| Asian citrus psyllid(ACP), Diaphorina citri Kuwayama(Hemiptera: Psyllidae),a phloem-feeding insect pest, is the only know insect vector of Huanglongbing bacteria,Candidatus Liberibacter asiaticus(Las) in Asia and North America. Huanglongbing(HLB)is the most destructive disease of citrus worldwide. Understanding the relationship between Las and ACP is critical for both ACP and HLB disease management. The technology of the normal PCR, q RT-PCR, fluorescence in situ hybridization(FISH) were used in the current study, and focus on the interaction of citrus plant-Candidatus Liberibacter asiaticusWolbachia-Diaphorina citri in view of molecule ecology. Based on the life history of Asian citrus psyllid on the different host plants, we studied effects of Candidatus Liberibacter asiaticus on the fitness of Diaphorina citri, and the underlying nutritional mechanisms.Meanwhile, this study investigated the differences of Wolbachia and Huanglongbing bacteria in the distribution and infection dynamics, tried to reveal Huanglongbing transmission mechanism involved in Wolbachia. The main results in this study are:1. Morphological characteristics of Asian citrus psyllid This study summarizes the age-specific typical morphological differences of Asian citrus psyllid. Size of citrus psyllid has obvious difference in different nymphs. 2nd instar nymph is biger than 1stinstar nymph, 0.145 mm longer and 0.166 mm wider; Compared 3rd instar nymph with 2nd instar nymph, 0.193 mm longer and 0.182 mm wider; Compared 4th instar nymph with 3rdinstar nymph, 351 mm longer and 0.279 mm wider; Compared 5th instar nymph with 4th instar nymph, 0.572 mm longer and 0.358 mm wider. And the age was determined based on the size of the nymph, further determined by a microscope.2. Biological characteristics of Asian citrus psyllid The life history of Asian citrus psyllid was different on the different host plants. The development of egg, 1st instar and total nymphs differed significantly on the different host plants. The survivorship and longevity of ACP immatures on the citrus maxima were the highest or longest, respectively. and were siginificant different on the Clausena lansium,which the survivorship and longevity was the lowest or shortest, respectively. The fecundity of ACP on the Clausena lansium was the lowest, differed siginificantly from other hosts. The intrinsic rate of population increase(rm) was the highest on the Murraya exotica, the lowest on the Citrus ’Suanju’; The net reproductive rate(R0) was the highest on the Citrus flamea Hort. ex Tseng shiyueju, the lowest on the Clausena lansium. Results indicate, in addition to the Murraya exotica, the Citrus flamea Hort. ex Tseng shiyueju is optimum host to the citrus psyllid.3. Effects of Candidatus Liberibacter asiaticus on the fitness of Asian citrus psyllid In the current study, the development, survivorship of ACP immatures and the longevity and fecundity of ACP female adults in four treatments(Las positive or negative ACP on Las infected and free citrus plants) were estimated. Our results revealed that Las positive ACP immatures developed significantly faster on Las infected citrus. The fecundity and longevity of Las positive female adults was also greater, or longer on Las infected citrus shoots, while the survival of Las positive immatures was significantly lower on Las infected citrus shoots. Similarly, the intrinsic rate of population increase(rm) was highest when Las positive ACP fed on Las infected citrus shoots and the lowest when the Las negative ACP fed on Las free citrus shoots. Our findings revealed that, compared to the infection of Las in ACP insects, the infection of Las in citrus shoots had more significant effects on the fitness of ACP.4. Influence of Huanglongbing infection on amino acid composition of host plant and Asian citrus psyllidThe kinds and contents of amino acids in phloem sap from citrus host changed with Huanglongbing infection, indicating the nutritional quality of phloem sap is involved in the citrus plant-Candidatus Liberibacter asiaticus-Diaphorina citri interaction.Free amino acids composition in honeydew excreted by ACP feeding on uninfected and Las-infected citrus plants differed significantly, especially for asparagine and proline,which increased obviously in the phloem sap of infected citrus plant. Those indicating the indirect effect of Huanglongbing increases asparagine and proline nutritional assimilation by ACP, and is beneficial to the nymphal development. The ACP adults feeding on Lasinfected citrus plants had a higher proportion of essential amino acids, which is beneficial to the citrus psyllid development.Free amino acids composition had no obvious difference in honeydew excreted by Las-infected and uninfected ACP feeding on Murraya exotica, indicating the direct effect of Huanglongbing have less impact for nutritional assimilation by Asian citrus psyllid.5. Localization and infection dynamics of Wolbachia during the development of Asian citrus psyllidSeven endosymbionts had different infection level in the ACP. The infection rate of Carsonella、Profftella and Wolbachia were 100%; Arsenophonus was 51.7%; Rickettsia、Cardinium and Hamiltonella endosymbionts were very low.All the developmental stages including egg, 1st-5th instar nymphs, male and female adults were infected with Wolbachia, but the infection rates varied during the different life stages. Wolbachia titer increased with the nymph development, and thoes in 4th and 5th instar nymphs were significantly higher than those in the 1st and 2nd instar nymphs. The Wolbachia titers in ACP adults were lower than those of 5th instar nymphs, but higher than those in other stages. FISH visualization showed that the localization of Wolbachia in ACP eggs moved along with the development. In ACP nymphs, Wolbachia showed a scattered localization pattern, but higher intensity was found in the U-shaped bacteriome in psyllid abdomen. In newly eclosion female and male adults, Wolbachia was confined and occupied two symmetrical organizations in abdomen. The potential mechanisms of Wolbachia infection dynamics are discussed.6. Localization and infection dynamics of Huanglongbing during the development of Asian citrus psyllidWhen newly emerged Asian citrus psyllid feeding on the infected citrus plant, the titer of Huanglongbing decreased with feeding time, but Wolbachia titer increased with feeding time(more age).Huanglongbing bacteria can be detected in the 3rd-5th instar nymphs, male and female adults by normal PCR, while, in the 2nd-5th instar nymphs, male and female adults by q RTPCR. The titer of Huanglongbing increased with the nymphal development, was the highest during the psyllid production period.By FISH, Huanglongbing bacteria can not be detected in the egg, 1st-3rd instar nymphs,and found in the U-shaped bacteriome in the 4th, 5th instar nymphs’ abdomen.Huanglongbing bacteria and Wolbachia distributed in U-shaped bacteriome, which indicating the two bacteria can be symbiotic in the bacteriome. Moreover, Huanglongbing bacteria scattered distribution, which make Huanglongbing bacteria for horizontal transmission.In conclusion, Huanglongbing is advantageous to the population growth of citrus psyllid; Huanglongbing can increase amino acids assimilation and utilization by Asian citrus psyllid, which affects fitness of ACP. Moreover, the titer of Huanglongbing and Wolbachia increased with the nymphal development. The two bacteria can be symbiotic in U-shaped bacteriome of ACP, which may be related to the high inoculation rate of Huanglongbing for nymph. We speculate the interaction of citrus plant-Candidatus Liberibacter asiaticus-Diaphorina citri may involve Wolbachia, but the specific molecular mechanism remains unclear. Further study is needed for the mechanism. |
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