 50S subunits, of ribosomes in prokaryotes [
19,
20]. There is limited information on the effects of neomycin on insects, whereas its physiological, biochemical, and molecular effects on the tissues and organs of higher eukaryotic organisms, including humans and animals, are well-known [
21,
22,
23,
24]. It was shown that higher levels of neomycin in both in vivo and in vitro treatments disorganized polytene chromosomes in the salivary glands of dipteran fourth instar
Chironomus sp. larvae, suggesting that neomycin may affect gene activity in insects [
25]. Based on general pro-oxidative effects, we demonstrated that antibiotics and anthelmintic exert oxidative damage and stimulate increased antioxidant and detoxification enzymes in some model insects [
26,
27]. Like other antibiotics, neomycin can also have unintended effects on animals and microorganisms in the environment when it is released through various uncontrolled routes [
18,
28]. This means that in addition to laboratory-controlled exposure to neomycin,
Drosophila may also be exposed to this antibiotic under natural conditions. Like all insects, fruit flies must adapt to the presence of different substances in the environment. Badyaev [
29] indicated that stressful and unsuitable environments may impair the integration of morphological regulatory systems, causing alterations of normal development. In this context, morphology provides an excellent opportunity to investigate the factors that may induce changes during growth. There is no information on the effects of antibiotics and other antibacterial substances on the developmental stability of insects, including
Drosophila spp.
Does adding neomycin to the medium cause developmental instability in
Drosophila? If so, can this be demonstrated by analyzing the index of fluctuation asymmetry? We predicted that more asymmetrical individuals with developmental instability might arise when insect larvae are reared on a diet containing antibiotics, in terms of nutritional stress, as observed for species that are forced to grow in an unfavorable habitat. Such asymmetry has been reported as a sign of environmental stress by several researchers [
30,
31]. In this paper, we measured fluctuating asymmetry (FA) in the size of anal papillae of larvae, as well as in wing-vein length of imagoes, to examine the hypothesis that breeding insects with a neomycin-containing diet under laboratory condition may create a stressful environment for the
D. melanogaster causing developmental instability. FA is often treated as a general biomarker of environmental and/or genetic stress [
32,
33,
34]. Anal papillae are clearly distinguishable structures of dipteran larvae. They are important structures for the regulation of osmotic conditions, due to their modified epidermis, e.g., lack of exocuticle and modifications of the size and ultrastructure of epithelial cells [
35]. Therefore, they can be influenced by various factors and may be early markers of stress. They can be easily observed and measured, and, to the best of our knowledge, the FA index has not been determined in
D. melanogaster larvae using these structures to date. Various studies have shown differences in the morphology of wings reflected in the FA are sensitive indicators of environmental stress [
36,
37,
38]. Moreover, Debat et al. [
36] indicated that wing shape can vary in more flexible ways and be more informative than simple traits such as the size of other body parts when investigating deteriorated larval survival, development, and adult fitness of insects exposed to environmental stressors (temperature, insecticides, and other chemical pollutants, dietary additives such as antimicrobial agents). Since the method of FA analysis is easy to apply, fast, and relatively cheap, it can be routinely used to compare numerous potential stressors on various species, worldwide.
This method was therefore applied to determine the possible stressful effects of sublethal concentrations of neomycin on D. melanogaster after rearing larvae on diets amended with the aminoglycoside antibiotic neomycin.
4. Discussion
Fluctuating asymmetry (FA) is a measure of developmental instability expressed as a difference in accurate bilateral symmetry, caused by environmental stresses, and genetic problems during development. Environmental stressors that may induce FA in insects, usually include temperature extremes, oxygen deficiency, poor diet, and chemical pollution [
49,
50,
51,
52]. There is no information on the effects of antimicrobial substances such as antibacterials, antivirals, and antihelminthics on the fluctuating asymmetry (FA) of insects, including
D. melanogaster. In many studies, the effect of various pesticides on the FA of numerous insect groups has been investigated [
3,
4,
49,
50,
51,
52]. Antipin and Imasheva [
52] examined the effects of a chlorine–organic insecticide, endosulfane (thiodan), on phenotypic and genetic variation in wing length, thorax length, the number of orbital bristles, and the number of sternopleural bristles of
D. melanogaster. Experiments carried out on
Ceriagrion sp. larvae suggest that FA may be regarded as an indicator of pesticide stress. [
50]. The usefulness of FA has also been demonstrated by investigating the effect of temperature and pesticide exposure on larval development in
Copera annulata (Selys), among other studies. Insecticide treatment did not significantly affect the mortality of the larvae of damselfly, but the FA values of three traits decreased at lower concentrations, and then increased slowly with increased insecticide doses [
49]. The results of these studies indicated that traits related to body size, expressed by the FA index, could be used as a suitable universal indicator of the environmental stress caused by chemical pollutants in populations of insects. A previous study reported the effects of chemicals such as arsenic and lead on FA in
Drosophila [
53]. It is known that nutrients available in the larval period are effective in all developmental stages of the insect [
54]. For this purpose, the content and quality of artificial diets used for growing insects in the laboratory environment must be balanced [
55,
56,
57,
58,
59]. Various antifungal, antibacterial, and antihelminthic agents are frequently used to improve diet quality. In this study, we used an asymmetry indicator to determine whether neomycin impaired the nutritional quality of the diet and led to nutritional stress. We checked whether the relationship between asymmetry parameters and the quality of the artificial diet could be determined. The effect of nutritional stress during development on FA has been addressed in a range of organisms [
60,
61]. Vijendravarma and coworkers (2011b) observed that larval malnutrition of
Drosophila melanogaster had a significant effect on all wing traits at both the plastic and evolutionary levels. When raised on poor larval food, both control and selected populations had smaller wings than when raised on standard food [
62]. On the other hand, experiments with
Drosophila ananassae showed that the level of fluctuating asymmetry was similar in flies reared on poor and standard media [
54]. This means that drawing general conclusions based on the use of fluctuating asymmetry as an indicator of nutritional stress should be done with caution.
Symmetry is important for the movement of an animal, reveals its physiological state, and it also plays an important role in reproduction, courtship, and reproductive behavior [
63,
64,
65]. The results of our research indicated that neomycin causes significant stress for larvae and imagoes of
D. melanogaster. Both examined life stages revealed significant differences in the FA indices of measured parameters. The results obtained for larvae appeared to be concentration-dependent; the FA index increased with the concentration of neomycin. Interestingly, there was also a distinct difference between effects caused by lower (150 and 300 mg/L) and higher (600 and 900 mg/L) concentrations of the antibiotic. Although the lowest FA values were observed in the control group, low levels of antibiotics did not lead to a significant increase in asymmetry. The highest FA index, observed in the group exposed to 900 mg/L neomycin, showed that the larvae had a pronounced nutritional stress response. The Pearson correlation coefficients for anal papillae (0.65 and 0.71) indicated a correlation between neomycin concentration and asymmetry. The strong effect for larvae may be due to their softer cuticle, and the inability to avoid the exposed environment (the larvae cannot emigrate from the substratum). Following this stage, they undergo processes that lead to fast growth, and development of tissues and organs that prepare them for the pupal stage. Therefore, any stress may significantly affect their morphology. For measurements of the length of anal papillae, the results were inconclusive. Although the addition of neomycin reduced their size compared to the controls, which was consistent with our previous observations [
3], there was no clear tendency to decrease in length as the concentration of the antibiotic increased. Interestingly, the lowest and highest concentrations did not cause the most distinct effects. It is possible that the low concentrations of the antibiotic did not stimulate the insects’ detoxifying pathways intensively enough, and neomycin remained in the body. On the other hand, the highest concentration might have been too high to be neutralized completely and some amounts of the antibiotic were still present in the environment.
The main role of the anal papillae is to regulate osmotic conditions. Neomycin has been described as a factor that affects concentration of potassium ions and blocks ryanodine RyR2 receptor [
66,
67,
68]. The ryanodine receptors are also responsible for calcium storage within cells [
69]. Therefore, due to altered concentrations of ions, this antibiotic may disturb osmotic conditions and, in consequence, lead to morphological malformations of anal papillae, which manifested in the measured FA. The ryanodine receptors are necessary for proper muscle physiology in fruit flies [
70]. Hence, neomycin may significantly affect the development of insects and cause lethal and sublethal effects. These receptors are also present in insects’ photoreceptor cells, including in
Drosophila [
71]. However, to our best knowledge, the effect of the antibiotic on the physiology of
D. melanogaster cuticle-forming epithelium and anal papillae, in particular, have not been reported to date. Therefore, we plan to carry out further studies on the ultrastructure and morphology of organs and tissues exposed to antibiotics in the future.
The wing FA indices indicated a large but variable correlation. The Pearson correlation coefficients varied from 0.52 to 0.87. However, there was no strict rule and the veins did not show the same pattern of the FA value alterations. On the other hand, the mean length of veins was the highest for the control group, with neomycin-exposed groups showing lower values. That suggests that the antibiotic may decrease the size of some morphological/anatomical organs. This result was in line with results obtained by Ventrella et al. [
3] and Chowański et al. [
4], where decreased size and shortened time of development of
D. melanogaster exposed to some natural substances was observed. It is possible that the insects speed up their development to the imaginal stage in order to decrease the time of exposure to toxic substances. In consequence, some processes and metabolic pathways may malfunction; the growth may be decreased, which results in asymmetry and the limited size of morphological features. However, due to the longer period of development, the presence of the pupal stage (when the insect does not feed and reorganizes its internal structures), the ability to move away from the neomycin-containing substratum, and a stronger cuticle, the malformations may be less significant than in the case of larvae. Additionally, the process of detoxification may be more intensive in imagoes than in larvae. In consequence, FA indices were not as significant as in the case of larvae. However, asymmetrical veins may significantly affect flight ability in imagoes, since veins play a crucial role in wing strengthening and in conditioning the surface of wings. Therefore, the increased asymmetry may affect imagoes in their behavior, locomotion, and perhaps reproductive success.
Although not lethal in the tested range of concentrations, neomycin may cause significant sublethal changes that manifest in FA. In consequence, it may lead to lower vitality of flies exposed to the antibiotic and limit the population size. It is possible that similar results would be observed not only in our model species, but also in others. The harmful activity of neomycin on insects was previously reported for
Pimpla turionellae [
72]. Most probably, the effect is caused by reactive oxygen species (ROS), which are produced during exposure to neomycin and other aminoglycosides [
73,
74]. Additionally, an increased level of O
2− and H
2O
2, together with a decreased level of Cu, Zn superoxide dismutase was reported in cells exposed to gentamicin [
75]. An imbalance between the level of ROS and antioxidant enzymes leads to oxidative stress within exposed organisms. If the cells are unable to achieve homeostasis, the imbalance will cause physiological abnormalities and morphological defects, which will lead to developmental errors and imperfections, which correlates with an increase in FA [
76]. We plan to continue this research in the future, focusing on other antibiotics added to the media used to rear fruit flies, especially since
Drosophila melanogaster is becoming increasingly recognized as a model organism in food and nutrition research.