| Coccoidea (Hemiptera: Coccoidea) commonly are called scale insects. According to statistics of theScaleNet, there are about7500described species in1050genera belonging to48families around theworld. Most scale insects happen commonly and damage severely to agriculture and forestry except asmall number of resource insects. They have highly adaptablility and great variation in shapes. Also,they can transmit virus and induce the sooty to host plant. Rapid and accurate identification of scaleinsects is a necessary precondition for the effective prevention and control of them. For a long time,classification based on morphological characteristics always encounters variety of complex situationswhich limite rapid and accurate identification. Such as, immatures (including egg, larva/nymph andpupa), cryptic species, species complex and sample conditions. In the present study, DNA barcodingtechnology was developed to apply in identification of scale insects. Based on known COI sequencespublished on GenBank, a pair of species-specific PCR primers (SS-COI) was designed for molecularidentification of Phenacoccus solenopsis Tinsley at the same time. The main results are as follows.(1) Application of DNA barcoding technology for species identification of scale insects. Scaleinsects are a large taxonomic group, belonge to the superfamily Coccoidea and include a number ofagronomic pests. Their morphology varies greatly amongst members of different families, so higherclassification is controversial. In the present study, the DNA barcoding method was used in a morecomprehensive way, by testing its performance on21species of scale insects in three families whichcollected from12different locations of seven provinces in China. We recovered barcode records from89specimens. Then, we calculated sequence divergences using the Kimura2-parameter (K2P) model, andconstructed a neighbour-joining (NJ) tree by MEGA. The results indicated that based on the COIsequences phylogenetic tree of21species scale insects was consistent with identification bymorphologically characters. Three distinct clusters were generated in the COI neighbour-joining (NJ)tree. The GC content was very low in most species, averaging just18.25%. Sequence divergences (K2P)between inter-specific averaged27.94%, while intra-specific variation averaged0.14%.No overlap wasobserved between inter-specific and intra-specific genetic distances. Our study indicates that the scaleinsects can be efficiently identified using the standard barcode region of COI gene.(2) Establishment of DNA barcoding system for scale insect identification. The databases includedthe specimen database, the knowledge database and DNA barcoding sequence database. The specimendatabase contained voucher specimens and DNA samples. The information for voucher specimenincluded sample coding, host plant speceis, collector, location, latitude and longitude. The DNA sampleswere stroed at-20oC. The information for30scale insect species and179DNA samples were recorded inthe specimen database. The knowledge database contains the species’ Chinese name, Latin name,morphological characteristics, biological characteristics, host plant speceis, distribution, controlmeasures and so on. The knowledge information of29scale insect species was recorded in theknowledge database. A total of437barcoding sequences from40scale insect species were recorded inthe database. One part of these sequences obtained from our present study (154sequences in6families, 24genus,30species), and the others collected from BOLD (283sequences in8families,25genus,35species). The interface of barcoding system includes COI sequence identification, sequence inquires,sequence submitting and my sequence management. The barcoding system can identify40scale insectspecies, which is of great significance in realizing the remote recognition and effective monitoring ofscale insects.(3) Species-specific COI primers for identification of Phenacoccus solenopsis Tinsley (Hemiptera:Pseudococcidae). Phenacoccus solenopsis Tinsley, a newly invasive species in China, is a worldwidepest, causing serious threat on the production of agriculture and forestry. Morphological identification ofthis mealybug species is limited by high degree of similarity and polymorphism. In the present study, amethod was described for the development of DNA marker to rapidly identify P. solenopsis. A pair ofuniversal primers based on published mitochondrial DNA cytochrome c oxidase subunit I gene (mtDNACOI) sequences of mealybugs in GenBank was designed. The mtDNA COI gene of P. solenopsis andseven other mealybug species, including Pseudococcus comstocki Kuwana, Planococcus lilaciusCockerell, Maconellicoccus hirsutus Green, Saccharicoccus sacchari Cockerell, Dysmicoccusneobrevipes Beardsley, Planococcus minor Maskel and Phenacoccus solani Ferris, common in Chinawere amplified and sequenced. And then one pair of species-specific PCR primers (SS-COI) wasdesigned. The SS-COI primers amplified a single band of546bp of P. solenopsis. The specificity of theprimer pair was validated using the seven mealybug species mentioned above. All P. solenopsisspecimens were detected, and no cross reactions with other mealybugs were observed. The method wastested on single individuals in the egg,1st-,2nd-and3rd-instar nymphs and female adult, anddemonstrated to be applicable for all life stages. Furthermore, the primer set was tested on one P.solenopsi population from Pakistan and fourteen P. solenopsis populations from invaded areas in Chinaand proved to be applicable for all geographic populations. These results suggest that the diagnostic PCRassay provides a quick, simple and reliable molecular technique for the identification and monitoring ofP. solenopsis. The technique should be useful in blocking and intercepting the further spreading of P.solenopsis.In this study, the molecular identification technology of scale insects was established based onbarcoding system. The technology provided an effective support for rapid and accurate identification ofscale insects in quarantine at port of entry, seedling transportation and so on, and built a remoterecognition and monitoring services platform. The designed SS-PCR primers for rapid identification of P.solenopsis proved to be useful in blocking and intercepting the further introduction and spreading of P.solenopsis. |
