Development of a Bio-Hybrid Mosquito Stinger-Based Atomic Force Microscopy Probe

Summary

Quantitative and controlled investigations into insect biting behaviors are crucial for devising effective strategies to combat vector-borne diseases. In this context, a method for fabricating a bio-hybrid atomic force microscopy (AFM) probe is introduced.

Abstract

Mosquitoes, notorious as the deadliest animals to humans due to their capacity to transmit diseases, pose a persistent challenge to public health. The primary prevention strategy currently in use involves chemical repellents, which often prove ineffective as mosquitoes rapidly develop resistance. Consequently, the invention of new preventive methods is crucial. Such development hinges on a thorough understanding of mosquito biting behaviors, necessitating an experimental setup that accurately replicates actual biting scenarios with controllable testing parameters and quantitative measurements. To bridge this gap, a bio-hybrid atomic force microscopy (AFM) probe was engineered, featuring a biological stinger - specifically, a mosquito labrum - as its tip. This bio-hybrid probe, compatible with standard AFM systems, enables a near-authentic simulation of mosquito penetration behaviors. This method marks a step forward in the quantitative study of biting mechanisms, potentially leading to the creation of effective barriers against vector-borne diseases (VBDs) and opening new avenues in the fight against mosquito-transmitted illnesses.

Introduction

World Health Organization (WHO) reported that vector-borne diseases (VBDs) account for over 17% of all infectious diseases, which cause more than 7,00,000 deaths per year globally. For example, as the deadliest animal in the world, mosquitoes spread numerous pathogens, such as dengue, malaria, and Zika, through blood-feeding arthropods, resulting in 700 million infections each year¹. Explorations toward the development of effective measures to prevent VBDs are of crucial importance, including mimicking the penetration behaviors of mosquitoes to investigate their biting mechanisms and studies of potential barriers to prove their efficacy in prev....

Protocol

The mosquito species used for this protocol is an uninfected adult female Aedes aegypti (A. aegypti), received frozen and stored in a -20 °C degree freezer. The species was provided by the NIH/NIAID Filariasis Research Reagent Resource Center for distribution through BEI Resources, NIAID, NIH: Uninfected Aedes aegypti, Strain Black Eye Liverpool (Frozen), NR-48920. The reagents and equipment used for the study are listed in the Table of Materials.

Discussion

Step 1 of the protocol is meant to clean the biological sample of the unwanted labium. To achieve this, an incision is made on the labium, but not on the fascicle, which rests directly beneath the labium (Figure 1). Because the fascicle and labium are not joined together at their interface (i.e., the labium is free to slide along the fascicle and is only kept in place by its attachment to the mosquito head), the performed incision is meant to separate part of the labium from the mosquito'.......

Materials

Name	Company	Catalog Number	Comments
5-SA-SE Straight Tapered Ultra Fine-Pointed Tweezers	Excelta	N/A	For manipulating/dissecting the proboscis.
C-4D Probe station	Everbeing Int’l Corp	N/A	Used for AFM assembly.
Tipless Tapping Mode Cantilever	NanoAndMore USA	TL-NCH	AFM cantilever used for mounting the labrum. Specs are shown here: Shape: Beam Force Constant: 42 N/m (10 - 130 N/m) Resonance Frequency: 330 kHz (204 - 497 kHz) Length: 125 µm (115 - 135 µm) Width: 30 µm (22.5 - 37.5 µm) Thickness: 4 µm ( 3 - 5 µm)
UV Expoxy	Let's resin	ALR00146	For stinger attachment.