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Mosquitoes: A Laboratory Manual


Subject Area(s):  Biotechnology; Laboratory Techniques

Edited by Laura B. Duvall, Columbia University; Benjamin J. Matthews, The University of British Columbia

Download a Free Excerpt from Mosquitoes: A Laboratory Manual:

Preface
Methods to Assess Blood and Nectar Meals in Aedes aegypti Mosquitoes
Index

Due May 2024 • 771 pages, illustrated (111 color and 7 B&W), index
Paperback •
ISBN  978-1-621824-41-1

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  •     Description    
  •     Contents    

Description

Mosquitoes have been studied for centuries by entomologists, epidemiologists, biologists, geneticists, and ecologists. Early investigations primarily focused on their anatomy, life cycles, and feeding habits. As scientific knowledge progressed, however, the research scope expanded and so has our understanding of the underlying biology of mosquito genetics, physiology, behavior, and pathogen transmission dynamics.

This laboratory manual provides a comprehensive collection of experimental procedures for research using mosquitoes. It includes basic methods for mosquito rearing, egg collection, scoring mating status, scoring reproductive and host-seeking behaviors, imaging RNA and proteins, and evaluating physiological responses to stimuli. The collection also features protocols for genetic manipulation of mosquitoes using RNAi and CRISPR–Cas9 genome editing. Methods to evaluate insecticide resistance are also described.

The methods in this manual focus primarily on Aedes and Anopheles mosquitoes, although we also provide rearing protocols for Culex, and many of the protocols included may also work well in multiple species, with minor modifications. This manual aims to serve both as a critical reference for laboratories already working with mosquitoes and as a comprehensive guide for those starting to use mosquitoes in their experimental work.

Contents

Preface
SECTION 1. REARING AND HUSBANDRY
CHAPTER 1
INTRODUCTION
Overview of Aedes aegypti and Use in Laboratory Studies
Margot P. Wohl and Conor J. McMeniman
PROTOCOLS
1 Batch Rearing Aedes aegypti
Margot P. Wohl and Conor J. McMeniman
2 Single-Pair and Small-Group Crosses of Aedes aegypti
Margot P. Wohl and Conor J. McMeniman
CHAPTER 2 INTRODUCTION
Considerations for Rearing and Maintaining Anopheles in the Laboratory
Laura N. Leite, Priscila Bascuñán, Ellen M. Dotson, and Mark Q. Benedict
PROTOCOLS
1 Anopheles Egg Collection, Disinfection, and Hatching
Laura N. Leite, Priscila Bascuñán, Ellen M. Dotson, and Mark Q. Benedict
2 Anopheles Larval Rearing
Laura N. Leite, Priscila Bascuñán, Ellen M. Dotson, and Mark Q. Benedict
3 Anopheles Adult Anesthesia, Feeding, and Sex Separation
Laura N. Leite, Priscila Bascuñán, Ellen M. Dotson, and Mark Q. Benedict
4 Anopheles Pupa Collection and Sex Identification
Laura N. Leite, Priscila Bascuñán, Ellen M. Dotson, and Mark Q. Benedict
CHAPTER 3 INTRODUCTION
Points to Consider When Establishing and Rearing Culex Mosquitoes in the Laboratory
Megan E. Meuti, Alden Siperstein, and Matthew Wolkoff
PROTOCOLS
1 Establishing a Culex Colony from Field-Collected Eggs
Megan E. Meuti, Alden Siperstein, and Matthew Wolkoff
2 Rearing and Maintaining a Culex Colony in the Laboratory
Megan E. Meuti, Alden Siperstein, and Matthew Wolkoff
CHAPTER 4 INTRODUCTION
Establishing Colonies from Field-Collected Mosquitoes: Special Accommodations for Wild Strains
Noah H. Rose, John J. Shepard, and Diego Ayala
PROTOCOLS
1 Collecting, Storing, and Hatching Aedes aegypti Eggs
Noah H. Rose, John J. Shepard, and Diego Ayala
2 Mosquito Larvae and Pupae Transport from the Field
Diego Ayala, John J. Shepard, and Noah H. Rose
SECTION 2. FEEDING ANALYSIS
CHAPTER 5 INTRODUCTION
Membrane Feeding Devices to Blood Feed Mosquitoes in the Laboratory
Andre Luis Costa-da-Silva
PROTOCOL
1 Artificial Membrane Feeding Mosquitoes in the Laboratory with Glytube
Andre Luis Costa-da-Silva
CHAPTER 6 INTRODUCTION
Trypsin, the Major Proteolytic Enzyme for Blood Digestion in the Mosquito Midgut
Andrew B. Nuss and Monika Gulia-Nuss
PROTOCOL
1 Analyzing Blood Digestion: Estimation of Active Trypsin Levels in the Mosquito Midgut
Jeremiah Reyes, Andrew Nuss, and Monika Gulia-Nuss
CHAPTER 7 INTRODUCTION
Methods to Assess Blood and Nectar Meals in Aedes aegypti Mosquitoes
Krithika Venkataraman, Veronica Jové, and Laura B. Duvall
PROTOCOL
1 Size Quantification of Blood and Sugar Meals in Aedes aegypti Mosquitoes
Krithika Venkataraman, Veronica Jové, and Laura B. Duvall
CHAPTER 8 INTRODUCTION
Characterizing Mosquito Biting Behavior at High Resolution
Gregory P.D. Murray, Emilie Giraud, and Felix J.H. Hol
PROTOCOL
1 Characterizing Mosquito Biting Behavior Using the BiteOscope
Gregory P.D. Murray, Emilie Giraud, and Felix J.H. Hol
CHAPTER 9 INTRODUCTION
Determination of Mosquito Probing and Feeding Time to Evaluate Mosquito Blood Feeding
Ines Martin-Martin, Adeline E. Williams, and Eric Calvo
PROTOCOLS
1 Evaluation of Aedes aegypti Feeding Status
Ines Martin-Martin, Adeline E. Williams, and Eric Calvo
2 Evaluation of Aedes aegypti Penetration, Probing, and Feeding Times on Mice
Ines Martin-Martin, Adeline E. Williams, and Eric Calvo
CHAPTER 10 INTRODUCTION
Mosquito Pollination and Sugar Detection Methods: An Overview
Daniel A.H. Peach
PROTOCOLS
1 Sugar Detection in Mosquitoes via Anthrone Tests
Daniel A.H. Peach
2 Assessment of Mosquito Pollination
Daniel A.H. Peach
CHAPTER 11 INTRODUCTION
Mosquito Blood Meal Analysis
Lawrence E. Reeves and Nathan D. Burkett-Cadena
PROTOCOLS
1 Preservation of Field-Collected Mosquito Blood Meals
Lawrence E. Reeves and Nathan D. Burkett-Cadena
2 Extracting DNA from Preserved Mosquito Blood Meals
Lawrence E. Reeves and Nathan D. Burkett-Cadena
3 Amplification and Identification of Vertebrate Host Cytochrome c Oxidase Subunit I (COI) DNA Barcoding Templates from Mosquito Blood Meals
Lawrence E. Reeves and Nathan D. Burkett-Cadena
SECTION 3. HOST-SEEKING EXPERIMENTS
CHAPTER 12 INTRODUCTION
Quantifying Mosquito Attraction Behavior Using Olfactometry
John S. Castillo, Anthony J. Bellantuono, and Matthew DeGennaro
PROTOCOLS
1 Building a Uniport Olfactometer to Assess Mosquito Responses to Odors
John S. Castillo, Anthony J. Bellantuono, and Matthew DeGennaro
2 Quantifying Mosquito Attraction Using a Uniport Olfactometer
John S. Castillo, Anthony J. Bellantuono, and Matthew DeGennaro
CHAPTER 13 INTRODUCTION
Quantifying Aedes aegypti Host Odor Preference Using a Two-Port Olfactometer
Hillery C. Metz, Jessica L. Zung, and Carolyn S. McBride
PROTOCOL
1 An Assay for Quantifying Aedes aegypti Host Odor Preference Using a Two-Port Olfactometer
Hillery C. Metz, Jessica L. Zung, and Carolyn S. McBride
CHAPTER 14 INTRODUCTION
Quantifying Mosquito Host Preference
Diego Giraldo and Conor J. McMeniman
PROTOCOLS
1 A Behavioral Assay to Quantify Odor-Guided Thermotaxis with Anopheles gambiae under Semi-Field Conditions
Diego Giraldo and Conor J. McMeniman
2 Quantification of Anopheles gambiae Olfactory Preferences under Semi-Field Conditions
Diego Giraldo and Conor J. McMeniman
CHAPTER 15 INTRODUCTION
Key Features and Considerations for Using Automated Long-Term Monitoring of Heat-Seeking Behavior of Mosquitoes
Chisako Sakuma, Emi Maekawa, and Hirotaka Kanuka
PROTOCOL
1 Automated Long-Term Monitoring of the Heat-Seeking Behavior of Mosquitoes
Chisako Sakuma, Emi Maekawa, and Hirotaka Kanuka
SECTION 4. MATING ASSESSMENT
CHAPTER 16 INTRODUCTION
Acoustic-Related Mating Behavior in Tethered and Free-Flying Mosquitoes
Garrett P. League, Catalina Alfonso-Parra, Hoover Pantoja-Sánchez, and Laura C. Harrington
PROTOCOL
1 Recording and Analysis of Mosquito Acoustic-Related Mating Behavior
Hoover Pantoja-Sánchez, Garrett P. League, Laura C. Harrington, and Catalina Alfonso-Parra
CHAPTER 17 INTRODUCTION
Insemination in Aedes aegypti and Aedes albopictus
Dhwani Parsana, Ferdinand Nanfack-Minkeu, and Laura K. Sirot
PROTOCOL
1 Determining the Insemination Status of Aedes Mosquitoes
Dhwani Parsana, Ferdinand Nanfack-Minkeu, and Laura K. Sirot
SECTION 5. EGG AND OVIPOSITION STUDIES
CHAPTER 18 INTRODUCTION
Mosquito Egg Development and Eggshell Formation
Jun Isoe, Michael A. Riehle, and Roger L. Miesfeld
PROTOCOLS
1 Visualization of Apoptotic Ovarian Follicles during Aedes aegypti Mosquito Egg Maturation by Fluorescent Imaging Studies
Jun Isoe, Roger L. Miesfeld, and Michael A. Riehle
2 Identification of Mosquito Eggshell Proteins from Aedes aegypti by Liquid Chromatography with Tandem Mass Spectrometry (LC–MS/MS) Proteomic Analysis
Jun Isoe, Krishna Parsawar, Cynthia L. David, Michael A. Riehle, and Roger L. Miesfeld
CHAPTER 19 INTRODUCTION
Measuring Oviposition Preference in Aedes aegypti Mosquitoes
Yunusa Garba Mohammed, Nicholas Kristoff Tochor, and Benjamin J. Matthews
PROTOCOLS
1 Evaluating Egg-Laying Preference of Individual Aedes aegypti Mosquitoes
Nicholas Kristoff Tochor, Yunusa Garba Mohammed, and Benjamin J. Matthews
2 Automated Egg-Counting Approaches for Aedes aegypti Oviposition Experiments
Nicholas Kristoff Tochor, Yunusa Garba Mohammed, and Benjamin J. Matthews
SECTION 6. FLIGHT AND NAVIGATION ANALYSIS
CHAPTER 20 INTRODUCTION
Retrospective Review of and Introduction to the Analysis of Mosquito Optomotor Responses
Clément Vinauger and Jeffrey A. Riffell
PROTOCOL
1 Tethered Preparation for the Analysis of Mosquito Visual-Motor Responses Using Modular Visual Displays
Clément Vinauger and Jeffrey A. Riffell
CHAPTER 21 INTRODUCTION
Experiments and Analysis of Mosquito Flight Behaviors in a Wind Tunnel: An Introduction
Diego Alonso San Alberto, Claire Rusch, and Jeffrey A. Riffell
PROTOCOL
1 Conducting an Analysis of Mosquito Flight Behaviors in a Wind Tunnel
Diego Alonso San Alberto, Claire Rusch, and Jeffrey A. Riffell
CHAPTER 22 OVERVIEW
Wind Tunnels and Airflow-Driven Assays: Methods for Establishing the Cues and Orientation Mechanisms That Modulate Female Mosquito Attraction to Human Hosts
Ring T. Cardé
CHAPTER 23 INTRODUCTION
Using Videography to Study the Biomechanics and Behavior of Freely Moving Mosquitoes
Andrew K. Dickerson, Florian T. Muijres, and Remco Pieters
PROTOCOLS
1 Designing a Generic Videography Experiment for Studying Mosquito Behavior
Florian T. Muijres, Andrew K. Dickerson, and Remco Pieters
2 Real-Time Tracking of Multiple Moving Mosquitoes
Andrew D. Straw, Remco Pieters, and Florian T. Muijres
3 Tracking the Body, Wing, and Leg Kinematics of Moving Mosquitoes
Tyson L. Hedrick, Andrew K. Dickerson, Florian T. Muijres, and Remco Pieters
4 Quantifying and Analyzing Mosquito Movement from Video Tracking Results
Florian T. Muijres
SECTION 7. PHYSIOLOGY
CHAPTER 24 INTRODUCTION
Mosquito (Diptera: Culicidae) Vision and Associated Electrophysiological Techniques
Daniel A.H. Peach and Adam J. Blake
PROTOCOL
1 Measuring Mosquito Spectral Sensitivity Using Electroretinograms
Daniel A.H. Peach and Adam J. Blake
CHAPTER 25 INTRODUCTION
Mosquito Electroantennography
Chloé Lahondère
PROTOCOL
1 Mosquito Electroantennogram Recordings
Chloé Lahondère
CHAPTER 26 INTRODUCTION
Taste Sensory Responses in Mosquitoes
Adriana Medina Lomelí and Anupama Arun Dahanukar
PROTOCOL
1 Single-Sensillum Taste Recordings in Mosquitoes
Adriana Medina Lomelí and Anupama Arun Dahanukar
CHAPTER 27 INTRODUCTION

Identification of Bioactive Volatile Organic Compounds Using Combined Gas Chromatography and Single-Sensillum Recording
Rickard Ignell, Majid Ghaninia, and Sharon Rose Hill
PROTOCOL
1 Combined Gas Chromatography and Single-Sensillum Recording for Mosquitoes
Rickard Ignell, Majid Ghaninia, and Sharon Rose Hill
CHAPTER 28 INTRODUCTION
GCaMP Imaging in Mosquitoes: Central Nervous System
Zhilei Zhao, Lukas Weiss, and Carolyn S. McBride
PROTOCOL
1 Two-Photon Calcium Imaging in the Brain of Aedes aegypti Mosquitoes
Zhilei Zhao, Lukas Weiss, and Carolyn S. McBride
CHAPTER 29 INTRODUCTION
Genetically Encoded Calcium Indicators for Functional Imaging of Mosquito Olfactory Neurons
Ali Afify and Christopher J. Potter
PROTOCOL
1 Calcium Imaging of Anopheles coluzzii Mosquito Antennae Expressing the Calcium Indicator GCaMP6f
Ali Afify and Christopher J. Potter
CHAPTER 30 INTRODUCTION
Acoustic Physiology in Mosquitoes
Matthew P. Su, Marta Andrés, Marcos Georgiades, Judit Bagi, and Joerg T. Albert
PROTOCOLS
1 Immunohistochemical Staining of the Mosquito Ear
Marta Andrés, Judit Bagi, and Joerg T. Albert
2 Electrophysiological Measurements of Compound Action Potential Responses from the Antennal Nerve in Response to Stimulation
Matthew P. Su and Joerg T. Albert
3 Recording and Extraction of Mosquito Flight Tones
Marcos Georgiades and Joerg T. Albert
4 Mosquito Phonotaxis Assay
Marcos Georgiades and Joerg T. Albert
CHAPTER 31 INTRODUCTION
Physiological and Behavioral Characterization of Larval Olfaction in the Malaria Vector Mosquito Anopheles coluzzii
Feng Liu, Huahua Sun, and Laurence J. Zwiebel
PROTOCOLS
1 Examining Peripheral Electrophysiological Responses of the Larval Antennal Sensory Cone of Anopheles coluzzii to Volatile Odorants
Feng Liu, Huahua Sun, and Laurence J. Zwiebel
2 Cup and Pan Behavioral Assays for Assessing Anopheles coluzzii Larval Volatile Responses
Feng Liu, Huahua Sun, and Laurence J. Zwiebel
SECTION 8. GENETIC TECHNIQUES
CHAPTER 32 INTRODUCTION
Mosquito Embryo Microinjection
Robert A. Harrell II
PROTOCOL
1 Mosquito Embryo Microinjection under Halocarbon Oil or in Aqueous Solution
Robert A. Harrell II
CHAPTER 33 INTRODUCTION
Mosquito Transposon-Mediated Transgenesis
Vanessa Bottino-Rojas and Anthony A. James
PROTOCOL
1 Generating and Validating Transgenic Mosquitoes with Transposon-Mediated Transgenesis
Vanessa Bottino-Rojas and Anthony A. James
CHAPTER 34 INTRODUCTION
Design and Validation of Guide RNAs for CRISPR–Cas9 Genome Editing in Mosquitoes
Ivan Hok Yin Lo and Benjamin J. Matthews
PROTOCOL
1 Validating Single-Guide RNA for Aedes aegypti Gene Editing
Ivan Hok Yin Lo and Benjamin J. Matthews
CHAPTER 35 INTRODUCTION
Oral RNAi for Gene Silencing in Mosquitoes: From the Bench to the Field
Keshava Mysore, Limb K. Hapairai, Jacob S. Realey, Longhua Sun, Joseph B. Roethele, and Molly Duman-Scheel
PROTOCOL
1 Sugar-Baited Delivery of Small Interfering RNA for Gene Silencing in Adult Mosquitoes
Keshava Mysore, Limb K. Hapairai, Jacob S. Realey, Longhua Sun, Joseph B. Roethele, and Molly Duman-Scheel
CHAPTER 36 OVERVIEW
Genetic Toolbox Approaches in Mosquitoes
Olena Riabinina, Matthew Quinn, and Joe P. Whitehead
CHAPTER 37 INTRODUCTION
CRISPR–Cas9 Methods and Key Considerations in the Production of Aedes aegypti Mutant Strains
Ruichen Sun, Robyn Raban, and Omar S. Akbari
PROTOCOL
1 Generating Aedes aegypti Mutant Strains with Transgenic Cas9
Ruichen Sun, Robyn Raban, and Omar S. Akbari
CHAPTER 38 INTRODUCTION
Techniques for Identifying and Sorting Transgenic Mosquito Larvae
Eric Marois
PROTOCOLS
1 Screening Mosquito Larvae under a Fluorescence Binocular Microscope
Eric Marois
2 Sorting Mosquito Larvae with a COPAS Machine
Eric Marois
3 Selecting Transgenic Mosquito Larvae with Puromycin
Eric Marois
SECTION 9. IMAGING AND ANALYSIS OF GENE EXPRESSION
CHAPTER 39 INTRODUCTION
The Golgi Method
Rickard Ignell and Sharon Rose Hill
PROTOCOL
1 Golgi Silver Staining of Mosquito Central Neuropils
Rickard Ignell and Sharon Rose Hill
CHAPTER 40 INTRODUCTION
Introduction to Techniques Used to Study Mosquito Neuroanatomy and Neural Circuitry
Florence V. Guerina, Ameya P. Patkar, and Meg A. Younger
PROTOCOLS
1 Whole-Mount Immunofluorescent Labeling of the Mosquito Central Nervous System
Meg A. Younger
2 Dextran Amine–Conjugated Neural Tracing in Mosquitoes
Meg A. Younger
CHAPTER 41 INTRODUCTION
Mapping Transcript Cell-Specific Localization and Protein Subcellular Localization in the Adult Mosquito Aedes aegypti
Farwa Sajadi, Salwa Afifi, Britney Picinic, and Jean-Paul V. Paluzzi
PROTOCOLS
1 Examining Cell-Specific Localization in Aedes aegypti Tissues with Fluorescence In Situ Hybridization
Farwa Sajadi and Jean-Paul V. Paluzzi
2 Examining Protein Localization in Aedes aegypti Cells, Tissues, and Organs: Whole-Mount Immunohistochemistry
Salwa Afifi, Farwa Sajadi, and Jean-Paul V. Paluzzi
3 Examining Apical or Basolateral Protein Localization in Aedes aegypti Tissues: Cross-Section Immunohistochemistry
Farwa Sajadi, Britney Picinic, Salwa Afifi, and Jean-Paul V. Paluzzi
4 Membrane Protein Fractionation and Analysis through Western Blot in Aedes aegypti Malpighian Tubules
Britney Picinic and Jean-Paul V. Paluzzi
CHAPTER 42 INTRODUCTION
Performing Immunohistochemistry in Mosquito Salivary Glands
Ines Martin-Martin, Thiago Luiz Alves E. Silva, Adeline E. Williams, Joel Vega-Rodriguez, and Eric Calvo
PROTOCOL
1 A Simple Method for Immunohistochemistry and Imaging of Mosquito Salivary Glands
Ines Martin-Martin, Thiago Luiz Alves E. Silva, Adeline E. Williams, Joel Vega-Rodriguez, and Eric Calvo
CHAPTER 43 INTRODUCTION
RNA In Situ Hybridization and Immunohistochemistry to Visualize Gene Expression in Peripheral Chemosensory Tissues of Mosquitoes
Margaret Herre and Chloe Greppi
PROTOCOLS
1 Cryosectioning and Immunohistochemistry of Peripheral Tissues of Mosquitoes
Chloe Greppi
2 Immunostaining Whole-Mount Olfactory Tissues of Mosquitoes
Margaret Herre
3 RNA In Situ Hybridization of Whole-Mount Mosquito Olfactory Tissues
Margaret Herre
CHAPTER 44 INTRODUCTION
Osmoregulatory Challenges Faced by Mosquitoes and Techniques for Studying the Transporters Involved in Their Adaptations
Andrea C. Durant and Andrew Donini
PROTOCOL
1 Localizing Protein Expression of Transporters in Epithelial Cell Membranes of Mosquitoes
Andrea C. Durant and Andrew Donini
CHAPTER 45 INTRODUCTION
Visualization of the Linear and Spatial Organization of Chromosomes in Mosquitoes
Jiangtao Liang, Simon M. Bondarenko, Igor V. Sharakhov, and Maria V. Sharakhova
PROTOCOLS
1 Obtaining Polytene, Meiotic, and Mitotic Chromosomes from Mosquitoes for Cytogenetic Analysis
Jiangtao Liang, Simon M. Bondarenko, Igor V. Sharakhov, and Maria V. Sharakhova
2 Visualization of Polytene Chromatin in Mosquito Cell Nuclei Using Three-Dimensional Fluorescence In Situ Hybridization
Simon M. Bondarenko, Jiangtao Liang, Maria V. Sharakhova, and Igor V. Sharakhov
SECTION 10. INSECTICIDE RESISTANCE
CHAPTER 46 INTRODUCTION
Assays to Measure Insecticide Toxicity and Insecticide Resistance in Mosquitoes
Nannan Liu, Yifan Wang, and Ting Li
PROTOCOLS
1 Mosquito Larval Bioassays
Yifan Wang, Ting Li, and Nannan Liu
2 Mosquito Adult Bioassays
Ting Li, Yifan Wang, and Nannan Liu
3 Synergism Study for Investigating Possible Mechanisms of Insecticide Resistance in Mosquitoes
Ting Li, Yifan Wang, and Nannan Liu
Appendix
General Safety and Hazardous Material Information

Index