| Summary: | 博士 === 國立中興大學 === 昆蟲學系所 === 100 === The symbiotic bacterium of the entomopathogenic nematode, Steinernema abbasi, isolated from Taiwan, was determined to be the genus Xenorhabdus based on physiological and biochemical characteristics. It was further identified to be similar to Xenorhabdus indica of S. abbasi Oman isolate as identified by the sequence analyses of its 16S rDNA. The cultured filtrates of Xenorhabdus indica from only the primary form were toxic to Sf21 and S2 cell lines, while those of both forms were not toxic to a mammalian cell line. The necrotic rates of Galleria mellonella hemocytes at 24 h after treating with the cultured filtrates of symbiotic bacteria were significantly different from those of the control, whereas the rates treated with X. indica lipopolysaccharide (LPS) were similar to those of the control. These results indicate that necrosis in G. mellonella hemocytes occurs at 24 h after treatment with the filtrates, while erythrocytes as treated with filtrates were not significantly different, revealing that the culture filtrates do not contain hemolytic substances. Inactivated bacterial cells (primary form) caused serious paralysis in G. mellonela larvae and eventually killed insects. This symptom was found to be similar to that injected with LPS extracted from the primary form. Therefore, it is suggested that LPS is neurotoxic to G. mellonella larvae. Both hemolytic rates of mammalian and insect hemocytes treated with inactivated bacteria (primary form) were capable of causing necrosis. In in vivo assays, the inactivated bacterial cells were capable of causing necrosis and subsequently killed hemocytes of both G. mellonella and Spodoptera litura larvae; however, they were comparatively lesser destructive to S. litura hemocytes. In in vitro assays, LPS from X. indica (primary form) was not markedly detrimental to G. mellonella hemocytes compared with the control groups, suggesting that LPS is not a major factor affecting insect immune system. Therefore, it is speculated that LPS and certain substances when released into insect hemocoel from symbiotic bacteria could hamper insect immune system, resulting in proliferation of symbiotic bacteria and nematodes, and subsequently causing septicemia to rapidly kill their insect hosts. Xenorhabdus indica caused ca. 95% mortality of Galleria mellonella mature larvae at 72 h after culturing, indicating that this bacterium secreted insecticidal substances in its culture medium. The cultured filtrates could also inhibit nine kinds of human pathogens and plant pathogenic fungi. The cultured filtrates screened through 10 or 100 kDa molecular sieves could inhibit the growth of Bacillus subtilis and Botrytis cinerea while those through 3-kDa sieve could inhibit B. subtilis only. However, only the filtrates through 10-kDa sieve resulted in 96.67% mortality of G. mellonella larvae at 24 h. It is thus indicated that both insecticidal and antimicrobial substances are present in the 10-kDa sieved filtrates. Proteins in the cultured filtrates were analyzed using SDS-PAGE electrophoresis. A protein band with 85 kDa of molecular weight was detected in the 100-kDa sieved filtrates while two bands with 22 and 25 kDa were found in the 10-kDa sieved filtrates. On the basis of coloration tests, most of the separated molecules showed amino acid structures. Furthermore, both exo- and endo-chitinases in the filtrates through 10-50 kDa sieves could be detected after reacting with different substrates, emitting fluorescence under the UV microscope. The concentration of LPS isolated from X. indica was ca. 3 x 105 EU/ml, causing ca. 93% mortality of G. mellonella larvae at 36 h, respectively. The LPS from X. indica resulted in ca. 7.67 mm of inhibition zone against a bacterium, B. subtilis and a fungus, B. cinerea, whereas that from Xenorhabdus nematophila caused ca. 8.00 and 5.33 mm of inhibition zone, respectively. In contrast, LPS from Escherichia coli which is also an intestinal bacterium produced only ca. 1.33 mm of inhibition zone against B. subtilis. Therefore, the LPS from X. indica could inhibit both bacterial and fungal growth. Electron micrographs showed a circular form of DNA structure in X. indica plasmids, its size being ca. 5,167 bp, with GC=39%, AT=61%, indicating an AT-rich DNA sequence. As a result from BLAST analysis, only 7 fragments contained similarly functional genes. It was also speculated that X. indica plasmids seem to be involved in cellular membrane formation, pathogenic hemolysin, glucose metabolism, spore germination, and others. However, further studies on many other unknown functional genes which are related to syntheses of amino acid sequence leading to these functions remain to be undertaken.
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