Professor Niu Changying's team has made new progress in the regulation mechanism of symbiotic bacteria on the feeding properties of fruit flies

Nanhu News Network News (correspondent Ren Xueming) Recently, the team of Professor Niu Changying of the College of Plant Science and Technology published the latest results "Gut symbiotic bacteria are involved in nitrogen recycling in the tephritid fruit fly Bactrocera dorsalis"
in BMC BIOLOGY 。 In this paper, the nitrogen nutrient acquisition strategy mediated by intestinal commensal bacteria was systematically explained from physiological, biochemical and molecular levels, and the dominant commensal bacteria that mediated the nitrogen nutrient acquisition pathway of larvae were screened, and the regulatory mechanism of nitrogen cycle of orange small fruit flies mediated by commensal bacteria was revealed, which provided a new understanding
for the nutritional interaction mode of host insects and commensal bacteria.

The feeding, development and reproduction of plant-eating insects depend on plant nutrition
.
For insects, usually the host plant has a high carbon-nitrogen ratio, nitrogen nutrient deficiency, and a large amount of nitrogen is excreted by insects in the form of nitrogen-containing metabolic waste, so nitrogen nutrient stress is a key factor
limiting the growth and development of plant-eating insects.

In this study, nitrogenase activity was measured in adults, larvae, pupae and 13 potential nitrogen-fixing bacteria in the intestines of Orange fruit flies, and it was shown that only Klebsiella oxytoca acid-producing bacteria had nitrogen-fixing enzyme activity, and other samples did not express nitrogenase activity, suggesting that nitrogen-fixing bacteria could not stably provide nitrogen nutrition
to the host through biological nitrogen fixation under natural conditions 。 On the other hand, it was found that with the development of larvae in the fruit, the urea content in the fruit gradually accumulated.
The results showed that the δ15N and 15N values in different tissues of orange fruit flies were significantly increased after feeding adults and larvae with 15N-labeled urea, and the δ15N and 15N values in the tissues of orange fruit flies after antibiotic treatment were significantly lower than the corresponding δ15N and 15N values in the untreated group, indicating that the intestinal commensal bacteria could convert urea nitrogen into nitrogen nutrients available to fruit flies and increase the nitrogen content in tissues
。

The expression of key genes in the biotic nitrogen fixation and urea hydrolysis pathway in the genome of commensal bacteria was analyzed by metagenomic and metatranscriptome sequencing technology, indicating that the transcription of nitrogenase-coding genes (nifH, nifK) was deleted, resulting in the blockage of nitrogenase synthesis and blocking the electron transport process, so nitrogen could not be fixed
by intestinal commensal bacteria.
On the other hand, the URE, ureA, ureB, ureC, ureAB genes encoding the urease complex were successfully transcribed, which in turn catalyzed the hydrolysis of urea to produce ammonia
。 Four potential urea hydrolyzed bacteria Morganella morganii, Citrobacter freundii, K.
oxytoca and K.
pneumonia were excavated from the intestines of orange fruit flies by selecting medium, and their growth curves, urease activity and urease gene expression were measured.
Morganii had stronger urease activity under acidic conditions, and the other three strains of bacteria were more suitable for expressing urease activity under neutral conditions, indicating that the above dominant bacteria played a key role
in the assimilation of urea nitrogen in orange fruit flies.

A summary map
of nitrogen-containing metabolic waste degradation and essential amino acid synthesis pathway mediated by commensal bacteria was further constructed.
Among them, nitrogenous metabolic wastes such as purine, uric acid, allantoin, allantoic acid, urea, etc.
are hydrolyzed to ammonia after catalysis by a series of biological enzymes; Ammonia is catalyzed by glutamate dehydrogenase or oxidoreductase for the synthesis of glutamate (GLU) or aspartate (ASP); GLU and ASP are catalyzed by aminotransferases to participate in the synthesis
of essential amino acids as amino donors.

A total of 115 functional genes were involved in the essential amino acid synthesis pathway mediated by commensal bacteria, and the matching results showed that the argF gene mediating arginine synthesis was missing in the metagenomic annotation results, and the remaining functional genes were present in the genomes of adult and larval intestinal commensal bacteria of Orange small fruit fly, indicating that the intestinal commensal bacteria had the ability to
synthesize essential amino acids (except arginine).
。 From the degree of participation of commensal bacteria in the synthesis of essential amino acids, Enterobacterales and Lactobacillales bacteria mainly mediate the synthesis of essential amino acids in larvae.
Enterobacterales, Orbales and Lactobacillales bacteria mainly mediate the synthesis
of essential amino acids for adults.

Ren Xueming, a doctoral student at Huazhong Agricultural University, is the first author of the paper, and Professor Niu Changying is the corresponding author of the paper, which was supported
by the National Natural Science Foundation of China International (Regional) Cooperation and Exchange Program (31661143045) and the National Natural Science Foundation of China (31972270).

Reviewed by: Niu Changying

【English Summary】

Background

Nitrogen is considered the most limiting nutrient element for herbivorous insects.
To alleviate nitrogen limitation, insects have evolved various symbiotically mediated strategies that enable them to colonize nitrogen-poor habitats or exploit nitrogen-poor diets.
In frugivorous tephritid larvae developing in fruit pulp under nitrogen stress, it remains largely unknown how nitrogen is obtained and larval development is completed.

Results

In this study, we used metagenomics and metatranscriptomics sequencing technologies as well as in vitro verification tests to uncover the mechanism underlying the nitrogen exploitation in the larvae of Bactrocera dorsalis.
Our results showed that nitrogenous waste recycling (NWR) could be successfully driven by symbiotic bacteria, including Enterobacterales, Lactobacillales, Orbales, Pseudomonadales, Flavobacteriales, and Bacteroidales.
In this process, urea hydrolysis in the larval gut was mainly mediated by Morganella morganii and Klebsiella oxytoca.
In addition, core bacteria mediated essential amino acid (arginine excluded) biosynthesis by ammonium assimilation and transamination.

Conclusions

Symbiotic bacteria contribute to nitrogen transformation in the larvae of B.
dorsalis in fruit pulp.
Our findings suggest that the pattern of NWR is more likely to be applied by B.
dorsalis, and M.
morganii, K.
oxytoca, and other urease-positive strains play vital roles in hydrolysing nitrogenous waste and providing metabolizable nitrogen for B.
dorsalis.

Links to papers: https://bmcbiol.
biomedcentral.
com/articles/10.
1186/s12915-022-01399-9#Abs1