| Panorpidae is the largest family of Mecoptera.Their larvae bear eight pairs of prolegs in addition to three pairs of thoracic legs.However,because the gene regulatory pathways of scorpionfly proleg development have not been determined,the homology and origin of the abdominal appendages of Mecopteran larvae remains unknown on the basis of morphological and anatomical evidence.This thesis mainly investigates the expression and functional analysis of some appendage-patterning genes that regulate appendage development in Panorpidae,hoping to provide evidence for the evolution of the Holometabola from the point view of proleg.In the present study,we identified the c DNA sequences of seven appendage-patterning genes and two inference genes in Panorpa liui Hua,1997 using transcriptome sequencing and bioinformatics analysis.To explore the developmental mechanism of abdominal prolegs,the expression patterns of abdominal Hox gene and its downstream gene Dll gene were analyzed by in situ hybridization,parental RNA interference and q PCR.Furthermore,the developmental pattern of the thoracic legs and prolegs was studied during embryogenesis in detail using scanning electron microscopy.Finally,to investigate the origin and homology of larval prolegs in Panorpidae,we compared the functional analysis of appendage-gap genes in the thoracic legs and prolegs by p RNAi and q PCR,and constructed character trees using pairwise differentially expressed genes at transcriptional level among nine tissues.The main research results are showed as follows:1.Based on a mix of all embryonic and larval stages of P.liui,the first de novo transcriptome currently available was condtructed by Illumina HiseqTM 2000.A total of8.8 Gb and 29454339 clean pair-end reads were generated after a stringent quality assessment and data filtering.The Q30 was more than 93.38%and the GC content was37.65%.We assembled those short reads into 62970 Unigenes（≥200 bp）with an N50 length of 1709 bp and the mean length of 813.31 bp using Trinity.BUSCO score assessed the transcriptome assembly to be 82.7%complete（S:40.4%,D:42.3%）and 15.0%as missing BUSCOs.A total of 133822 transcripts were additionally gained with an N50 length of 3786bp and the mean length of 1764.39 bp.The mapped reads were 23755576 and the mapped ratio was approximately 80.65%.The ORF predictor Trans Decoder found that 26622Unigene sequences（≥100 bp）were determined to contain complete ORFs.2.Candidate genes related to proleg development were cloned by RT-PCR and RACE,based on the next-generation transcriptome assembly.A total of nine candidate genes for P.liui were identified,including seven appendage-patterning genes（Distal-less、dachshund、extradenticle、Ultriabithorax、abdominal-A、Abdominal-B、paired）and two reference genes（β-actin、α-Tubulin）.3.The gene regulation of larval prolegs in Panorpidae was studied during embryogenesis based on the expression patterns and function of abdominal Hox genes and its downstream gene Dll.During early embryogenesis,Ubx,abd-A and Dll were co-expressed on the first abdominal segment.During post embryogenesis,Ubx and Dll were co-expressed in distal part of T1-A1 appendages;abd-A expression was solely restricted to a proximal ring of each proleg,with the most distal region lacking,whereas Dll expression was highly present in the distal tips of abdominal prolegs.Abd-B was mainly expressed on A7-A11.RNAi-mediated knockdown of Ultrabithorax（Ubx）changed the distributions of appendages of abdominal segment I（A1）and the downstream target Distal-less（Dll）,but did not affect the expression levels of Dll.Knockdown of abdominal-A（abd-A）resulted in malformed segments,abnormal prolegs,and disrupted Dll expression.Embryonic RNA interference of Abd-B resulted in a set of abnormalities,including developmental arrest,malformed suckers and misspecification of posterior segment identity.In addition,the expression of the appendage marker Dll was up-regulated and was additionally present on abdominal segments IX andⅩcompared with wild embryos.4.The origin and homology of larval prolegs in Panorpidae were investigated based on the evidence of embryonic,histological and appendage-pattern genes data.Using scanning electron microscope,we found that two pairs of processes develop chronologically on A1-A8 during scorpionfly embryonic development:the abdominal process primordia represent lateral processes in line with the thoracic legs,but then degenerate into triangular processes;the medial processes appear later along the mid-ventral line and form conical prolegs without segmentation.Parental RNAi-mediated knockdown of Dll and dac resulted in the thoracic legs being shorten,but did not affect proleg development.Comparative transcriptome shows that scorpionfly larval proleg was closer to the maxially endites than other tissues.In summary,the first de novo transcriptome for P.liui（all embryonic and larval stages）currently available was condtructed.Based on the expression and functional analysis of abdominal Hox genes（Ubx,abd-A,Abd-B）and appendage-patterning genes（exd,dac,Dll）,we found that changes in abdominal Hox gene expression and their target genes regulate abdominal appendage morphology during the evolutionary course of holometabolous larvae.Based on the expression and functional analysis of leg-gap genes data,the origin and homology of larval prolegs in Panorpidae are clarified:the prolegs are not serially homologous with the thoracic legs and may be the outgrowths origined from coxopodal endites of abdominal appendages.These results partly explain the developmental mechanism of larval prolegs and the different developmental mechanisms in regulating proleg development in various insect lineages further supported a model of evolutionary convergence,in which the re-evolution of larval prolegs is likely to be driven by the activation of particular appendage-patterning network in the abdomen. |
