# Complete mitochondrial genome pathological characteristics and scanning electron microscopic observations of Armillifer moniliformis isolated from Manis javanica

## TL;DR

This study reports the first case of Armillifer moniliformis in Malayan pangolins and provides insights into its mitochondrial genome and pathology.

## Contribution

First report of A. moniliformis in pangolins and the first complete mitochondrial genome assembly for this species.

## Key findings

- Armillifer moniliformis was found in Malayan pangolins, expanding its known host range.
- The complete mitochondrial genome of A. moniliformis was assembled, containing 13 protein-coding genes and 22 tRNA genes.
- Phylogenetic analysis placed Armillifer within the Pentastomida clade, supporting its classification in Oligostraca.

## Abstract

Armillifer moniliformis is a multi-host parasite, with previous studies documenting various wild animals as well as humans serving as its intermediate hosts. However, to date, there have been no reports of pangolins being infected with this parasite. Necropsy revealed Armillifer moniliformis formed cysts on the abdominal greater omentum and multiple organs (lungs, liver, etc.). Scanning electron microscopy showed its typical structures (oral hooks, abdominal annuli), while histopathological examination indicated infected organs exhibited congestion, inflammatory infiltration, and calcification. At the molecular level, identification based on the 18S rRNA sequence demonstrated that this parasite shares 99.89 % homology with Armillifer moniliformis and has a close phylogenetic relationship with it. In addition, Armillifer moniliformis parasites were isolated from the host, and their DNA was extracted for Illumina and HiFi sequencing, resulting in the assembly of the complete mitochondrial genome of Armillifer moniliformis. The assembled circular mitochondrial genome had a total length of 16516 bp and comprised 13 unique protein-coding genes, 22 tRNA genes, and 2 rRNA genes. The overall nucleotide composition of Armillifer moniliformis was 62 % A + T and 38 % G + C. The protein-coding genes of the parasite encode a total of 3538 amino acids, utilizing four types of initiation codons (ATA, ATC, ATT, and ATG) and three types of termination codons (TAA, TAG, and T). The genome contained 4 simple sequence repeats (SSRs) and 158 dispersed repeats (≥20 bp). Phylogenetic trees based on 13 protein-coding genes showed Armillifer and Linguatula formed a Pentastomida clade (100 % bootstrap support), which together with Branchiura and Ostracoda constitutes Oligostraca; Crustacea and Hexapoda form a monophyletic Pancrustacea clade. In this study, an Armillifer moniliformis parasite was isolated and identified from the Malayan pangolin. This work not only expands our understanding of Armillifer moniliformis species but also provides a crucial foundation for further investigations into their taxonomy, diagnostics, and biological characteristics.

Image 1

•First report of Armillifer moniliformis infection in Malayan pangolins, expanding its known host range.•SEM shows oral hooks and abdominal annuli; pathology shows congestion, inflammation and calcification in lung and liver.•18S rRNA confirms A. moniliformis; the complete mitogenome (16,516 bp) was assembled and annotated.•Phylogeny using 13 PCGs supports monophyletic Pentastomida and places it within Oligostraca.

First report of Armillifer moniliformis infection in Malayan pangolins, expanding its known host range.

SEM shows oral hooks and abdominal annuli; pathology shows congestion, inflammation and calcification in lung and liver.

18S rRNA confirms A. moniliformis; the complete mitogenome (16,516 bp) was assembled and annotated.

Phylogeny using 13 PCGs supports monophyletic Pentastomida and places it within Oligostraca.