Infection Cycle Of Spiroplasma Melliferum And Replication Sites In Apis Mellifera

Spiroplasmas isolated from honeybees are characterized by helical, motile and lack of cell wall. They usually parasitize in the young bees and are one of the main pathogens which caused "crawling bee disease" in China. Since the1980s, spiroplasmosis has spread widely all over the country, and often occurs mixed with sporidiosis, palsy disease and so on. It has caused severe economic losses to the beekeeping. It is reported that spiroplasma could be detected in honeybees, plant flowers and other insects such as Hymenoptera, Diptera, Lepidoptera in the onset of spiroplasmosis, but fail to be detected in the non epidemic period, especially in winter and hot summer. Based on the findings, researchers have speculated several infection cycle ways of honeybee spiroplasmas in nature, but still lack of systematic research and direct evidence. In this research, a rapid molecular detecting method was developed and used for the detection of Spiroplasma melliferum which caused "crawling bee disease" in our country. Its host range, possible routes of transmission was systematicly studied. Basic biological characteristics and pathogenicity of spiroplasmas isolated from different hosts during the same period were compared. In addition, we preliminarily studied the location and infection characteristics of S. melliferum in midgut and chest muscles of the honeybee Apis mellifera.A molecular detecting approach, based on the Chelex-100chelating resin DNA extraction method was developed and used for the detection of spiroplasma in plant flowers, honrybees and their living environment. The detection was carried out in2-4hours and its minimum detectable concentration was5-6spiroplasmas per mL. Compared with conventional isolation culture, this method was more sensitive, rapid and efficient. This laid the foundation for the rapid diagnosis of spiroplasmosis and the resource survey of spiroplasmas.In order to explore the possible routes of transmission of honeybee spiroplasmas in nature, we detected honeybees, flowers, other insects and the living environment of honeybees regularly by using molecular detection and isolation culture methods. A total of1339honeybees,131kinds of insects,51kinds of plant flowers and77samples from beehives were collected from March2010to January2012, from which92samples were detected to contain spiroplasmas, and54spiroplasma isolates were obtained totally. Spiroplasmas can be detected in honeybees throughout the year (including the epidemic period and non epidemic period). The detection rate of diseased honeybees collected in spring and the epidemic period was highest. In addition, spiroplasmas were detected in honeybee larvae, pupae, plant flowers, other insects, hive, nest door and honeycomb which were collected in the epidemic period while spiroplasmas only found in few honeybees in the non epidemic period. It was thought that spiroplasmas persisted in honeybees and were transmitted by horizontal transmission in nature.To further confirm spiroplasmas could spread through horizontal transmission between honeybees and plant flowers, we explored its routes of transmission though artificial inoculation of spiroplasmas to honeybees and plant flowers, respectively. The results showed that spiroplasmas in honeybees could spread from honeybees to flower surface, and the spiroplasmas in flower surface aslo could spread to the honeybees and other plant flowers though feeding of honeybees. The honeybee infected by spiroplasma showed symptoms of "crawling bee disease", and plant flowers did not show any symptoms. This result provided direct evidence to study the route of transmission of spiroplasmas in nature.Morphology, motility, basic biological characteristics, serological and molecular biological characteristics of seven spiroplasma isolates isolated from different hosts (diseased Apis mellifera, healthy A. mellifera, dead A. mellifera, Calystegia hederacea, Melia azedarach, Erigeron annuus, Polygonaceae) for the same period and the same location were studied and compared. The result showed that all traits above of7spiroplasma isolates accorded completely with the descriptions of genus Spiroplasma. The growth velocity of isolates MF1006and LK1001was the fastest, their doubling time were1.8h and2.4h, respectively. MF1008was the slowest, its doubling time was7.8h. Isolates MF1006, YNP1001and LK1001could not grow at37℃, the optimum temperature was slightly lower than the remaining four strains. The results of metabolic inhibition test, deformation test and ELISA were consistent. S. melliferum CH-1antiserum could not inhibit MF1006, YNP1001, LK1001. But it showed much stronger reaction with the other four strains and could inhibit the growth of the strains. ZHUF0901antiserum and MF0905antiserum showed strong reaction with isolates MF1006, YNP1001and LK1001, respectively. Phylogenetic analysis based on the16S rDNA and ITS revealed that MF1006and YNP1001had a close relationship with Spiroplasma apis, and LK1001was close to Spiroplasma clarkii. Other four strains were all close to S. melliferum. These results suggest that the honeybees and flowers of the same area during the same period contained more than one kind of spiroplasma. The relationship of spiroplasmas isolated from different hosts was close, which further confirmed that spiroplasmas spread through horizontal transmission in nature.In summary, we proposed the route of transmission of honeybee spiroplasmas in nature. First, in the epidemic period (usually in April and May for the spring), spiroplasmas were spread from honeybees to the living eviroment or plant flower surface outside the beehives. Healthy honeybee or other insects were infected by spiroplasmas during feeding in the spiroplasma-infected places or flowers, and then transported spiroplasmas to other places within the beehives or other flower surface. This aslo provided the pathogens for the re-infection of other honeybees and insects. Second, in the non epidemic period, there was few crawling bees could be seen and the detection rate of spiroplasma was aslo very low. We speculated that it was difficult for spiroplasmas to traverse the midgut barrier to enter the hemolymph, and then multiply in the hemolymph and result in the death of honeybees at this period. A few spiroplasmas may be existed in honeybees for a long time or infect other healthy honeybees though gastrointestinal excretion. With the weather changed or other factors resulted in the honeybee resistance decline, spiroplasmas would pass through the midgut barrier, and then cause the death of honeybees. This may be the possible reason why few crawling bees occasionally appeared in the apiary during the non epidemic period.In addition, the pathogenicity of isolates MF1006, YNP1001, MF1008to Apis mellifera was investigated by feeding spiroplasma cultures. The spiroplasma-infected honeybees showed symptoms of spiroplasmosis around5days after feeding. At the same experimental conditions, the incidence speed of honeybees infected MF1006and YNP1001was faster. After9days, the mortality rates of honeybees fed by spiroplasmas were significantly higher than those fed with fresh medium. Compared with the honeybees in the control group, the honeybees in the experimental group were very significant at1%significance level. Spiroplasmas could be isolated from dead bees in the experimental group and positive control group, but could not be in the control group with fresh medium.16S rDNA sequences comparison showed that the homology of re-isolates and the feeding spiroplasma strains were the highest. It is aslo indicated that isolates MF1006, MF1008, YNP1001were indeed the cause of honeybee death. The discovery of pathogenic strains MF1006and YNP1001enriched the understanding of the pathogen of "crawling bee disease" in China. This was the second pathogenic spiroplasma to honeybees discovered in China. Finally, spiroplasmas were detected in the midgut, lymph and chest muscles of diseased honeybees by using molecular biology methods, but no one was detected in healthy bees. S. melliferum CH-1distribution, infection mechanisms and cytopathological effects in the intestines and chest muscles of Apis mellifera were investigated by transmission electron microscopy. Spiroplasmas usually occurred in membrane-bound cytoplasmic vesicles that often were located near the nuclear at the top and internal of intestinal epithelial cells, between the plasma membrane and basal lamina at the basal part. In addtion, spiroplasmas aslo found in the basal lamina and accumulated at high numbers in chest muscle cells. Compared to the tightly aligned fiber bundles in healthy muscle cells, bundles in spiroplasma-containing muscle cells appeared fragmented and loosely arranged. These symptoms were likely to be the reasons leading to the "crawling bee" and the death of honeybees.