Identification Technology Of Two Quarantine Mealybugs And Their Morphologically Allied Species

Dysmicoccus neobrevipes and Planococcus minor belonging to Insecta,Hemiptera,Coccoidea,Pseudococcidae,are quarantine pests in China.However,in import it is always difficult to distinguish them based on morphological characters,because of their morphologically allied species existed.The allied species for D.neobrevipes is Dysmicoccus brevipes,a species in the same genus.The allied species for P.minor is Planococcus citri in the same genus.In this study,some experiments were conducted in the two quarantine mealybugs and their morphologically allied species,to investigate the identification technology,based on the combine morphological and molecular characters.Research contents（1）Biological experiment:A rearing program was developed for the four maelybugs in lab,and their biology was observed.（2）Morphological technology:Morphological characters of the four female mealybugs in different developmental stages(from 1^st instar nymph to adult)were observed,measured,counted,described and illustrated.（3）Molecular technology:110 specimens from different geographic populations of the four mealybugs were used.DNA barcoding was implied to distinct them.The four molecular makers（28S,ITS,COI,EF-1α）were tested and selected,amplified and sequenced to identify the morphologically allied species,D.neobrevipes and D.brevipes,P.minor and P.citri.Research results（1）Biological differenceD.neobrevipes and D.brevipes were reared on Cucurbita moschata and their populations were establisted.The results showed that the two species of mealybugs both have gathering habit.The female developmental period was about 39 days in one generation.The reproduction,survivorship and longevity of D.neobrevipes were higher than D.brevipes.P.minor and P.citri were reared on Citrus medica and their populations were establisted,and their biology is similar.（2）Identification based on Morphological charactersDiagnosis between D.neobrevipes and D.brevipes1^st instar nymph:diameter of eyes/body length;diameter of anal ring/body length;claw length of hind leg/hind leg length;dorsal cerarius and circulus present or lack;the type of discoidal pore.2^nd instar nymph:diameter of eyes/body length;mouthparts length/body length;diameter of anal ring/body length;claw length of hind leg/hind leg length;1^st flagellomere length of antenna/antenna length;2^nd flagellomere length of antenna/antenna length;scape length of antenna/antenna length;different numbers of trilocular between dorsum and venter;distribution range of discoidal pore on venter;the type of discoidal pore.3^rd instar nymph:spiracle length/spiracle width;claw length of hind leg/hind leg length;body seta length/body length;4th flagellomere length of antenna/5m flagellomere length of antenna;different numbers of trilocular between dorsum and venter;the type of discoidal pore.Female adult:spiracle length/spiracle width;claw length of hind leg/hind leg length;dorsal seta length/body length;4th flagellomere length of antenna/5th flagellomere length of antenna;different numbers of multilocular disc pore between dorsum and venter;oral collar tubular duct present or lack in 3^rd and 8th abdominal segments;the type of discoidal pore.Diagnosis between P.minor and P.citri1^st instar nymph:The morphological characters in 1^st instar nymph were not provided,because too few slide specimens of P.minor can be used.2^nd instar nymph:spiracle length/body length;different numbers of trilocular between dorsum and venter;distribution range of simple pore and oral collar tubular duct.3^rd instar nymph:1^st flagellomere length of antenna/antenna length;different numbers of trilocular between dorsum and venter;simple pore present or lack in 6th abdominal segments;oral collar tubular duct present or lack.Female adult:different numbers of trilocular between dorsum and venter;different numbers of simple pore between thorax and abdomen on dorsum;distribution range of multilocular disc pore and oral collar tubular duct.（3）Identification based on Molecular charactersPrimer:Primers 28S_S3660_F/28S_A335_R,ITS_n44_F/ITS_n55_R,Pco_F/C1-12342_R and EF_scutA_F/EF_rcM52.6_R were tested and a single,stable and clear band was given in PCR products in all samples from the four mealybugs.Sequencing:All PCR products with a single band were sequenced in this study.Results showed that 28S,ITS and COI sequences were successfully obtained in the four mealybugs.Sequencing EF-1α succesed in D.neobrevipes,P.minor and P.citri,but sequencing EF-1α failed in D.brevipes,because its single band was produced by co-amplification of similar-sized fragments.Barcode gap:The divergence gap between the interspecific minimum and the intraspecific maximum is barcode gap.The barcode gap between D.neobrevipes and D.brevipes existed in the three regions（28S,ITS,COI）,and they were 2.13%,12.51%,4.50%,respectively.Barcode gap of P.minor and P.citri existed in the two regions（28S,ITS,COI）,and they were 0.28%,0.54%,1.69%,respectively.In the EF-1αregion,a phenomenon of sharing same haplotype was found in the two mealybus,so that it is without barcode gap between P.minor and P.citri.Phylogenetic NJ tree:The NJ trees inferred from 28S,ITS and COI showed that sequences of D.neobrevipes and D.brevipes clustered correctly with their respective species and fell into two reciprocally monophyletic clades in confidence 89%-100%.The NJ trees inferred from ITS and COI showed sequences of P.minor and P.citri clustered correctly with their respective species and fell into two reciprocally monophyletic clades.However,the confidence of NJ trees inferred from 28S and EF-1α were under 70%.Conclusion:DNA makers for identification D.neobrevipes and D.brevipes were COI（about 750 bp）,ITS（about 1300 bp）and 28S（about 750 bp）.DNA makers for identification P.minor and P.citri were COI（about 750 bp）and ITS（about 1300 bp）.