Molecular Detection Of Endosymbionts Wolbachia And Cardinium And Its Reproductive Manipulation On Psocids

The psocids, Liposcelis tricolor and L. bostrychophila Badonnel belonging to Liposcelididae, Psocoptera, are worldwide and commonly found in various processed and unprocessed dry foods. Wolbachia and Cardinium are maternally inherited intracellular bacteria that infect a wide range of arthropods and are associated with various reproductive abnormalities in their hosts, including cytoplasmic incompatibility, thelytoky or parthenogenesis, male-killing, and feminization, by which reproduction manipulations, these endosymbionts add their infection frequency in nature. In this paper, the infections of Wolbachia and Cardinium in L. tricolor and L. bostrychophila were investigated using Long PCR and hemi-nested PCR, respectively. In addition, molecular and ecology methods were used to investigate the relationship between psocids and the endosymbionts. The results will rich the infections of Wolbachia and Cardinium in nature, and offer the theory supports for the application of these endosymbionts in pest control. The project is supported by the National Natural Science Foundation of China (30471173), and lasted about 4 years, the main results are as follows:1 Molecular detection of Wolbachia and Cardinium in psocids1.1 Molecular detection of Wolbachia in psocidsTotally 12 geographical populations of 7 species of psocids were prepared for molecular detection of the Wolbachia infection through Long PCR. The results showed that a 60S bp Wolbachia wsp gene fragment was amplified from Heze, Shandong geographical population of L. tricolor. The resuts of the sequence BLAST in GenBank suggested that it belongs to Wolbachia wsp gene sequence. Phylogenetic tree of Wolbachia in different hosts was built using PHYLIP3.6a, The topology structure showed that the Wolbachia in L. tricolor belonged to B subgroup and possessed the closest evolutional relationship to Wolbachia Pip in Culex quinquefasciatus and Aedes albopictus.1.2 Molecular detection of Cardinium in psocidsThe results of hemi-nested PCR amplification of 16S rDNA suggested that L. bostrychophila from Xian Shangxi, Tongliang Chongqing, and Zhengzhou Henan were infected by Cardinium. Three 1462bp nucleic acid sequences were achieved through DNA clone. BLAST in GenBank showed that they belong to Cardinium. The phylogenetic tree based on the 16S rDNA sequences of Cardinium in different hosts using PHYLIP3.6a sofeware was constructed. The topology structure showed that these three Cardinium in different L. bostrychophila geographical population have the closest evolutional relationship, and possessed the closest evolutional relationship to endosymbiots in Acanthamoeba spp.2 Development and reproduction of L. tricolor as a function of temperatureThe psocid L. tricolor is a new recorded species in China. To date, the information on its biology and ecology is lacking. The development, survival, and reproduction of the psocid L. tricolor were evaluated at eight constant temperatures (20,22.5,25,27.5,30,32.5,35,37.5℃). The colonies were maintained under scotoperiod of 24 h and the humidity was controlled with saturated NaCl solution at 75-80%. The developmental rate, survival, intrinsic rate of increase (r_m), net reproductive rate (R₀), mean generation time (T), and population doubling time (t) of L. tricolor population were calculated at different temperature to provide an experimental basis for further research the relationship between I. tricolor and Wolbachia.2.1 Development of L. tricolor at different temperaturesThe population reared at 37.5℃failed to develop and reproduce, and then the data from 37.5℃was excluded from analysis. Within 20 to 35℃, the developmental periods of egg, nymphs, and whole generation were generally shortened with the increasing of temperature, but the high temperature (35℃) caused a decline in developmental rate of immature stage of L. tricolor.Within the range of the tested temperatures, the population reared at 30℃had the highest egg survival rate and at 27.5℃had the highest nymphal survival rate. Extreme high temperatures had a detrimental effect on the survivorship of nymphal stages, particularly for 1st stadia.The fecundity and oviposition period of L. tricolor are greater. Liposcelis. tricolor reproduced the most eggs (53.91) at 27.5℃and the fewest (17.58) at 35℃. L. tricolor had the longest adult longevity (262.15 d) at 20℃and the shortest (67.37 d) at 35℃.2.2 Life table analysis of L. tricolor at different temperaturesThe relationship between life table parameters of L. tricolor and temperatures were analyzed by constructing the age-specific and time-specific life table. Liposclis tricolor populations reared at 30℃had the largest r_m (0.0367), shortest t (18.89 d), the largest trend index of population (I) (22.17), and R₀ (19.55). The Weibull frequency distribution gave a good fit to the data set of age-specific survivorship for all test temperatures. Liposcelis tricolor populations reared at 30℃have a type I survivorship curve (c > 1.0); whereas populations reared at other temperatures have a type HI survivorship curve (c < 1.0) based on Weibull frequency distribution.3 Influence of Wolbachia on development and reproduction of L. tricolor Antibiotic treatment could remove the infection of endosymbionts from L tricolor, so Wolbachia-ftst female (♀^w-) and male (♂^w-) of L. tricolor were obtained. Crosses of Wolbachia-free L. tricolor with Wolbachia-infected strains (♀^w+,♂^w+) were carried out, the results would help to understand the relationship between Wolbachia and its host. In addition, the life history parameters such as mean developmental rate, survivorship, and intrinsic rate of increase of the Wolbachia-free strain were compared between first and second generations to clarify the influence of Wolbachia on the development and reproduction of L tricolor.3.1 Removal of Wolbachia from L. tricolorAfter 30d treatment with the artificial diets containing 0.3%, 0.03%, 1% rifampicin, the infection of Wolbachia in L. tricolor was checked with the Long PCR. The results showed that the Wolbachia infection was successfully removed by treating with 1% rifampicin for 30d. Lower concentrations of rifampicin (0.3% or 0.03%) failed to remove the Wolbachia infection completely.3.2 Crosses of Wolbachia-free with infected strains of L. tricolorFour crosses (♀⁺×♂⁺,♀^-×♂⁺,♀⁺×♂^-, and♀^-×♂^-) were tested in this experiment. The total egg numbers within 60 days were counted for each treatment. Following the theory of cytoplasmic incompatibility (CI), if Wolbachia cause the CI of I. tricolor, then the egg producted by cross of♀^w-×♂^w+ will show high embryonic mortality. In this experiment, the cross of♀^w-×♂^w+ reproduced more eggs (25.89) than cross of♀^w-×♂^w-(8.23), which suggested Wolbachia can not cause the CI in L. tricolor. In this study, egg production of♀^w-×♂^w+,♀^w+×♂^w- were all significantly less than the♀^w+♂^w+, suggested that for optimal reproduction, Wolbachia must also be present in the male.3.3 Influence of Wolbachia removal on fitness of L. tricolorIn order to elucidate the relationship between Wolbachia and L. tricolor, the bionomics of Wolbachia-free strain were observed for two consective generations. The results showed that the removal of Wolbachia infection had the negative effects on the fitness of L. tricolor, expressed as control had the highest r_m values, because of its faster development, higher survivorship of immature stages, greater total progeny production, as well as its considerably higher rate of oviposition. In contrast, Wolbachia-free strain (both in 1st and 2nd generation) had a prolonged developmental time, reduced survivorship of immature stages, as well as reduced fecundity and longevity period, resulted decline of r_m values. The Wolbachia-free strain was calculated by r_m to have a fitness value of 0.45 in F₁ and 0.27 in F₂ relative to control.4 Influence of Cardinium on development and reproduction of L. bostrychophila 4.1 Removal ofCardinium from L. bostrychophilaLiposcelis bostrychophila is a parthenogenetic species, treated with 1% rifampicin diet for 30 d could remove the Cardinium from the host completely. After observation for two consective generations of the Cardinium-free L. bostrychophila, no heritable male individual was found.4.2 Influence of Cardinium removal on fitness of L. bostrychophilaIn order to elucidate the relationship between Cardinium and L. bostrychophila, the bionomics of Cardinium-free strain were observed for two consective generations. The results showed that the removal of Cardinium infection had the negative effects on the fitness of L. bostrychophila, expressed as Cardinium-infected population had the highest r_m values, because of its faster development, higher survivorship of immature stages, greater total progeny production, as well as its considerably higher rate of oviposition. In contrast, Cardinium -free strain (both in 1st and 2nd generation) had a prolonged developmental time, reduced survivorship of immature stages, as well as reduced fecundity and longevity period, resulted decline of r_m values. The Cardinium-free strain was calculated by r_m to have a fitness value of 0.91 in F₁ and 0.74 in F₂ relative to control.