| Due to the fact that social insects live inside nest with high densities, high humidity and warm temperature condition, it is considered that social insects are susceptible to disease transmission in a colony. However, in the long process of natural evolution, the high pressure that pathogen inserted on social insects had shaped the complex social defensive behavior of social insects against the transmission of pathogen in the nest, which frustrates us to control them with entomopathogenic fungi. In order to efficiently manage this invasive ant with entomopathogenic fungi, their complex behaviors must be considered. Solenopsis invicta(Red imported fire ant), belonging to Hymenoptera, Formicidae, is a seriously invasive pest and also a social insect. In the present dissertation, the social behavior and underlying defensive mechanism of S. invicta against infection of pathogenic fungi was investigated by inoculating different worker castes with fungal spores, and then observing their behavior responses such as grooming, foraging and trophallaxis as well as analyzing chemical and molecular changes of infected ants. The main results are as follows: 1 Strains screeningSeven strains of Beauveria bassiana(Bb02 and Bb04), Metarhizium anisopliae(Ma01, SM076 and M09) and Isaria fumosorosea(IFCF-H and IFCF-L) were selected and screened based on their pathogenicity as well as non-avoidance or attractive effect against S. invicta workers. The results of bioassay showed that the LC50 of strains Bb04, M09, Ma01 and IFCFH were 3.04×106, 9.90×105, 6.13×106,1.20×107 spores/ml on10 days after inoculation, respectively, in which M09 had the lowest LC50 value and LT50 value(2.7 d) than other strains. But strains of Bb02, SM076 and IFCF-L had less pathogenic effect to S. invicta workers, and also had a longer lethal time against S. invicta. Furthermore, the results of feeding preference bioassay showed that Bassiana Bb04 had significantly repellent effect against S. invicta, while other strains had no such repellent effect on S. invicta. Combined these results of pathogenicity as well as repellency, Metarhizium M09 was finally chose as experimental strain for the following experiments for its relatively high pathogenicity and short lethal time. 2 Molecular identification of M09 and electronic observation of infection processThe M09 strain was identified based on sequence of internal transcribed spacer(ITS) of Ribosomal DNA(r DNA) and aligned it with BLAST to find its similar sequence in NCBI. Then a phylogenetic tree was constructed with Neighbor-Joining method with Mega software. Results showed that the M09 strain belongs to Metarhizium anisopliae var. anisopliae. Then the infection process of M09 against S. invicta was observed. Results of scanning electron microscope(SEM)showed that most of the spores were attached on the antennae, seta, setal veoli, spiracle and thorax, less spores were attached on the relative smooth area on the surface of S. invicta just post inoculation. Majority of spores germinated and formed germ tube at 12 hours post inoculation, some could form two germ tubes as well. 24 hours after inoculation, some germ tubes expanded at the end position and started to exude secretion onto the cuticle of S. invicta and followed by penetration into cuticle and cavity of host. The results of transmission electron microscope(TEM) showed that the spores invaded host and generated granular and filamentous mycelium. The epidermis of host began to wave under the pressure of mechanical force by mycelium development. 36 hours post inoculation, mycelium in epidermis began to invade muscle tissue along with mechanical pressure and secretion of active substance. And it is observed that the tissues were dissolved next to the growing mycelium. 72 hours after infection, the mycelium began to invade the intestinal cells, and destructed the intestinal digestion function. 96 hours after infection, fungus generated great population in the host body by the way of fragmentation of mycelium hyphae and resulted in the dissolve of host organs and tissues. 3 Effect of M. anisopliae infection on brood care behavior of nurse workersIn social insects, broods as the most valuable resource of nest generation, have critical influence for the population development and growth of the whole nest. Therefore, the nurse workers usually pay more attention to protect the brood against potential infection. This research showed that when pupae were infected by M. anisopliae, nurse took 5.3 times more time to take care of the pupae compared to control. Brood care behavior could reduce the quantity of spores on the surface of pupae as well. The average spore numbers on the surface of pupae were 103.1, 51.6 and 31.3 when accompanied with 0, 2 and 10 nurse workers, respectively. In addition, the presence of ants could also inhibit spore germination. The average germination rates were 95.1%, 80.4% and 59.9% when accompanied with 0, 2 and 10 nurse workers, respectively. Finally, more pupal could emerge successfully with the increased nurse workers. Nurse workers protect brood against fungal infection guarantee the development of the nest and S. invicta population as well. 4 M. anisopliae infection accelerates the transition rate of nurse to forager and changes transcription profileThere is age-dependent worker division of labor in S. invicta. Specifically, younger worker ants engaged in the task of brood and queen care inside the nest, they gradually go outside of nest to forage and defense for the nest as they aged. This task transition generally occurs in about 2-3 weeks after emergence. It was found that nurse workers decrease the time spent in the brood chamber when they were infected by M. anisopliae. Infected nurse spent 103.4 seconds on day 1 and spent significant less time on day 3 of 38.5 seconds. The proportion of time specifically for brood care behavior also reduced, from 13.6% on day 1 to 3.5% on day 3 post inoculation. Furthermore, infected nurse decreased the contact frequency with queen on day 3 post inoculation. These behavior results indicate that fungal infection change the task of nurse workers and lead to an accelerate forager behavioral profile. Therefore, RNA-seq method was used to analyze the potential molecular mechanism for this accelerates transition from nurse to forager due to infection. Result showed that the expression levels of 289 genes were significantly changed due to infection, in which 190 genes were up regulated and 199 genes were down-regulated. These genes were significantly enriched to 17 GO terms. Comparison of the gene expression profile between forager and nurse indicated that 284 genes were differentially expressed and enriched to 16 GO terms. By comparing the two pairs of nurse-infected and nurse-forager, it was found that 43 differentially expressed genes were shared, including some oxidoreductase activity and ion binding. There was also 11 GO terms shared, including detoxing gene P450 and pheromone biding gene Gp-9. The cluster of these three samples showed that despite infected nurse behaved like a forager, the whole gene expression profile was still clustered together with normal nurse rather forager. The shared 43 genes and 11 GO terms might play potential roles in regulating the behavior of nurse to go outside as a forager. 5 M. anisopliae infection affects foraging and trophallaxis behavior of forager workersIn social insects, the foragers are responsible for collecting food and defending the nest. Since the foragers usually act outside of the nest and the chance to be infected by pathogens increased. Thus, the foraging behavior of foragers might be changed due to infection. The foods are always transferred through trophallaxis in social insects. Foragers collect the food back to the nest and share with other nestmates inside the nest. However, this kind of trophallaxis would also result in the transition of pathogens within the nest. Therefore, the foraging and trophallaxis behavior of fungal infected foragers was investigated in this chapter. Results showed that the infected foragers consumed significantly more food(0.78 ug/ant) than controls(0.62 ug/ant) on 4 day post inoculation, which might result from the fact that fungus took much energy from their host for their own development and reproduction. Food added with bitterness alkaloids(quinine) had repellent effect on forager workers. But when the foragers were infected by M. anisopliae, the repellent effect was decrease significantly. The tolerance indexes on day 1 and 2 post inoculation were-0.12 and 0.01 respectively, but it was 0.35 on day 3 post inoculation. Although the foraging behavior was activated after fungal infection, there was no evidence of higher expression level of forage gene(P>0.05). Thus, the forage gene seems to have no correlation with the alternated foraging behavior. Finally, the trophallaxis frequency of infected foragers increased on day 3 post inoculations, the frequencies for infected and control workers were 3.1 and 1.4 times/min, respectively. But the relative quantities of 16 cuticle hydrocarbons compounds was not changed, indicating that the nestmates seems not to use the CHCs to detect whether workers were infected by pathogens. 6 Grooming behavior alteration of worker under stress of M. anisopliae infectionGrooming behavior is a very important hygienic behavior in social insects. S. invicta showed significantly various resistances against M. anisopliae within different densities ants groups. The death ratio of isolated workers was 1.8 times higher than workers in a group. Fungal infected ants performed significantly more self-grooming behavior when isolated, the total time and duration of each time were 47.2 and 15.1 seconds in fungal infected workers, but there were only 6.5 and 2.4 seconds in controls. When in a group, the nestmates would perform significantly more mutual grooming behavior to infected workers, and the total time and duration were 39.2 and 14.84 seconds, respectively, but it was only21.6 and 5.5 seconds for controls. Furthermore, we counted the numbers of spores remained on the surface of workers and found that significantly more spores were removed from the surface of workers in a group(77.3%) than workers isolated(21.9%), indicating that mutual-grooming could remove more spores than self-grooming. Grooming behavior could increase the chance of nestmates to contact with pathogens and lead to infection of nestmates. However, results showed that exposure to little amounts of pathogenic fungi would not cause significant mortality of nestmates, but improved the immunity of nestmates against the pathogen during short time, indicating the social immunity in S. invicta. But whether this immunity can maintain long-term needs further research. 7 The impact of infected corpses on the necrophoric behavior of workerSince the microbial on the body of dead ants, the corpses threaten the survival of other nestmates. Such threat would be more serious in a high humidity, suitable temperature and closed ants nest. Therefore, in this chapter, how S. invicta deal with the naturally died corpses as well as fungal infected corpses of their nestmates were investigated. The results showed that the S. invicta showed different responses toward corpses from the same nest and other nest. S. invictaspent495 seconds to remove the nestmate corpses, but spent significantly less time(305 sec) to remove corpses from other colonies. They also showed different necrophoric behavior toward different developmental stages(adult workers and pupae) corpses from the same colony. Specifically, S. invicta removed almost all of the worker corpses to dead pile in 1 hour while took about 24-48 hours to recognize and remove the pupal corpses. S. invicta removed the worker corpses in one hour suggest that they could recognize the corpses quickly. However, the chemical alteration on the surface of corpses was not detected. S. invicta could recognize the pupal corpses at 24 hours post death. There was more accumulation of palmitic acid, linoleic acid and oleic acid on the infected pupal corpse’s surface than on controls. Specifically, for palmitic acid, linoleic acid and oleic acid, there were 0.62, 0.7 and 1.1 μg on each infected corpse, but only 0.38, 0.27 and 0.42 μg for control, respectively. When put three kinds of fatty acids on surface of freshly died pupal corpses, worker would treated them as long time died corpses and removed about 11.1%, 50.5% and 68.9% for palmitic acid, linoleic acid and oleic acid treated pupal corpses, respectively, to dead pile. The induced necrophoric behavior was more obvious when put combined fatty acid on pupal corpses. Such result suggests that these fatty acids are responsible for the necrophoric behavior of S. invicta. Accelerated removing of fungal infected pupal corpses would with no doubt reduce the risk of infecting other healthy brood and queen by pathogens, and therefore minimized the risk of outbreak of a pandemic disease in the nest. Conclusion: With the stress of pathogenic fungi infection, nurse, forager and hygienic workers showed various defensive behaviors to prevent the growth and spread of fungi in the nest. When broods were inoculated with fungal spores, nurse showed more frequent brood care behavior, resulting in the reduction of spores on the brood surface and decreased germination and survival of fungal spores as well. Furthermore, when the nurse themselves were infected with fungi, they transfer from inside nest of caring the brood and queen to outside of nest, which would apparently reduce the opportunity of queen and brood infected by fungi. While foragers would increase their feeding behavior as well as trophallaxis behavior after 3 days post fungal inoculation. The hygienic workers would perform a frequent grooming behavior toward spores inoculated nestmates, and also acceralated the necrophoresis behavior toward fungal infected pupal corpses to outside of the nest. The various defensive behavior performed by these three worker castes could significantly reduce the survival and spread of spores in the nest, which challenge the use of entomopathogenic fungi for the biological control of S. invicta. |
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