The Biology, Ecology And Molecular Evolution Study Of Chetoneura Shennonggongensis

The adaptable characteristics of cave insects include transparent body, degenerate vision, acute sense, low metabolism and long period of development. Thus, they are the ideal materials to study animal evolution and geographic distribution. Chetoneura shennonggongensis Amorim & Niu is a recently described cave-dwelling insect species, which belongs to Diptera, Keroplatidae. The larvae of C. shennonggongensis live in the moist and dark cave and construct long vertical, sticky threads to capture prey. However, they are not luminescent species. Together with the laboratory rearing and field observations, the biological character of C. shennonggongensis has been studied. The population distribution and status of C. shennonggongensis in cave food web were documented. In addition, the phylogenetic relationships of cave Keroplatidae insects have been analyzed by using 16S rDNA sequences. Finally, the homologous luciferase gene in C. shennonggongensis has been cloned by Nested-PCR, and being compared with other luciferase gene. The main results are as follows:1) MorphologyThe adult of C. shennonggongensis has the flattened flagellomeres, the presence of only two ocelli, reduced mouthparts, presence of anepisternal setae, no postspiracular setae, laterotergite bare, regular rows of tibial setulae, and unbranched R₄₊₅. All these features are listed in the genus Chetoneura. The features that differ from the genus Chetoneura are the absence of "strong bristles dorsally" on the "metanotum", the short fusion of M₁₊₂ with Rs, and the absence of a distal spine on the male gonostyle.2) BiologyUnivotine. The larval stages usually last 8-10 months. Early instar larva has low livability. Larvae inhabit dark and moist Karst cave ceiling. They often hang long sticky silk threads with a series of mucous droplets to capture prey. The larvae are the only feeding stage in the life cycle. Although they are not selective in their predation, the larvae rejected dehydrated prey. Large amounts of pupae are observed at late June, where they remain suspended horizontally or vertically from the silk threads, differing from other keroplatidae species. Sexual differentiation is evident from the pupal stage. The pupal stage is 6-7 days. July is the peak time of adult emergence. Mating usually takes place immediately upon female emergence if adult males are available. The adult life span is short, 3-5 days for females and 5 -7 days for males. Males mated several times, while females mated only once. The average number of the eggs is 85 per female. Egg development time varies from 20-30 days.3) EcologyThe spatial pattern of the larvae of C. shennonggongensis is conferred to assemble distribution in Shennonggong Cave. The most important physical factors controlling the larvae population are relative humidity and water pH in the cave. Additionally, the cave harvestman, parasite and human activities also make important roles in the larvae population distribution. To construct the cave food web by systematic investigation of Shennonggong Cave, the middle consumers in the cave food web of C. shennonggongensis has been defined. The larvae of C. shennonggongensis mainly prey on the small flying invertebrates. Cannibalism of the larvae is observed in the field.4) PhylogenyThe fragments of 16S rDNA gene were sequenced for 3 species from genus Chetoneura, family Keroplatidae., and 19 homologous sequences were downloaded from the GenBank data library. The molecular phylogenetic trees were reconstructed using Pseudobrazypeza sp.and Culex pipiens quinquefasciatus as complex outgroups with NJ, MP and ME methods. There were 103 variable sites and 86 parsimoney informative sites in the 400 and the nucleotides of the data, and the relative frequencies of the four nucleotides were unequal. The average content of A, T, C, G were 40.4%, 40.0%, 12.8% and 6.8%, respectively. The base compositions had A + T bias. Among the 20 kinds of amino acid, the Phenylalanine, Isoleucine, Lysine, Leucine and Serine are preference. The phylogenetic trees showed that the ingroup were divided into 5 clades. They were subgenus Campara, subgenus Arachnocampa, A.luminosa in New Zealand, genus Orfelia and genus Chetoneura, respectively. Among them, genus Chetoneura was more familiar with Orfelia than other relatives.5) Detection of homologous luciferase geneA homologous luciferase gene which contained 717 base pair was cloned in C. shennonggongensis. It encoded 218 amino acids and had Leucine, Serine, Alanine preference. The identity of amino acid sequence between C. shennonggongensis and A. flava is 20.7%, which is lower than D. melanogaster and A. flava (21.5%), but is higher than Photinus pyralis and A. flava (19.2%). The identity of amino acid sequence between C. shennonggongensis and A. luminosa, A. richardse is 21.0% and 21.3%, respectively. Both higher than that of D. melanogaster (20.4%, 18.1%) and P. pyralis (19.2%, 17.8%). We inferred that C. shennonggongensis do not produce light possible because of unexpression of the luciferase gene or expression without activity. The existing homologous luciferase gene may participate in other biochemical reaction in vivo.Based on the above results, we deduce that C. shennonggongensis belong to the middle-evolutionary group in the long evolutionary history of Keroplatidae insects, which provide the scientific proof for the evolution of Keroplatidae insects. This study deepens the research of Chinese cave insects, also benefit for cave biodiversity protection.