Taxonomic Status of a Native Species of the Genus Feltiella Rübsaamen (Dip.: Cecidomyiidae) in Iran

 The idea of sustainable agriculture has been considered recently due to increasing knowledge and concerns about the destructive effects of chemical pesticides. Biological control is an ecologically based pest management strategy with an important role in achieving sustainable agriculture. The success of this beneficial method closely depends on taxonomy, since accurate identification of pests and their natural enemies has a great importance in biocontrol project’s achievement. The gall midges of the genus Feltiella are cosmopolitan species known as highly effective predators of tetranychid mites. Despite the high potential of Feltiella species as a biological control agent, F. acarisuga is the only species commercially available among eleven species of the genus. These predators are difficult to distinguish from each other because of the high similarity and low information about them. Comprehensive taxonomic studies are needed to identify promising species for the control of tetranychid mites. The aim of this study is to determine the status of the native Feltiella species in Iran emphasizing their molecular characteristics.

The native predatory gall midges larvae and pupae were collected periodically from the spider mites colony on various host plants (Urtica dioica, Lactuca scariola and Rubus sp.) in countrysides around Mashhad during 2018-2019 and maintained in a growth chamber (LD 16:8, 21±1°C, RH 75±5%) until emerging adults. Adults were preserved in ethanol for further analysis and identified morphologically based on male genitalia and other structures used in taxonomic treatments of the genus. The molecular genetic analysis was included DNA extraction using the Chelex 100 method, PCR amplification of the mitochondrial COI gene using the LCO/HCO universal primer pair, sequencing the gene, and matching the sequence with those of the related species using BLAST. Nucleotide divergence between sequences was estimated by Maximum Composite Likelihood model and by the Pairwise deletion method in MEGA-X software. Intra- and interspecific distances were calculated using ExcaliBAR software and their frequency distribution histogram was plotted using Excel software. The sequence data were analyzed through the neighbor-joining method using MEGA-X software. Evolutionary distances for the NJ method were computed by Kimura’s two-parameter distances. The resulting tree was subjected to bootstrap analysis with 1000 pseudoreplications. The cecidomyiid genus Endaphis was employed as an outgroup taxon to construct the phylogenetic tree.

Based on morphological studies, specimens of the native acarivorous gall midges from various localities in Mashhad were identified as Feltiella acarisuga Vallot. In spite of the morphological result, the DNA sequence of the native species was relatively different from the corresponding sequence of F. acarisuga available in GenBank. The sequence match between the two species was 92.74% in maximum. The match with F. acarivora sequences was also low (maximum 91.84%). Whereas the BLAST results of the indigenous species sequence matched the corresponding sequence of F. tetranychi with more than 99% homology. Comparing the nucleotide differences between the specimens of the present species with F. acarisuga and F. tetranychi also showed that our Feltiella is a distinct species from F. acarisuga, despite of morphological identification. In the histogram of nucleotide distances, intra and inter specific distances in the COI gene overlapped with each other which were related to the nucleotide distances between individuals of F. tetranychi species in the gene bank and individuals of the species collected in the present study. Based on the neighbor-joining tree inferred from partial sequences of the COI gene related to Feltiella species, Iranian indigenous species and F. tetranychi species were in the same ancestor, while individuals of F. acarisuga species were in separate ancestors from the native gall midges. Therefore, according to our molecular studies, the specimens of the native gall midges of Mashhad were F. tetranychi. The possible interpretation for the difference between morphological and molecular identification results in this study is the difficulty of distinguishing the two species from each other, due to their great morphological similarity. F. tetranychi has been mentioned as a possible synonym for F. acarisuga so far, because of the high morphological resemblance. Personal correspondence with international experts revealed that there are two taxa named F. tetranychi, one named by Rubsaamen and introduced as one of the synonymous names of F. acarisuga, and the other named by Kieffer which is an unknown species and mentioned as a possible synonym of F. acarisuga. To prove or disprove the hypothesis whether F. tetranychi is synonymous with F. acarisuga or completely separate from it, it is necessary to study voucher specimens of Feltiella species. Studying further populations of the gall midges on various hosts around the world through sequencing more than one molecular marker is also needed.

 In this study specimens of the native gall midges were identified as Feltiella acarisuga Vallot based on morphological identification, while molecular studies identified them as F. tetranychi. Since molecular identification is more accurate than morphological one, the present study can show how different the indigenous species is from the well-known commercial species F. acarisuga. The present native species probably has little ability to settle in artificial and manipulated environments despite of its activity in the nature of Mashhad. Its usage as a biological control agent for tetranychid mites requires further bio-ecological studies in the laboratory and its genetic comparison with known species in the world.