Isolation of salivary glands from Hippobosca equina for vector potential analysis

Abstract

Louse flies, including Hippobosca equina (Linnaeus, 1758), are hematophagous insects that infest livestock and are considered potential vectors of various pathogens. Investigating their vector potential requires a detailed examination of their salivary glands, which plays an important role in pathogen transmission. The present work details a dissection and isolation technique for salivary glands, adapted from methodologies established for other Hippoboscidae species, such as deer keds (Lipoptena spp.). The method allow the abdominal region to be dissected, enabling salivary gland extraction with minimal contamination – a crucial step for molecular analysis. From the previously described protocol, certain aspects were simplified in terms of materials. Materials like plasticine was used to secure the fly, instead of wax. For dissection, 18G needles were used instead of scalpel blades, as they allowed for more precise incisions in the abdomen due to their smaller size. Additionally, H. equina has wings, unlike Lipoptena species, which may cause difficulties in the procedure. The legs weren’t removed, contrary to the method described in the original protocol. Specimens of Hippobosca equina were collected from dairy cattle on São Miguel Island, Azores, and preserved in RNAlater for subsequent analysis. Dissections began with the flies being immobilised under a stereomicroscope. The abdominal cuticle was removed to expose the internal organs. The salivary glands were located and excised. Once isolated, the glands were stored for subsequent pathogen analysis to assess their role in transmitting infectious agents. The main difficulty found was dissecting the salivary glands of flies containing large larvae. Additionally, isolating the salivary glands was easier in males than in females. This adapted technique is crucial to advancing our understanding of the biological mechanisms underlying the vector potential of Hippobosca equina and its role in disease ecology. This study aims to refine the process of salivary gland isolation to obtain high-quality samples with minimal contamination and a higher DNA yield for molecular analyses. This will enable the capacity of this fly to transmit pathogens to be evaluated. The results provide a foundation for future investigations into pathogen transmission by Hippobosca equina, contributing to a better understanding of its impact on livestock health.