Effect of Lactobacillus Plantarum on the Growth and Development and Gut Microbial Diversity of Spodoptera Litura

Background: The main challenge for agricultural research is the determination of the best method 6 for efficient and economical management of insects. Research on the possibility of regulating insect 7 development by targeting the intestinal microbial community is in its early stages; however, the use of 8 microorganisms to control the composition of the host's intestinal microbes and to affect its 9 physiological functions has garnered considerable attention. 10 Results: This study evaluated the effects of probiotics ( Lactobacillus plantarum ) isolated from the 11 intestine of Spodoptera litura (Fabricius) on the growth and development of the host and the diversity 12 of its intestinal microbes. The results of the study showed that the larvae of S. litura fed with artificial 13 diets with different feed units could proceed towards the development of generations normally. Compared with the control responses, after ingesting L. plantarum , larval gut sucrase and other 15 digestive enzyme activities increased, growth and development accelerated, fecundity generally 16 increased, and there was a significant change in the female-male ratio. Additionally, differences in microbial abundance and diversity were found in the gut of S. litura larvae fed with diets supplemented 1 with L. plantarum and without L. plantarum . 2 Conclusions: These findings indicate that supplementation of feed with L. plantarum can 3 effectively affect the growth and development of the host and the composition of the intestinal flora, 4 thereby providing useful applications in research regarding pest management.


Background 8
There are several types of insects with different morphologies, and insects are the most widely   insect management, they are insufficient; therefore, the use of probiotics to manipulate the composition 7 of intestinal microbes to achieve ecological control of the host may be a potential supplement to these 8 existing technologies and other insect management methods. Considering the manipulability of 9 probiotics on the intestinal flora, we used the endogenous probiotic L. plantarum that is found in the 10 intestine of S. litura, to regulate the composition of the host intestinal microbial community to varying 11 degrees, and explored the influence of L. plantarum on the intestinal microbial diversity, growth, and 12 development of S. litura under laboratory conditions. As a major pest, S. litura has been widely used in 13 various studies; however, because large-scale breeding of S. litura is mainly conducted using 14 approaches that include the provision of artificial diets, this study aimed to evaluate the influence of 15 L. plantarum on the growth, development, and intestinal microbial diversity of S. litura fed with an 16 artificial diet, to provide a theoretical reference for the subsequent screening of insect-specific 17 microecological agents and biological controls.

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Insect culture 20 The S. litura larvae were provided by the Key Laboratory of Natural Pesticide and Chemical Biology of the Ministry of Education, P. R. China. The larvae were reared on an artificial diet as per 1 methods recommended by Zhu et al. (2001) with slight modifications for subsequent generations, 2 and under controlled temperature and humidity conditions of 26 ± 2 °C and 80% ± 5%, respectively.

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The main ingredients of the artificial diet were organic corn flour, cooked soy flour, nine vitamin 4 pills, methyl-p-hydroxybenzoate, sorbic acid, and cholesterol. The rearing was performed in a 5 transparent plastic box (17 × 11.5 × 6 cm 3 ) with daily changes in diet. The mature larvae were 6 placed in a ten-well plate for pupation. After the pupae were collected, they were placed in a 7 transparent plastic box in which the bottom portion was moistened with wet filter paper. Freshly 8 emerged adults were transferred to transparent plastic buckets (22 × 28 × 26 cm 3 ) containing 10% 9 honey water at the bottom to prepare the adults for laying eggs. The transparent plastic buckets were 10 lined with butter paper to facilitate egg laying. Larvae from the fourth generation of laboratory 11 cultures were used for experimental purposes.

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Observations were performed daily on various biological parameters of S. litura, including single Determination of nutritional index of larvae 1 The larvae of S. litura that underwent molting to transition into the fourth-instar stage and showed 2 consistent healthy development during which they were fed with artificial diets with different bacterial 3 loads, were subjected to starvation overnight. 4 1) Twenty S. litura larvae reared on artificial diets with different bacterial loads (including the 5 control group) were selected, and their fresh weight was recorded; then, they were subjected to drying 6 conditions in an oven at 80 °C to achieve a constant weight, and the dry-wet ratio was determined. 2) Twenty larvae were selected and their fresh weight was recorded. According to the dry-wet 8 ratio of the tested larvae, the dry weight of the larvae before the test (C) was inferred, and then they 9 were fed with artificial feed with different bacterial loads (including the control group). After 48 h, the 10 residual feed and feces were removed, and the larvae and feces were subjected to drying conditions at 11 80 °C to achieve a constant weight to determine the dry weight of the feces (E) and the dry weight of 12 the larvae (D).

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3) Based on the same method as that mentioned above, the dry weight of feed with different 14 bacterial loads was measured before (A) and after (B) execution of the tests.

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Nutritional indices were calculated as per methods prescribed by Wheeler & Isman (2001) using the 16 following formulae: In the formula, T represents the number of experimental days.

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Determination of digestive enzyme activity in the larval midgut   Table 1, the relative feeding rates of the different larval feeding groups were in the 5 order CD6 > CK ≥ CD4 > CD5 > CD7. There was a significant difference between rates of the CD6 6 group and the CD7 group, and there was no significant difference between the other groups. The 7 relative food conversion rates of the larval groups were in the order CD6 > CD5 > CD7 > CK > CD4.

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The food conversion rate of the CD6 group was significantly higher than that of the other groups, and 9 the food conversion rates of the larvae in the other groups were not significantly different from those of 10 the CK group. The relative food utilization rates of the different larval groups were in the order CD6 > 11 CD7 ≥ CD5 ≥ CK > CD4. There was no significant difference between the food utilization rates of the 12 bacteria-fed larval groups and the control; however, the food utilization rate of the CD6 group was 13 significantly higher than that of the CD7 and CD4 groups. The relative growth rates of the larval 14 groups were in the order CD6 ≥ CK > CD5 >CD4 > CD7, and relative growth rates of the CD6 group 15 were significantly higher than those of the other bacteria-fed groups; however, none of the bacteria-fed 16 groups exhibited a significant difference compared with the control group. The relative approximate 17 digestibility of the larval groups were in the order CK > CD4 > CD5 > CD7 > CD6 , and that of the 18 CD6 group was significantly lower than that of the control group.

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As shown in Table 2, the average fresh weights of the larvae fed with artificial diets containing 6 different bacterial loads were in the order CD6 > CD7 > CK > CD4 ≥ CD5, and those of the CD4 7 group and CD5 group were significantly lower than those of the CD6 group; however, none of 8 bacteria-fed groups presented a significant differences compared with the control group.
9   Table 3, the effects of artificial diets with different bacterial loads exerted on the 8 activity of digestive enzymes in the midgut of the fourth-instar larvae of S. litura were different. The 9 relative amylase activity of the fourth-instar larvae fed with artificial diets with different bacterial loads 10 was in the order CD5 > CD4 > CD7 > CD6 > CK , and all groups that were fed with a feed 11 supplemented with bacteria exhibited significantly higher activity than the control group. The relative 12 trehalase activity of the fourth-instar larvae fed with artificial diets with different bacterial loads was in 13 the order CD4 > CD5 > CD7 > CD6 > CK; except for the CD6 group, the other bacteria-fed groups 14 exhibited significantly higher activity than the control group. The relative sucrase activity of the fourth-instar larvae fed with artificial diets with different bacterial loads was in the order CD4 > CD5 > 1 CD7 ≥ CD6 > CK, and the activities of the CD4 and CD5 groups were significantly different from 2 those of the other groups.
3   Table 4, regarding the effect of artificial feeds with different bacterial loads exerted 11 on the pupae of S. litura, the relative pupation rates of S. litura larval groups were in the order CD6 > that of the control group and there was no significant differences between the other groups. The relative 1 ratios of female to male were in the order CD5 > CK > CD7 > CD6 > CD4, and both the CD5 and CD4 2 groups were significantly different from the control group. The relative weights of female pupae were 3 in the order CD6 > CD5 > CK > CD4 > CD7, and there were no significant differences between the 4 groups. The relative male pupae weights were in the order CD6 > CD4 > CD5 > CK > CD7, and those 5 of the CD6 and CD4 groups were significantly different from those of the control group. The relative 6 pupal emergence rates were in the order CD6 > CK > CD5 > CD4 > CD7, and all larvae fed with 7 bacteria-supplemented diets exhibited significant differences compared with the control group.  (Firmicutes) to Ralstonia (Proteobacteria) and Bacillus (Firmicutes) (Fig. 1), and there was a negative 7 correlation between Enterococcus (Firmicutes) and Ralstonia (Proteobacteria). group. One color represents a species, and the length of the color block (bar graph) represents the relative abundance ratio of the species.

Population differences 1
According to the Venn diagram, there were 116 OTUs in the CK and four treatments of larvae in 2 all, but only 65 OTUs were shared by all of them (Fig. 2). Of these, one OTU was unique to CK, CD4, 3 or CD7, and no OTU was unique to CD5 or CD6. A multi-sample comparison tree was constructed and 4 used to compare the community similarity of each sample at the OTU composition and phylogenetic more similar is the composition of the two; therefore, the bacterial communities of the three treatment 10 groups (CD4, CD5, and CD6) were similar, as they were clustered in the same area of the PCA (right 11 or lower right) (Fig. 3).

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The relative abundance and diversity of gut bacteria in S. litura larvae fed with L. plantarum are 13 shown in Fig. 2. The top ten genera were distributed into three phyla and eight families.   indicating that the larvae of S. litura had higher adaptability to CD6. Although the food conversion rate 8 and food utilization rate of the larvae of the CD5 and CD7 groups were higher than those of the control 9 group, their relative growth rates and relative feeding rates were lower than those of the control group.

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Additionally, the relative feeding rate, food conversion rate, and food utilization rate of the larvae of 11 the CD4 group were lower than those of the control group. This indicates that the three treatments 12 using bacteria-supplemented feed, CD4, CD5, and CD7, are less suitable for application as feed for S.

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litura larvae. Moreover, the high feeding rate, food utilization rate, food conversion rate, and insect 14 weight, along with low approximate digestibility, of the larvae of the CD6 indicate that certain 15 substances in the artificial diet of the CD6 group could not be digested well by the larvae; however, 16 substances that were digested resulted in higher food conversion and utilization rates, which could be 17 better transformed into insect tissues (Zhigang et al. 2005).

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Changes in the activities of digestive enzymes, such as sucrase and amylase, in insect intestines

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usually reflect the effect of food on insect host feeding and fitness to a certain extent ( Yuzhu et al.

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conversion utilization of sugars, even though their growth rate was relatively low. Additionally, it was 1 also determined that there was a significant positive correlation between the trehalase and sucrase 2 activities of the larvae and the larval period of each treatment group, and the larvae feeding on these 3 two groups of food did not show a high pupation rate，suggesting that this might be related to an good developmental quality during the larval stage (Zhu et al. 2005b); furthermore, the larvae of the 10 CD6 group did not exhibit a higher male to female ratio, which might be related to changes in the gut 11 microbes. The present study found that the larvae of the CD5 group had a relatively high egg hatching 12 rate, which might be related to its relatively high amylase activity and sugar conversion utilization rate.

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Concurrently, the larvae of the CD7 group had relatively low pupal weights and egg hatching rates,

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The present study revealed that the addition of L. plantarum to the artificial diet of larvae altered

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This study showed that after the larvae were fed with L. plantarum, the activity of digestive enzymes 18 such as amylase also increased; however, the abundance of L. plantarum in the gut was less than 1%,

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which might be related to a shorter colonization time in the gut. As the number of bacteria per unit of 20 feed increased, the abundance of Ralstonia decreased, which was consistent with the trends of the 21 enzyme activities, such as those of sucrase. Therefore, it is speculated that Ralstonia may participate in the induction of digestive enzymes.

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The larvae of the CD6 group showed the best performance, with the feed promoting their physiological  Relative abundance of the detected gut bacteria at the genus level. The abscissa indicates the sample name; the ordinate indicates the relative abundance percentage. CD4−CD7: four treatment groups fed with an arti cial diet with different bacterial loads; CK: control group. One color represents a species, and the length of the color block (bar graph) represents the relative abundance ratio of the species.   Linear discriminant analysis effect size cladogram (LEfSe). CD4−CD7: four treatment groups fed with an arti cial diet with different bacterial loads; CK: control group. The circles radiating from the inside to the outside of the clade map represent the classi cation level from phylum to species. Each small circle at different classi cation levels represents a classi cation at that level, and the diameter of the small circle is proportional to the relative abundance. The coloring principle includes the uniform coloration of the species name, with no signi cant differences highlighted in yellow, and other different species are colored according to the group with the highest abundance of the species. Different colors indicate different groups, and nodes of different colors indicate the microbial groups that play an important role in the group represented by the color.