Evaluation of different concentrations antibiotics on gut bacteria and growth of Ectropis obliqua Prout (Lepidoptera: Geometridae)

The use of antibiotics to remove gut bacteria is a commonly used method to study gut function of insects. We assessed the impact of the artificial diet made from tea powder with different concentrations of antibiotics mixture (containing tetracycline, gentamicin, penicillin, and rifampicin) on Ectropis obliqua Prout (Lepidoptera: Geometridae) survival, growth, and reproduction. Antibiotic-induced bacterial clearance was monitored by Polymerase Chain Reaction (PCR) and by Colony-counting Methods. The results indicated that administration of the antibiotic mixture at a concentration of 200μg/ml caused the increase of gut bacteria, while a concentration of 300μg/ml was more effective in clearing gut bacteria but the concentration of 400μg/ml caused a large number of larval deaths. The concentration of 300μg/ml had no significant effect on the growth and development of E. obliqua, but had impact on fecundity. Therefore, we could use 300μg/ml concentrations of the antibiotic mixture to obtain sterile E. obliqua to study the function of intestinal microbes.


Introduction 26
Insects are the most abundant class of animals worldwide, with more than one million 27 species. More than half of insect species feed on plants; therefore, insects are the most 28 important herbivores in the world [1]. Insect guts contain a substantial amount of microflora[2, food source, the adult flies only mate with members of the opposite sex that were raised on 34 the same food source, But these preferences all disappeared after the antibiotic treatment [9]. 35 By participating in the nitrogen cycle of the host to recycle the nitrogen from waste excreted, 36 termite intestinal microorganisms can maintain the host's nitrogen source balance [10]. 37 Metagenomic analysis of the gut bacterial community of Nasutitermes revealed that the 38 termites, which feed on plant xylem and phloem, host a large number of bacteria that can 39 Hamiltonella defense, which colonizes aphids, directly kills the larvae of aphids '  with antibiotics to remove its gut bacteria, therefore, and is unable to complete its life cycle 50 [15]. When antibiotics were used to remove the indigenous midgut bacteria of the gypsy 51 moth, Bacillus thuringiensis was unable to kill the gypsy moth larvae [16]. It is found that 52 exposure to antibiotics significantly altered the honeybee gut microbial community structure The first-instar larval of the E. obliqua were randomly selected and divided into 5 groups.    survival rate of the larval was no effect (Fig 1). PCR-Agarose gel electrophoresis analysis 152 confirmed that treatment with 300μg/ml or 400μg/ml of the antibiotic mixture more lowly 153 gene expressed and significantly reduced the number of gut bacteria in E.obliqua larvae (Fig   154   2). However, treatment with 200μg/ml of the antibiotic mixture resulted high gut 155 microorganisms gene expression. CFU analysis revealed that the number of cultivable gut 156 bacteria significantly reduced, as increasing the concentration of antibiotic mixture. When using 300μg/ml of the (Table 1)antibiotic mixture diet treated, the gut bacteria was effectively 158 eliminated by antibiotics.

159
Culturing 4th-instar E.obliqua larvae gut homogenates on LB plates showed that 160 treatment with antibiotics almost completely eliminated the cultivable bacterial in the gut of 161 E.obliqua compared with the groups that received TL or TP only (Fig 3). Quantitative  (Fig 4). There was no significant difference between the weights of the female pupae and the 175 male pupae in the four groups, but no matter which groups, the female pupae were heavier 176 than the male pupae (Fig 5). Next, the eclosion rate, hatching rate, pupation rate, and 177 fecundity rate were compared between the four groups. There was no significant difference in 178 eclosion rate, egg hatching rate, or pupation rate between the groups that received antibiotics 179 and those that did not, indicating that antibiotics had no significant effect on these indicators.

180
The largest oviposition number of tea powder group was compared with other groups.

181
However, the treatment with antibiotic mixture made the oviposition number notably reduced, 182 but it did not effect the entire growth cycle (Fig 6). So antibiotic treatment was significant 183 effected on fecundity of E.obliqua.

185
Our results indicated that 300μg/ml of antibiotic mixture to treat E.obliqua could effectively 186 remove gut microflora and obtain germ-free insect, as well as the treatment had no effect on However, a similar study of P. xylostella [12] concluded that a tea powder diet could be easily 205 digested without the help of gut bacteria, so treatment with antibiotics did not affect normal 206 feeding and larval development.  Wolbachia was eliminated by the antibiotic treatment, which significantly reduced the 213 fecundity of E.obliqua. Previous studies that used antibiotics to eliminate gut bacteria[15, 16,214 18] mainly focused on the effect of the antibiotics, but few of these studies investigated 215 whether these antibiotics were toxic before they were applied. Our results showed that a 216 specific concentration of the antibiotic mixture could be used to effectively eliminate gut 217 bacteria with almost no effect on the larvae, so obtaining sterile insect.