Gammaproteobacteria are crypt-dwelling primary bacterial symbionts of the shield bug, Graphosoma lineatum
عنوان دوره: دومین کنگره بین المللی حشره شناسی ایران
نویسندگان
چکیده
Many insects maintain symbiotic bacteria inside their body and are depend on the symbionts for normal growth and fecundity, particularly those feeding on restricted diets. Symbionts provide many biological and physiological benefits to their host like digestion of diet and detoxification as well as nutritional contributions. Many members of suborder Heteroptra such as the shield bug, Graphosoma lineatum (Hemiptera: Pentatomidae), are sucking insects feeding on leaves, stems, and fruits of their host plants, therefore, many of these insects harbor heritable symbiotic bacteria. G. lineatum harbors extracellular bacterial symbiont in the enclosed crypts around the fourth section of the midgut and is dependent on the gut symbiotic bacteria for normal growth and fecundity. Here we investigated the gut microbiota of the midgut in G. lineatum by using real-time PCR. To do this, DNA was extracted from the fourth-section of the midgut (V4). To assess the composition of bacteria within this section of the gut, taxon-specific primers including (α682F and 908αR), (Eub338F and Bet680R), (1080γF and γ1202R), (Act920F3 and Act1200R), (Lgc353F and Eub518R), (798cfbF and Cfb967R) and (906F and 1062R) respectively targeting Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and eubacterial, were used in qPCR assays. Real-time PCR analyses showed the Gammaproteobacteria occupied the V4 and have the most population, whereas the other bacterial taxon were not detected or detected at the very low levels. Also in this study we used specific qPCR primers to estimate relative density of the bacterial symbiont in different developmental stages of the insect. To do this the specific qPCR primers of the bacterial groEL gene based on groEL sequence was used to quantify population of the Gammaproteobacteria in the insect. Also specific qPCR primers based on conserved regions of 18S rRNA gene in Pentatomidae insects was designed and used as the reference gene. Stage-specific detection of bacterial symbionts showed that the first and second nymphal stages harbored the lowest population of the symbiont compared with other stages; however, the symbiont population highly increased thereafter and reached the highest population in the adult insects. In conclusion, we showed that Gammaproteobacteria persistently colonized midgut-crypts of G. lineatum during its developmental stages highlighting the biological importance of Gammaproteobacteria symbionts in this insect.
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