The complete mitochondrial genome of Nycteribia formosana (Diptera, Nycteribiidae)

Abstract The family Hippoboscidae is an ectoparasite that primarily inhabits bats and relies on the host’s blood for sustenance. This research provides the first complete mitochondrial genome of Nycteribia formosana, which shares similar characteristics with other dipteran insects. The circularized mitochondrial genome, spanning 15,107 bp, encompasses 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNA), two ribosomal RNA genes, and a control region. The nucleotide composition of A, C, G, and T is 40.4%, 10.9%, 6.7%, and 42.0%, respectively. The findings from the phylogenetic analysis suggest that the species under investigation forms a cluster with other species belonging to the family Nycteribiidae. Consequently, this study provides valuable insights for the identification of N. formosana.


Introduction
The family Nycteribiidae, which falls under the order Diptera and the superfamily Hippoboscoidea, consists of highly specialized blood-feeding flies that exclusively associate with bats (Mammalia: Chiroptera).These flies, known as bat flies along with the family Streblidae, are obligate body surface parasites of bats.They parasitize the host's fur and wing membranes, primarily feeding on the host's blood (Dick and Dittmar 2013).Nycteribiids exhibit distinct morphological features: this group is characterized by their complete lack of wing and spider-like appearance, the thorax is dorsoventrally flattened, head size is reduced, and eyes are either reduced or completely absent (Dick and Patterson 2006).
Bats are widely believed to serve as the primary source of transmission for numerous zoonotic pathogens.Among these pathogens, bat flies have been identified as potential carriers and significant contributors to the transmission and persistence of bat borne diseases.In recent decades, a substantial number of pathogens have been detected in bats and their ectoparasites on a global scale (Han et al. 2021) Based on the available data, Nycteribia formosana (Karaman, 1939) is predominantly a parasite of the genus Myotis and was initially detected in Taiwan, China (Orlova et al. 2021).Other documented occurrences of this species have been reported in Korea and Russia (Mogi et al. 2023).Notably, N. formosana shares striking morphological similarities with Nycteribia uenoi, thereby posing difficulties in their taxonomic differentiation (Kim et al. 2012).Consequently, the accurate identification of N. formosana presents a considerable challenge.Thus, in light of these findings, our study has contributed to the comprehensive characterization of the mitochondrial genome of N. formosana, enhanced the knowledge regarding species within the Nycteribiidae family, and established a solid genomic resource base for future investigations.

Statement
The capture, procedures, and handling of Myotis fimbriatus were conducted in accordance with the regulations and guidelines established by the Ethics Committee of Dali University, under the approval number MECDU-202104-27.This study adhered rigorously to ethical standards and guidelines pertaining to animal handling.

DNA extraction, mitogenome sequencing, and annotation
First, the genomic DNA of N. formosana was extracted using the Tissue DNA Kit (Omega, Norcross, GA).The experiment utilized a single individual to complete the genome assembly.The complete mitochondrial genome of N. formosana was sequenced using the Illumina Novoseq 6000 sequencing platform.The type of flowcell used for Illumina is MiSeq flowcell, which has a read length of 150,150 and is a paired read.In this particular study, the MitoZ 2.3 software (https://doi.org/10.1093/nar/gkz173)was employed for the assembly of the mitochondrial genome (Meng et al. 2019), while MITOS (Bernt et al. 2013) was utilized for annotation purposes.Lastly, a circular mitochondrial genome map was generated using the online platform GenomeVx (Conant and Wolfe 2008).The nucleotide sequence of N. formosana has been deposited in the GenBank database under the accession number OQ675011.

Phylogenetic analysis
According to the phylogenetic tree depicted in Figure 2, N. formosana, Dipseliopoda setosa, Basilia ansifera, Phthiridium szechuanum, and Nycteribia parvula form a single clade for the family Nycteribiidae.The molecular phylogeny supports the placement of N. formosana in the family Nycteribiidae, and the close proximity of Nycteribia parvula provides evidence for the genus identification (Figure 3).

Discussion and conclusions
This study presents a comprehensive analysis of the gene sequence of N. formosana, marking the first investigation for this species.The complete mitochondrial genome information of this species was successfully obtained.Furthermore, the phylogenetic analysis provided additional evidence supporting the homology between N. formosana and other related species.Consequently, this study effectively addresses the present limited amount of molecular data on N. formosana, thereby furnishing crucial DNA molecular information for prospective research endeavors encompassing phylogenetic studies, evolutionary analysis, and species identification within the bat flies.

Figure 1 .
Figure 1.Representative of N. formosana collected in this study (the photo was taken by Xiaoyan Zheng).

Figure 2 .
Figure 2. The maximum-likelihood phylogenetic tree of 15 species was inferred by IQ-TREE based on nucleotide sequence of 13 protein-coding genes.'Square' indicates newly sequenced data in this study.The vertical row indicates species of the same family.

Figure 3 .
Figure 3.The circular mitochondrial genome map of N. formosana.