Invasion Genetics Of Procambarus Clarkii In China

The red swamp crayfish, Procambarus clarkii, is an important freshwater aquaculture species in China. The crayfish has caused serious damages for invasion areas as an invasive species although it has attributed greatly to our economy of China. Information on dispersal pattern and various impacts for invasion success would be useful for the establishment of possible methodologies for the prevention and control of their invasions. In order to identify the spread pattern, invasion route, and related impacts in the course of invasion for the prevention and control of the crayfish, the genetic diversity and population genetic structure etc. were investigated in this study to infer initial invasion place, retrace the dispersal routes, and study dispersal pattern of P. clarkii. The mitochondrial genome of P. clarkii were cloned, and its gene structure and adaptive evolution for mitochondrial gene were analyzed, to investigate what variations the mitochondrial gene of P. clarkii and crustacean would happen while enduring different environment pressure and evolution in molecular level. And the expression of the prophenoloxidase (proPO) gene was studied after challenges by varied stimuli to infer whether there is a correlation between immunogene and invasion success of P. clarkii. The results are summarized as follows:(1) An efficient protocol for DNA extraction from red swamp crayfish exoskeleton was described. If the samples are collected from crayfish exoskeleton, the crayfish exoskeleton samples are less invasive and can be easily preserved long. And crayfish exoskeleton samples showed less apparent DNA degeneration than muscle DNA samples. The crayfish exoskeleton samples could be used for the population genetic research and related applications in molecular biology.(2) The genetic structure and diversity of P. clarkii populations at37sampling sites (35from China, one from the USA and one from Japan) were analyzed using both mitochondrial gene sequences (COI and16S rRNA) and12nuclear microsatellites. And based on those analyses, the dispersal pattern of P. clarkii, and whether the introduction of P. clarkii in China was derived from a single or multiple event(s) were further analyzed. The results showed that:(â…°) relatively high genetic diversity was detected in Chinese populations,(â…±) the population from Japan and those collected from China, particularly from NanJing (BGt and XG) and its some neighboring sites (CJr, NT and NB), have similar genetic composition,(iii) human-mediated dispersal was one important dispersal route for P. clarkii in China, and (iv) multiple introductions and/or other unintentional introductions in China might exist.(3) Based on the study of genetic structure and diversity of P. clarkii populations using both mitochondrial gene sequences (COI and16S rRNA) and nuclear microsatellites, the sequences of mitochondrial control region and nuclear proPOx intron were used to infer the invasion history of P. clarkii in China in combination with historical records. The results revealed P. clarkii is most possible from Japan, which originated via introductions from an unsampled source, then mainly expanded and transferred in the middle and lower reaches of Changjiang River in China by natural expansion and human-mediated jump dispersal, and less spread from the upper reaches of Changjiang River to the middle and lower reaches of Changjiang River. Additionally, the results also show that the dispersal pathways for P. clarkii might be much more complex than previously assumed.(4) The mitochondrial genome of P. clarkii was obtained. And the base composition and gene arrangement of the mitochondrial genome were analyzed. We investigated adaptive evolution for mitochondrial COI gene of37P. clarkii populations, and13mitochondrial genes concatenated of92crustacean mitochondrial genomes. The results showed that mitochondrial genes were conserved, given priority to purifying selection in the course of evolution, and given priority to synonymous mutations for variation.(5) The expression of the prophenoloxidase (proPO) gene was investigated in nine tissues, including haemocytes, hepatopancreas, ovary, muscles, spermary, cuticular epidermis, branchia, intestines, and stomach of red swamp crayfish Procambarus clarkii by real-time PCR before and after challenges by CpG oligodeoxynucleotide (ODN), Aeromonas hydrophila and white spot syndrome virus (WSSV). The results showed that the highest level of mRNA was observed in haemocytes, followed by those in the hepatopancreas, ovary, muscles, spermary, cuticular epidermis, branchia, intestines, and stomach sequentially. The proPO gene may play an important role in the immune system of the crayfish, and proPO expression might be activated in different tissues by different mechanisms in response to various stresses, such as CpG ODN, A. hydrophila and WSSV challenges.