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Nonfunctional ingestion of plant miRNAs in silkworm revealed by digital droplet PCR and transcriptome analysis.

Jia L, Zhang D, Xiang Z, He N - Sci Rep (2015)

Bottom Line: The results of TA clone, Sanger sequencing and droplet digital PCR demonstrated that several mulberry-derived miRNAs could enter to silkworm hemolymph and multiple tested tissues.Mulberry miRNAs are convincingly transferred to the silkworm orally and no physiological process associated with the miRNAs was demonstrable.The results provided a new aspect of cross-kingdom miRNA transfer.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Beibei, Chongqing 400715, P. R. China.

ABSTRACT
Since a plant miRNA (miR168) cross-regulating a mammalian transcript was reported, miRNA-mediated cross-kingdom communication has become one of the most compelling but controversial topics. In the present study, we used silkworm and mulberry, which is a model for studies on the interactions between the insect and its host plant, to address whether miRNA-mediated cross-kingdom communication is a common phenomenon. The results of TA clone, Sanger sequencing and droplet digital PCR demonstrated that several mulberry-derived miRNAs could enter to silkworm hemolymph and multiple tested tissues. Synthetic miR166b was also detected in hemolymph and fat body. However, the ingestion of synthetic miR166b did not play roles in silkworm physiological progress, which was revealed by RNA-seq analyses, RT-PCR, and phenotypic investigations. Mulberry miRNAs are convincingly transferred to the silkworm orally and no physiological process associated with the miRNAs was demonstrable. The results provided a new aspect of cross-kingdom miRNA transfer.

No MeSH data available.


The phenotypic investigation of silkworm larvae fed on mulberry leaves with/without synthetic miR166b.The phenotypic investigation including larva weight (A), rate of wandering (B), rate of survival (C), pupa and cocoon weight (D) were measured before (0 h) and after silkworm ingested synthetic miR166b (24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, and 192 h). Fold change of weight means the ratio of lavae weight after silkworm ingested synthetic miR166b (24 h, 48 h, 72 h) divided the larvae weight at 0 h. Rate of wandering indicates the percentage of silkworm developed into the wandering stage. FED-control represents the silkworm larvae were fed only on mulberry leaves. FED-miR166b means the silkworm larvae were fed on a piece of mulberry leaf containing synthetic miR166b. Statistical significance was determined by Student’s t test (*p < 0.05).
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f4: The phenotypic investigation of silkworm larvae fed on mulberry leaves with/without synthetic miR166b.The phenotypic investigation including larva weight (A), rate of wandering (B), rate of survival (C), pupa and cocoon weight (D) were measured before (0 h) and after silkworm ingested synthetic miR166b (24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, and 192 h). Fold change of weight means the ratio of lavae weight after silkworm ingested synthetic miR166b (24 h, 48 h, 72 h) divided the larvae weight at 0 h. Rate of wandering indicates the percentage of silkworm developed into the wandering stage. FED-control represents the silkworm larvae were fed only on mulberry leaves. FED-miR166b means the silkworm larvae were fed on a piece of mulberry leaf containing synthetic miR166b. Statistical significance was determined by Student’s t test (*p < 0.05).

Mentions: We also investigated the phenotypic changes of silkworm feeding on mulberry leaves with and without synthetic miR166b. As shown in Fig. 4, neither the weight (Fig. 4A) nor their wandering rate (Fig. 4B) showed significant difference between two groups. Similar observations had been made in lethality (Fig. 4C). The fat body and silk gland are two major organs in silkworm. The developmental growth of silk gland and fat body can be reflected by the weight of cocoon and pupae, respectively. Since the synthetic miR166b was detected in fat body and not silk gland, we then measured the pupa and cocoon weight at 10 days after ingestion of synthetic miR166b. Results showed that there was no significant difference of pupa and cocoon weight between two groups (Fig. 4D).


Nonfunctional ingestion of plant miRNAs in silkworm revealed by digital droplet PCR and transcriptome analysis.

Jia L, Zhang D, Xiang Z, He N - Sci Rep (2015)

The phenotypic investigation of silkworm larvae fed on mulberry leaves with/without synthetic miR166b.The phenotypic investigation including larva weight (A), rate of wandering (B), rate of survival (C), pupa and cocoon weight (D) were measured before (0 h) and after silkworm ingested synthetic miR166b (24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, and 192 h). Fold change of weight means the ratio of lavae weight after silkworm ingested synthetic miR166b (24 h, 48 h, 72 h) divided the larvae weight at 0 h. Rate of wandering indicates the percentage of silkworm developed into the wandering stage. FED-control represents the silkworm larvae were fed only on mulberry leaves. FED-miR166b means the silkworm larvae were fed on a piece of mulberry leaf containing synthetic miR166b. Statistical significance was determined by Student’s t test (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4508662&req=5

f4: The phenotypic investigation of silkworm larvae fed on mulberry leaves with/without synthetic miR166b.The phenotypic investigation including larva weight (A), rate of wandering (B), rate of survival (C), pupa and cocoon weight (D) were measured before (0 h) and after silkworm ingested synthetic miR166b (24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, and 192 h). Fold change of weight means the ratio of lavae weight after silkworm ingested synthetic miR166b (24 h, 48 h, 72 h) divided the larvae weight at 0 h. Rate of wandering indicates the percentage of silkworm developed into the wandering stage. FED-control represents the silkworm larvae were fed only on mulberry leaves. FED-miR166b means the silkworm larvae were fed on a piece of mulberry leaf containing synthetic miR166b. Statistical significance was determined by Student’s t test (*p < 0.05).
Mentions: We also investigated the phenotypic changes of silkworm feeding on mulberry leaves with and without synthetic miR166b. As shown in Fig. 4, neither the weight (Fig. 4A) nor their wandering rate (Fig. 4B) showed significant difference between two groups. Similar observations had been made in lethality (Fig. 4C). The fat body and silk gland are two major organs in silkworm. The developmental growth of silk gland and fat body can be reflected by the weight of cocoon and pupae, respectively. Since the synthetic miR166b was detected in fat body and not silk gland, we then measured the pupa and cocoon weight at 10 days after ingestion of synthetic miR166b. Results showed that there was no significant difference of pupa and cocoon weight between two groups (Fig. 4D).

Bottom Line: The results of TA clone, Sanger sequencing and droplet digital PCR demonstrated that several mulberry-derived miRNAs could enter to silkworm hemolymph and multiple tested tissues.Mulberry miRNAs are convincingly transferred to the silkworm orally and no physiological process associated with the miRNAs was demonstrable.The results provided a new aspect of cross-kingdom miRNA transfer.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Beibei, Chongqing 400715, P. R. China.

ABSTRACT
Since a plant miRNA (miR168) cross-regulating a mammalian transcript was reported, miRNA-mediated cross-kingdom communication has become one of the most compelling but controversial topics. In the present study, we used silkworm and mulberry, which is a model for studies on the interactions between the insect and its host plant, to address whether miRNA-mediated cross-kingdom communication is a common phenomenon. The results of TA clone, Sanger sequencing and droplet digital PCR demonstrated that several mulberry-derived miRNAs could enter to silkworm hemolymph and multiple tested tissues. Synthetic miR166b was also detected in hemolymph and fat body. However, the ingestion of synthetic miR166b did not play roles in silkworm physiological progress, which was revealed by RNA-seq analyses, RT-PCR, and phenotypic investigations. Mulberry miRNAs are convincingly transferred to the silkworm orally and no physiological process associated with the miRNAs was demonstrable. The results provided a new aspect of cross-kingdom miRNA transfer.

No MeSH data available.