The effects of antibiotics on the reproductive physiology targeting ovaries in the Asian tiger mosquito, Aedes albopictus

Mosquitoes have adapted to various environmental conditions. Symbionts with mosquitoes impact this adaptation in different environments. In the field, mosquitoes could get exposed to antibiotics during their developmental period, which could reduce or eliminate their symbiotic microbes. However, the side effects of the antibiotics on the ovary and reproductive physiology of the Asian tiger mosquito, Aedes albopictus remains unknown. In this study, we investigated the effects of tetracycline and combinations of rifampicin and tetracycline at environmentally acceptable levels on the reproductive physiology of ovaries in Ae. albopictus. Rifampicin and tetracycline in combination reduced the hatching rate and fertility of Ae. albopictus compared to the untreated control group. These antibiotics induced histopathological damage and reactive oxygen species production in the ovaries. The combination of antibiotics decreased the expression of surface protein of Wolbachia (WSP) in Ae. albopictus. Additionally, the expression of Toll like receptor 2 (TLR2) and Myd88 were triggered by the combinations. The findings demonstrate the detrimental effects of antibiotics, particularly combinations of rifampicin and tetracycline, on the reproductive capacity of Ae. albopictus females.


Introduction
Mosquitoes of the genus Aedes transmit infectious diseases including dengue fever, chikungunya, and filariasis (Gubler 1998). Among them, the Asian tiger mosquito Aedes albopictus is an invasive vector species that is an emerging public health concern worldwide (Bonizzoni, Gasperi, Chen, & James 2013). In the Republic of Korea, Ae. albopictus could be the vector for various viruses (Chang, Kim, Ha, et al. 2018). To control mosquito populations, various strategies have been used including insecticides. However, increased resistance to insecticides has prompted efforts to develop alternative methods. A new approach using a symbiotic bacteria including Wolbachia has been tested and used in several countries (Hegde,  Mosquitoes could be exposed to antibiotics in various ways. In the field, mosquito may hatch in water contaminated by antibiotics and could be exposed throughout their development. (Endersby-Harshman et al. 2019). In another case scenario, when a mosquito bites the host, antibiotics can enter the mosquito body (Gendrin et al. 2015;Thayer 1973). In laboratory conditions, antibiotics have been used to remove the microbes from mosquito gut (Ballard & Melvin 2007;Mousson et al. 2010). Elimination of microbes from their host by antibiotics has been applied to examine the role of microbes (Ballard & Melvin 2007, Mousson et al. 2010. Antibiotics affect mosquitoes in various ways. They affect the lysis of red blood cells, delay protein digestion and reduce egg production in female mosquitoes (Gaio Ade et al. 2011). They are also known to suppress protein biosynthesis and to produce toxic effects in the host (Arenz & Wilson 2016). In addition, antibiotics affect the mosquito by interfering with the microbe-induced cytoplasmic incompatibility (CI) during reproduction (Endersby-Harshman et al. 2019). For example, the antibiotics tetracycline hydrochloride and chloramphenicol make the cross Ae. polynesiensis male × Ae. kesseli female incompatible (Trpis, Perrone, Reissig, & Parker 1981). The use of antibiotics is required for Wolbachia removal to avoid genetic depression in the outbreeding procedures (Calvitti, Moretti, Skidmore, & Dobson 2012). In addition, the cell structure of the ovary could be changed by feeding to formation of matured eggs (Greif 2016). Therefore, exposure to antibiotics might have an impact on the reproductive organ of mosquitoes. However, very little is known about the effect of antibiotics in the ovary and reproductive capacity of Ae. albopictus.
In this study, we investigated the effect of antibiotics (tetracycline and combinations of rifampicin) on reproductive capacity of Ae. albopictus. To compare the effects of antibiotics, the hatching rate and fertility were investigated. Additionally, the morphology of follicles and nurse cells of the ovary following treatment with antibiotics were confirmed histologically by hematoxylin and eosin (H&E) staining. After treatment with tetracycline and combinations of rifampicin with tetracycline, the distribution of reactive oxygen species (ROS) was visualized in the ovaries of Ae. albopictus. Our experimental results reveal the influence of antibiotics on the ovaries of Ae. albopictus.

Mosquito rearing
Aedes albopictus (Skuse) were collected from Cheongju-si, South Korea, and screened out following identification keys. They were reared and adapted under standard laboratory conditions. Mosquitoes were maintained on a 10% sugar solution at 27 ± 1°C and 65-75% humidity with a 12-h:12-h light/dark cycle. Eggs were hatched in water and larvae were reared in a 2 L plastic container. After pupation, the mosquitoes were transferred to insect rearing cages. Eggs were collected on filter paper with distilled water. This study was conducted strictly in accordance to the guidelines and recommendations provided in the 'Guide for the Care and Use of Laboratory Animals' of the National Institutes of Health (Korea NIH, Cheongju, Republic of Korea).

Antibiotic treatment to eliminate Wolbachia
Aedes albopictus mosquito samples were collected from the 10th generation (F10) under laboratory conditions. They were treated with tetracycline and rifampicin to obtain a Wolbachiafree strain as described previously (Mousson et al. 2010). First instar larvae and pupae were treated with 10 mg/L tetracycline (Sigma Aldrich, St. Louis, MO, USA) in the F10 generation. Subsequently, 20 mg/L tetracycline was treated in larval and pupal stages of F11 generation. Additionally, 40 mg/L tetracycline was used to treat the F12 and F13 generations during the larval and pupal stages. After the last larval and pupal treatment, only the F13 generation was treated with rifampicin (2.5 g/L) (Sigma Aldrich) dissolved in 10% sucrose solution. In this study, 10 mg/L of tetracycline-treated F10 generation and 40 mg/L of tetracycline and 2.5 g/L rifampicin-treated F13 generation were investigated for comparison of single and combination treatment. These treatment groups were compared with the untreated group.

Egg hatching and bleaching
After treatment with 10 or 40 mg/L tetracycline and 2.5 g/L rifampicin, blood-fed females laid eggs, which were used for egg hatching. A total of 100 eggs from each group on a small piece of filter paper were placed in a 400-mL plastic container. The mosquito eggs were flooded with a 0.25 g/L yeast solution in distilled water. The hatched larvae were counted for up to 24 h. All experiments were performed in triplicate. The eggs were bleached with 50% sodium hypochlorite solution containing approximately 6% chlorine for 20 min to determine whether the eggs were embryonated. The bleached eggs were viewed and counted under a microscope (Olympus, CKX53 culture microscope, Tokyo, Japan).

Histopathological examination
Mosquitoes were fixed in 10% formalin overnight at 4°C and 0.25% Triton X-100 at room temperature for 15 min. Fixed mosquitoes were washed three times with phosphate-buffered saline (PBS) and embedded in paraffin. Using a standard protocol (Kpclab, Gwangju, Gyeonggi, South Korea), the ovary section slices were stained with haematoxylin and eosin. The stained ovary sections were observed under a CKX53 Olympus inverted microscope with a 100 × or 400 × objective lens.

Detection of reactive oxygen species
For ROS detection, the ovary samples were dissected from mosquitoes anaesthetized at 4°C using a needle-like probe and forceps. Subsequently, the ovaries were rinsed in PBS with 0.0025% Triton X-100. The rinsed ovaries were stained with 30 μM concentration of CM-H 2 DCFDA (a chloromethyl derivative of H 2 DCFDA (5-(and-6)-chloromethyl-2′, 7′dichlorodihydrofluorescein diacetate). Subsequently, the ovaries were incubated for 5-15 min at room temperature (in dark conditions) using a 30 μM solution of CM-H -2 DCFDA in PBS. After rinsing in PBS, the intracellular ROS generation in the ovaries were observed with a fluorescent microscope. Fluorescence was visualized at excitation and emission wavelengths of 490 and 530 nm, respectively. The CM-H 2 DCFDA is a fluorescent probe that reacts with intracellular ROS (Eruslanov & Kusmartsev 2010) and reveals the distribution of ROS within follicles.

RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)
From the dissected ovaries, RNA was isolated using an RNA extraction kit (Qiagen, Hilden, Germany). RT-PCR was conducted using a DiaStar 2 × OneStep RT-PCR premix kit (SolGent, Daejeon, South Korea). Wolbachia surface protein (WSP) primers were used to confirm the presence of Wolbachia (Zhou, Rousset, & O'neil 1998). β-Actin was also detected using previously described primers (Park et al. 2016). TLR6 and Myd88 primers for Ae. albopictus were designed using the online Primer3 web-based interface (http://frodo. wi.mit.edu/primer3/) (Table 1). RT-PCR was performed under the following conditions: an initial step at 50°C for 30 min for reverse transcription and 95°C for 15 min for denaturation, followed by 30 cycles of 45 s at 95°C, 52 s at 52°C, and 45 s at 72°C, with a final extension step at 72°C for 10 min.
To determine the band intensity, relative expression changes were calculated between the intensity of samples normalized to the β-actin housekeeping gene using ImageLab software version 5.2 (Bio-Rad, Hercules, CA, USA).

Statistical analysis
Mean values were calculated in triplicate. The results are expressed as the mean ± standard deviation. All data were statistically analyzed using the t-test (P value) in Sigma Plot, version 12.5 (Systat Software, Inc., San Jose, CA, USA). Significance values of P < 0.05 were considered to indicate statistical significance.

Effects of antibiotics on egg hatching
To investigate the effect of antibiotics on egg hatching, 10 mg/L tetracycline or 40 mg/L tetracycline with 2.5 g/L rifampicin were treated. The average hatching rates were significantly affected by antibiotic treatment. The average hatching rates were approximately 80.0 ± 18.3% and 69.6 ± 19.2% in the untreated group and 10 mg/L tetracycline-treated group, respectively. In the group treated with 40 mg/L tetracycline with 2.5 g/L rifampicin, the average hatching rate was 7.0 ± 14.0% (Fig. 1). Therefore, when Ae. albopictus were treated with 10 mg/L tetracycline and 40 mg/L tetracycline with 2.5 g/L rifampicin, a respective decline in egg hatch by 13.0% and 91.2%, was observed compared to untreated group. This infers that the egg hatching rate was significantly impaired in the combination antibiotics treatment group when compared with untreated samples and treatment with 10 mg/L tetracycline (P < 0.05).

Effects of antibiotics on egg fertility
To determine the effect of antibiotics on fertility and infertility, the eggs were bleached and embryonated and unembryonated eggs were distinguished. Bleach digestion removes the egg chorion while leaving the serosal cuticle intact to observe the formation of embryogenesis. Figure 2A shows the unbleached eggs which were not treated with bleaching solution and antibiotics. After bleaching the eggs with sodium hypochlorite solution, the unembryonated eggs (Fig. 2B) and embryonated eggs (Fig. 2C) were visualized. The percentage of embryonated and unembryonated eggs was counted in antibiotics-treated and untreated groups. The percentages of  embryonated eggs were 89.43 ± 9.09%, 97.48 ± 2.39%, and 62.66 ± 3.92% in the untreated group, 10 mg/L tetracycline-treated group, and 40 mg/L tetracycline and 2.5 g/L rifampicin-treated group, respectively (Fig. 2D).
When Ae. albopictus were treated with 40 mg/L tetracycline with 2.5 g/L rifampicin, a respective decline in percentage of embryonated eggs by 30.0%, was observed compared to untreated group. This result indicates that the egg fertility  was significantly declined in the combination antibiotics treatment group when compared with untreated samples and treatment with 10 mg/L tetracycline (P < 0.05).

Histopathological effect of antibiotics in the ovary
The normal follicle forms the vitelline membrane between the oocyte and epithelial cells (Fig. 3A and B). The epithelial cell layer was thickened in the group treated with 40 mg/L tetracycline + 2.5 g/L rifampicin ( Fig. 3C and D). Further, the boundary between the oocyte and nurse cells was obscured compared to the untreated group. In the oocyte, degradation of the yolk granules was observed ( Fig. 3C and D). A light region at the periphery of the oocyte was more prominent within the epithelial follicles in the 40 mg/L tetracycline with 2.5 g/L rifampicin-treated group compared to in the untreated group. Hence, the combinations of antibiotics induced histopathological damages in the ovary of Ae. albopictus. Figure 4A and C show optical images of untreated and 40 mg/L tetracycline with 2.5 g/L rifampicin-treated ovaries. The follicles showed a weak fluorescence signal in the untreated group (Fig. 4B). In contrast, a strong signal was observed in the oocyte nucleus and nurse cell nucleus in the 40 mg/L tetracycline with 2.5 g/L rifampicin-treated group.

Detection of ROS in the mosquito ovary
The signal from the oocyte cytoplasm was much weaker compared to that in the nucleus (Fig. 4D).

Ovary-specific expression of Toll like receptors in the ovaries
Following exposure to antibiotics, Wolbachia surface protein (WSP) expression was decreased in a dose-dependent manner (Fig. 5). In the 10 mg/L tetracycline-treated group, TLR2 and Myd88 expression was up-regulated 1.5 and 6.1 times, compared to in the untreated group, respectively. TLR2, TLR6, and Myd88 expression was significantly up-regulated 1.2, 3.3 and 8.5 times in 40 mg/L tetracycline with 2.5 g/L rifampicin-treated ovaries compared to in the untreated group, respectively. In contrast, Cactus expression was downregulated about 0.7 times in the 40 mg/L tetracycline and 2.5 g/L rifampicin-treated group, compared with the 10 mg/L tetracycline-treated group and untreated group (Fig. 5).

Discussion
Mosquitoes have been able to adapt to various habitats and are considered vectors for viruses causing clinical manifestations in humans (Ricci et al. 2012). With the environmental contamination by antibiotics ever increasing, mosquitoes are at a greater risk to be exposed (Endersby-Harshman et al. 2019;Gaio Ade et al. 2011). In most cases, mosquitoes such as Ae. Images of 40 mg/L tetracyclinewith 2.5 g/L rifampicin-treated ovary (100x and 400x). Fc, follicular epithelial cell; Nc, nurse cell; Oo, oocyte; po, presumptive follicle.

The effects of antibiotics in mosquito
Entomological Research 51 (2021) 65-73 aegypti get exposed to greater antibiotic concentrations when they inhabit drains or underground water resources than when they breed in containers filled by rain or drinking water (Endersby-Harshman et al. 2019). Antibiotics such as tetracycline and rifampicin can be particularly devastating in their mode of action.
Tetracycline is a broad-spectrum antibiotic for Gram-positive and Gram-negative bacteria, chlamydiae, mycoplasma, protozoan parasites, or rickettsiae (Ballard & Melvin 2007). It can inhibit mitochondrial protein synthesis and delay the oviposition in insect (Kurlovs, Li, Cheng, & Zhong 2014;Zeh, Bonilla, Adrian, Mesfin, & Zeh 2012). Rifampicin is a lipid-soluble antibiotic that can penetrate membranes and concentrate in cells (Dhillon & Mitchison 1989). It influences bacterial diversity and richness that can be incompletely recovered even after discontinuing the antibiotic (Rosas et al. 2018). The presence of antibiotics including tetracycline in the environment has been  investigated in previous study. Tetracycline concentrations in reported field maxima in mosquito container habitats ranged from 0.04 to 0.97 μg/L (Curtis, Matzen, Neira Oviedo, et al. 2015). Its level in animal waste holding ponds is 1,000 μg/L (Campagnolo, Johnson, Karpati, et al. 2002). Tetracycline at a concentration of 1.0 to 5.0 × 10 6 μg/L was required to clear mosquitoes of Wolbachia infection in the laboratory (Li, Floate, Fields, & Pang 2014). This is significant as Wolbachia infected mosquitoes have been used in the dengue control international programs wherein these are introduced into endemic regions to replace the resident populations by means of CI (McMeniman et al. 2009;Walker, Johnson, Moreira, et al. 2011;Flores & O'Neill 2018). Mosquito control using Wolbachia could be affected by antibiotic pollution in the environment.
However, the chemical toxicity of antibiotics towards reproductive physiology of mosquitoes has still not been fully explored. In this study, we detailed the effects of the antibiotics tetracycline and rifampicin on egg hatching and fertility. Antibiotic treatment reduced the hatching of eggs with a lesser number of embryonated eggs. Hence, the results suggest a direct correlation relating to the influence of antibiotics on the reproductive capacity of mosquitoes. The reduced hatching rate and fertility in mosquitoes may be ascribed to the inhibition of amino acid absorption (Chopra & Roberts 2001;Thayer 1973). A total of ten amino acids, including arginine, valine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and histidine, have been found to be essential for egg production and larval development (Dimond, Lea, Hahnert, & Delong 1956). This substantiates the requirement of amino acid absorption towards reproductive capacity in mosquitoes. Another reason for reduced reproductive capacity in mosquitoes is the clearance of bacteria through antibiotics exposure. Especially, Wolbachia are intracellular bacteria and are maternally inherited as an infection by many arthropod species (Dobson et al. 1999). Wolbachia effects reproductive alterations, such as feminization, parthenogenesis, male killing, and CI in arthropods (Noda, Miyoshi, & Koizumi 2002). Interactions between Wolbachia and the host can influence fecundity, immune response, stress response, and metabolite levels (Brownlie et al. 2009;Caragata et al. 2013;Dutra et al. 2017). In this study, we found dose-dependent decrease in the expression of WSP that in turn stimulates increased transcription of immune genes in mosquitoes. In addition, Wolbachia downregulates apoptosis in the developing ovaries. Therefore, elimination of bacteria leads to the apoptosis of ovarian cells (Pannebakker, Loppin, Elemans, Humblot, & Vavre 2007;Werren, Baldo, & Clark 2008). Our results corroborate the earlier findings showing reduced hatching rates, fertility, and ovarian damage following antibiotics treatment with Wolbachia inhibition. Treatment with antibiotics may affect the innate immune system of mosquitoes. The major immune signaling cascades in mosquito are Toll, immune deficiency (IMD), and Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathways. RNA interference (RNAi) pathway is not a classical innate immune pathway, but plays an important role in antiviral defense (Sim, Jupatanakul, & Dimopoulos 2014). In a previous study, the innate immune responses to Wolbachia in Brugia malayi and Onchocera volvulus was found dependent on Toll like receptors (TLRs), such as TLR2, TLR6, and Myd88, but not TLR4 (Hise, Daehnel, Gillette-Ferguson, et al. 2007). In addition, Wolbachia induced ROS-dependent activation of the Toll pathway in Ae. aegypti (Pan et al. 2012). In our examination of the Toll pathway-regulated genes using RT-PCR, we found that the treatment of a high dose (40 mg/L) of tetracycline with rifampicin induces TLR2, TLR6, and Myd88 mRNA expression in the ovaries. Alternatively, the expression of Cactus was downregulated in the ovaries. The Toll signaling is triggered by the Toll receptors that are activated by ligands. Adaptor proteins such as Myd88 lead to the phosphorylation and degradation of Cactus. Subsequently, Cactus degradation activates the transcription of Toll pathway-regulated genes (Sim et al. 2014). From our results, it is understood that the Toll pathway may be directly and indirectly activated by antibiotics in the ovary of mosquitoes. However, further detailing of reproductive physiology of female mosquitoes is needed to explain the detailed effects of broad-spectrum antibiotics such as tetracycline and rifampicin.
In conclusion, our study provides an increased understanding of the ovarian physiology in Ae. albopictus mosquitoes following treatment with antibiotics such as tetracycline and rifampicin. The hatching rate, fertility rate, histopathological changes, and altered TLR expression triggered by antibiotics were investigated. To the best of our knowledge, this is the first study to report on the effect of antibiotics in the ovary and reproductive capacity of Ae. albopictus. Given the findings of this study, the level of antibiotics in rearing mosquitoes will assist in understanding the reproductive capacity of mosquitoes involved in the mosquito control.