| Pine wilt disease is one of the most serious forest diseases in the world caused by Bursaphelenchus xylophilus.It occurs seriously in China and causes huge economic losses.The latest announcement of the State Forestry & Grassland Administration of China shows that pine wilt disease has spread into Liaoning and Gansu provinces northward and westward in recent years,respectively.As an alien invasive species,the internal mechanism of B.xylophilus’ successful invasion and rapid epidemic is one of the scientific problems worthy of in-depth study.Considering the niche overlap and competition between B.xylophilus and native species,Bursaphelenchus mucronatus,in the process of invasion,the interspecific competition and molecular regulation mechanism between B.xylophilus and B.mucronatus was studied in the present paper from the perspective of mating interference,in order to reveal the biological invasion mechanism of B.xylophilus.The results are as follows:1.There was an obvious interspecific competition between B.xylophilus and B.mucronatus.In the condition of mixed cultivation(50 pairs of B.xylophilus + 50 pairs of B.mucronatus),B.xylophilus completely occupied the niche after 30 days,while B.mucronatus completely disappeared;When 10 pairs of B.mucronatus were mixed with 4 to 9 pairs of B.xylophilus,B.xylophilus could successfully eliminate B.mucronatus and occupy the niche.When 10 pairs of B.mucronatus were mixed with 1 to 3 pairs of B.xylophilus,B.xylophilus were eliminated by B.mucronatus;Further research found that there was obvious mating interference between the two nematodes.We found that when one pair of B.xylophilus was added to one B.mucronatus(whether female or male),the mating success rate of the former decreased significantly,and when two were added,the mating success rate further decreased significantly.When three were added,B.xylophilus could not mate normally due to the interference of B.mucronatus,and vice versa.However,under the interference of B.xylophilus,the mating success rate of B.mucronatus decreased more obviously,and the interference ability of male was stronger than that of female.2.The ratios of female to male of B.xylophilus and B.mucronatus were 2.77 and 2.21 respectively under the non competitive condition of single cultivation.But under the interspecific competition condition of mixed cultivation,the ratios of female to male of the two nematodes could rise to 4.87 and 3.88,respectively,which means that both nematodes have certain gender regulation abilitiesy.Considering the basic biological parameters of the two nematodes,the life cycle and average oviposition of B.xylophilus were 4 ± 0.17 days and 24.33 ± 1.16 grains respectively,while that of B.mucronatus were 4.5 ± 0.33 and 18.52 ± 0.97,respectively.When the ratios of female to male increase,the population growth of B.xylophilus would be much faster than that of B.mucronatus at the same periods.Therefore,we believed that increasing the proportion of females might be an important strategy for interspecific competition of B.xylophilus.The results of interspecific competition between the two nematodes were further simulated by mathematical model.It was found that when the two nematodes were mixed cultured with 1000 pairs,the B.xylophilus could eliminate the B.mucronatus after 8 generations,while when the B.xylophilus and the B.mucronatus were 100 and 1000 pairs respectively,the B.mucronatus could eliminate the B.xylophilus after 6 generations.3.The spatiotemporal dynamic expression characteristics and biological functions of sex pheromone receptor gene Bxy-octr-1 of B.xylophilus were studied for the first time.The sex pheromone receptor gene Bxy-octr-1 of B.xylophilus was expressed in various developmental stages,with a wide range of expression in body wall muscle,intestine,gonad and sexual organs.However,after entering the adult stage,it was mainly expressed in the reproductive system(ovary and seminal vesicle gland)and genital organs(male spicules and female vulva);In terms of relative expression,the expression of Bxy-octr-1 gene was relatively high in the early stage of ontogeny,in which the expression was the highest in J2 stage and the lowest in J3 stage,and then the expression increases in J4 and adult stage,indicating that the gene may participate in early ontogeny while completing biological functions such as signal transduction.RNAi results showed that the embryo hatching rate of B.xylophilus decreased significantly after Bxy-octr-1 gene was disturbed;After J2,J3 and J4 larvae were disturbed,the head swing frequency decreased significantly.After J2 larvae were disturbed,some larvae showed obvious bulge and swelling in the middle of the body,and obvious grooves in the pharynx and tail;After the virgin male and female nematodes were disturbed,the mating error rate increased significantly,and the effective mating rate and average oviposition decreased significantly,indicating that Bxy-octr-1 gene played a very important role in regulating the ontogeny and reproductive behavior of B.xylophilus.4.The spatiotemporal dynamic expression characteristics and biological functions of sex determination and differentiation gene Bxy-laf-1 of B.xylophilus were studied for the first time.The expression of sex determination and differentiation gene Bxy-laf-1 of B.xylophilus showed an obvious normal distribution in different developmental stages,that is,the expression was relatively low in embryonic stage,increased in J2 and J3 stage,then decreased significantly,and reached the lowest level in adult stage;In terms of expression sites,Bxy-laf-1 gene was expressed in all stages of the individual development of B.xylophilus,and the expression sites were relatively concentrated,especially in the adult stage,the gene was only expressed in the spicules of males and vulva of females.RNAi results showed that after the embryo and J2 larva were disturbed,the ratios of female to male of adults were 2.06 ± 0.08 and 2.05 ± 0.05,respectively,which was significantly lower than that of the control(2.64 ± 0.03 and 2.62 ± 0.04,respectively);After the Bxy-laf-1 gene of J2 and J3 larvae was disturbed,the larva body was abnormally twisted,curled into a circle,the head trembled slightly,lost motor ability,and the head swing frequency decreased significantly;After the adult Bxy-laf-1 gene was disturbed,the false mating rate increased significantly,indicating that the gene had a very important regulatory function in the process of sex determination and differentiation of B.xylophilus,and might be involved in the development of muscle or nervous system of B.xylophilus. |
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