Complete Mitochondrial Genome Sequence Analysis and Phylogenetic Location Determination of Hydropsyche Fryeri (Insecta: Trichoptera)

Hydropsyche fryeri belongs to the Trichopteridae family and builds nests in clean and unpolluted streams using stones. It also can be used as an indicator of water quality. Here, we describe the complete mitochondrial genome sequence of Hydropsyche fryeri. The mitochondrial genome is 15,676 bp long and contains 13 protein-coding genes, 22 tRNAs, 2 rRNAs and an AT-rich control region. Phylogenetic tree analysis shows that Hydropsyche fryeri is more closely related to the family Hydroptera than other Trichoptera.


Introduction
Hydropsyche fryeri is a member of the Trichopteridae family. It has a fusiform head with white stripes and a brown or light-green body of about 1.9 ± 0.4 cm (Fig. 1). Hydropsyche fryeri live in clear rivers and can be used to detect water quality [1] and heavy metal [2] pollution. Hydropsyche fryeri has extremely strict requirements for water temperature and cannot survive below 20°C. However, Hydropsyche Fryeri can tolerate a turbulent water environment.
Most of the previous classi cation methods for insects of Trichoptera are morphological classi cation [3] and the phylogenetic data at the molecular level of the caddis y still requires further research.
Mitochondrial DNA (mtDNA) is a circular structure DNA that exists in the mitochondria of eukaryotic cells [4] . Mitochondrial DNA is inherited by the maternal line and its primary structure shows signi cant interand intraspecies variation and thus can be used for molecular classi cation [5] . In general, metazoan mitochondrial genomes consist of a non-coding sequence called the control region (CR) and 37 genes, including 13 protein-coding genes (PCGs), 22 tRNAs and 2 rRNAs [6] . Mitochondrial cytochrome oxidase (COX 1) and (COX 2) [7] and the tRNA gene sequences [8] are used for the classi cation of species [9] and infraspeci c category identi cation [10] . In this study, we determined the mitochondrial DNA sequence of Hydropsyche fryeri and analysed the phylogenetic relationships between this species and other related species.
Of the 22 tRNAs, only tRNA-Ser (AGA) at position 6124-6183 had a structure without a TWC arm, and an A + T content of 85.0%, while the other 21 tRNAs had typical clover structures with an A + T content of 72.9-92.2% (Table 1). Eight of the   (Fig. 4).
We compared other families of Trichoptera with Lepidoptera, which are closely related to Trichoptera. We selected eight families (Pryganeidae, Limnephilidae, Apataniidae, Uenoidae, Pryganopsychidae, Sericostomatidae, Leptoceridae and Hydropsychidae) from Trichoptera and 22 species of Hepialidae from Lepidoptera for the construction of an evolutionary tree. Hydropsyche fryeri was most closely related to Hydropsyche orris, Hydropsyche simulans and Hydropsyche pellucidula of the genus Arctopsyche of Hepialidae and was relatively closely related to Potamyia ava and Hydromanicus wulaianus of Hydropsychidae. However, Hydropsyche fryeri was distantly separated from Sericostoma personatum of Sericostomatidae and Triaenodes tardus of Leptoceridae.

Discussion
The basic composition of the Hydropsyche fryeri mitochondrial genome is consistent with the common composition of metazoans. The entire genome is 15,676 bp long and contains both overlapping and spaced gene segments. There is one structurally abnormal tRNA of 22 tRNAs while the remaining 21 have normal clover structures.
The evolutionary tree constructed by the maximum likelihood method showed that the genomic sequence and the protein-coding sequences were consistent (Fig. 5). Therefore, Hydropsyche fryeri was identi ed as being a member of Hydropsychidae, which forms a sister population with Pryganeidae and Limnephilidae.

Samples
This study was conducted without harming protected or endangered species, and all research activities were authorized. Samples were collected from of Shiwandashan River system (21°49' 33" N, 107°59' 119" E) in Fangchenggang City, Guangxi Zhuang Autonomous Region (China). The collected samples were transported through a cold chain to the Key Laboratory of Biodiversity, College of Marine Sciences, Beibu Gulf University (China). The samples were dissected in vivo under normal saline to remove the intestinal tract and head. The specimen has been deposited in Ocean college marine specimen showroom of Beibu Gulf University (Voucher No. BBGC 00014).

Mitochondrial DNA Extraction
Total mitochondrial DNA was extracted according to the method of Roehrdanz (1997) with partial modi cations. Cells were disrupted, proteinase K and RNase were added for enzymatic digestion in a water bath for 5 h at 56 ° C. DNA was extracted in a phenol: chloroform: isoamyl alcohol (25:24:1) solution and then centrifuged with a cold isopropanol precipitate and 70% ethanol wash with a dissolved TE buffer.

Gel Electrophoresis
The mitochondrial genome was obtained by gel electrophoresis and sequenced by high-throughput sequencing [11] . A 1.0% agarose gel was prepared to separate the total mitochondrial genes, and the electrophoresis conditions were a voltage of 120 V and current of 40 Ma for 20 min. The gel was observed on a Tanon3500 gel imaging system (Shanghai Tianneng Technology Co., Ltd, China) and compared with a 10,000 bp DNA marker to preliminarily identify whether the DNA size was in the insect mtDNA size range. DNA Recovery And Puri cation DNA was cut from agarose gels, weighed in 2 ml centrifuge tubes, and the DNA was recovered according to the Tiangen universal DNA puri cation recovery kit protocol (Tiangen, Beijing, China). The mitochondrial genome of Hydropsyche fryeri was sequenced and assembled using Illumina highthroughput sequencing technology and Spades v.3.5.0 software.

Figure 2
The mitochondrial ring structure of Hydroptyche fryeri.  Relative synonymous codon usage analysis of Hydropsyche Fryeri.
Page 15/16 The clover structure of 22 tRNA.