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Gammaproteobacteria as essential primary symbionts in the striped shield bug, Graphosoma Lineatum (Hemiptera: Pentatomidae)

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ABSTRACT

Many members of suborder Heteroptra harbor heritable symbiotic bacteria. Here we characterize the gut symbiotic bacterium in Graphosoma lineatum (Hemiptera: Pentatomidae) by using molecular phylogeny, real-time PCR analysis as well as light and electron microscopy observations. The microscopy observations revealed the presence of a large number of rod-shaped bacterial cells in the crypts. A very high prevalence (98 to 100%) of the symbiont infection was found in the insect populations that strongly supports an intimate association between these two organisms. Real-time PCR analysis also showed that the Gammaproteobacteria dominated the crypts. The sequences of 16sr RNA and groEL genes of symbiont showed high levels of similarity (93 to 95%) to Pantoea agglomeranse and Erwinia herbicola Gammaproteobacteria. Phylogenetic analyses placed G. lineatum symbiont in a well-defined branch, divergent from other stink bug bacterial symbionts. Co-evolutionary analysis showed lack of host-symbiont phylogenetic congruence. Surface sterilization of eggs resulted in increased pre-adult stage in the offspring (aposymbionts) in comparison to the normal. Also, fecundity, longevity, and adult stage were significantly decreased in the aposymbionts. Therefore, it seems that the symbiont might play a vital function in the host biology, in which host optimal development depends on the symbiont.

No MeSH data available.


Egg surface sterilization for elimination of the symbiotic bacterium.(a) Surface sterilization of G. lineatum eggs with hypochlorite sodium to eliminate bacterial symbionts. Bacterial population declined after sterilization of eggs (b) qPCR analysis of DNA extracted from untreated eggs (control) and surface-sterilized eggs of G. lineatum. Bacterial symbiont population dramatically declined after sterilization of eggs (c) qPCR analysis for bacterial symbiont intensity in first nymphs of G. lineatum hatched from control and surface-sterilized eggs with hypochlorite sodium for 15 s. Egg sterilization decreased bacterial symbiont intensity in treated nymphs in comparison with the controls.
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f8: Egg surface sterilization for elimination of the symbiotic bacterium.(a) Surface sterilization of G. lineatum eggs with hypochlorite sodium to eliminate bacterial symbionts. Bacterial population declined after sterilization of eggs (b) qPCR analysis of DNA extracted from untreated eggs (control) and surface-sterilized eggs of G. lineatum. Bacterial symbiont population dramatically declined after sterilization of eggs (c) qPCR analysis for bacterial symbiont intensity in first nymphs of G. lineatum hatched from control and surface-sterilized eggs with hypochlorite sodium for 15 s. Egg sterilization decreased bacterial symbiont intensity in treated nymphs in comparison with the controls.

Mentions: Given that we demonstrated the constant presence of the symbiont in the crypts of G. lineatum, it was envisaged that there must be an efficient transmission route for delivering bacterial symbiont to the offspring. Since the most common symbiont transmission mode in true bugs is through egg surface contamination, different surface sterilization methods were used to see if the symbiont would be eliminated. DNA was extracted from each egg mass separately, and subjected to PCR detection. The results showed that ethanol treatment couldn’t eliminate the symbiont. Washing the eggs with 12% hypochlorite sodium for 14 min almost completely eliminated the bacterial symbiont, however, the egg hatching rate was quite low, reaching nearly zero (Data not shown). To improve the egg hatching rate, the washing time was cut short which resulted in less stringent sterilization (Fig. 8a), but enhanced the hatching rate almost to the level of normal eggs. These results confirmed that vertical transmission of symbiont to the progeny is only via egg surface contamination. To further confirm reduction of the bacterial symbionts in the aposymbiont insects, we estimated the relative density of the bacterial symbionts in the aposymbiont and the control insect. Our results indicate that density of the bacterial symbiont in the sterilized eggs was dramatically reduced when compared to the control eggs (Fig. 8b). The level of symbiont density in aposymbiont first-instars was about 38 folds lower than that of control insects (Fig. 8c).


Gammaproteobacteria as essential primary symbionts in the striped shield bug, Graphosoma Lineatum (Hemiptera: Pentatomidae)
Egg surface sterilization for elimination of the symbiotic bacterium.(a) Surface sterilization of G. lineatum eggs with hypochlorite sodium to eliminate bacterial symbionts. Bacterial population declined after sterilization of eggs (b) qPCR analysis of DNA extracted from untreated eggs (control) and surface-sterilized eggs of G. lineatum. Bacterial symbiont population dramatically declined after sterilization of eggs (c) qPCR analysis for bacterial symbiont intensity in first nymphs of G. lineatum hatched from control and surface-sterilized eggs with hypochlorite sodium for 15 s. Egg sterilization decreased bacterial symbiont intensity in treated nymphs in comparison with the controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016839&req=5

f8: Egg surface sterilization for elimination of the symbiotic bacterium.(a) Surface sterilization of G. lineatum eggs with hypochlorite sodium to eliminate bacterial symbionts. Bacterial population declined after sterilization of eggs (b) qPCR analysis of DNA extracted from untreated eggs (control) and surface-sterilized eggs of G. lineatum. Bacterial symbiont population dramatically declined after sterilization of eggs (c) qPCR analysis for bacterial symbiont intensity in first nymphs of G. lineatum hatched from control and surface-sterilized eggs with hypochlorite sodium for 15 s. Egg sterilization decreased bacterial symbiont intensity in treated nymphs in comparison with the controls.
Mentions: Given that we demonstrated the constant presence of the symbiont in the crypts of G. lineatum, it was envisaged that there must be an efficient transmission route for delivering bacterial symbiont to the offspring. Since the most common symbiont transmission mode in true bugs is through egg surface contamination, different surface sterilization methods were used to see if the symbiont would be eliminated. DNA was extracted from each egg mass separately, and subjected to PCR detection. The results showed that ethanol treatment couldn’t eliminate the symbiont. Washing the eggs with 12% hypochlorite sodium for 14 min almost completely eliminated the bacterial symbiont, however, the egg hatching rate was quite low, reaching nearly zero (Data not shown). To improve the egg hatching rate, the washing time was cut short which resulted in less stringent sterilization (Fig. 8a), but enhanced the hatching rate almost to the level of normal eggs. These results confirmed that vertical transmission of symbiont to the progeny is only via egg surface contamination. To further confirm reduction of the bacterial symbionts in the aposymbiont insects, we estimated the relative density of the bacterial symbionts in the aposymbiont and the control insect. Our results indicate that density of the bacterial symbiont in the sterilized eggs was dramatically reduced when compared to the control eggs (Fig. 8b). The level of symbiont density in aposymbiont first-instars was about 38 folds lower than that of control insects (Fig. 8c).

View Article: PubMed Central - PubMed

ABSTRACT

Many members of suborder Heteroptra harbor heritable symbiotic bacteria. Here we characterize the gut symbiotic bacterium in Graphosoma lineatum (Hemiptera: Pentatomidae) by using molecular phylogeny, real-time PCR analysis as well as light and electron microscopy observations. The microscopy observations revealed the presence of a large number of rod-shaped bacterial cells in the crypts. A very high prevalence (98 to 100%) of the symbiont infection was found in the insect populations that strongly supports an intimate association between these two organisms. Real-time PCR analysis also showed that the Gammaproteobacteria dominated the crypts. The sequences of 16sr RNA and groEL genes of symbiont showed high levels of similarity (93 to 95%) to Pantoea agglomeranse and Erwinia herbicola Gammaproteobacteria. Phylogenetic analyses placed G. lineatum symbiont in a well-defined branch, divergent from other stink bug bacterial symbionts. Co-evolutionary analysis showed lack of host-symbiont phylogenetic congruence. Surface sterilization of eggs resulted in increased pre-adult stage in the offspring (aposymbionts) in comparison to the normal. Also, fecundity, longevity, and adult stage were significantly decreased in the aposymbionts. Therefore, it seems that the symbiont might play a vital function in the host biology, in which host optimal development depends on the symbiont.

No MeSH data available.