Traditional Morphometrics of Adult Mosquitoes

Summary

This protocol details a method for dissecting and measuring key morphological traits using an image analysis software to assess mosquito fitness.

Abstract

Mosquitoes are major vectors of pathogens that affect human and animal health. Measuring morphological traits of mosquitoes enables researchers to assess the impact of treatments (e.g., insecticide application, transgenesis) or ecological factors (e.g., nutritional stress, habitat changes) on fitness. Fitness in mosquito species has been linked to vectorial capacity, and thus directly impacts risk of vector-borne pathogen transmission. Here, we use an image analysis software to measure key morphological traits of mosquitoes collected during mosquito surveillance as a proxy for fitness.

Introduction

Fitness represents an individual organism’s capacity to survive and reproduce within its environment. Under ideal conditions, fitness research involves the assessment of environmental exposures on total reproductive capacity. However, measuring fecundity in ecological studies is often difficult, or even impossible, because the age of individuals in surveyed populations cannot be reliably controlled for. As such, ecologists measure functional traits, including morphological, physiological, and phenological traits, which have been shown to impact or be representative of an individual’s fitness^1,2. Such assessments are particularly valuable when fitness metrics can inform population dynamics and the potential for disease transmission in mosquitoes³.

Immature mosquitoes are aquatic, and different genera and species exhibit distinct habitat preferences. For example, Aedes aegypti, the primary vector of dengue worldwide, is known to develop in a wide variety of man-made containers, such as flowerpots, cans, bottle caps, tires, and gutters^4,5,6. On the other hand, some species, such as Aedes triseriatus, prefer to oviposit in natural habitats such as tree holes⁷. This variability in developmental habitats results in larval exposure to different stressors, including temperature, predation, and intra-specific competition for resources, which can influence the fitness of the resulting adults. For instance, while environmental temperature is positively correlated to the rate of larval development, temperature variability during larval development can negatively affect adult mosquito longevity and fecundity^8,9,10,11. Similarly, resource scarcity during development caused by low food availability or high levels of larval competition can result in smaller adults with shorter lifespans^12,13,14.

Many factors that influence mosquito reproductive potential also affect their capacity to transmit pathogens¹⁵. Mosquito-borne pathogen infection imposes a fitness cost, as the mosquito vector must allocate resources to resisting infection, which are no longer available to fulfill other functions^16,17,18,19. Larger and fitter individuals have been shown to ingest larger quantities of nectar and blood during feeding and to sequester resources more effectively than smaller individuals, resulting in increased longevity and fecundity²⁰. In addition, these individuals tend to be more resistant to pathogens²¹. This can be seen in multiple species in food stress and larval competition studies. Aedes aegypti provided a high quantity and quality larval diet, has exhibited lower dissemination and transmission rates for Zika and Sindbis virus^22,23. Similarly, Aedes triseriatus reared on a nutritionally deprived diet were more susceptible to La Crosse virus than their well-fed counterparts²⁴, and a comparable pattern was reported in the Anopheles gambiae-Plasmodium falciparum infection model²⁵. Increased inter- and intra-specific larval competition in Aedes aegypti and Aedes albopictus has also been associated with greater susceptibility to the dengue virus²⁶. Finally, heat stress has been shown to increase West Nile virus transmission in the members of the Culex pipiens species complex¹⁷, and malaria transmission in Anopheles stephensi²⁷.

Given the impacts of environmental factors on mosquito fitness and vectorial capacity, researchers need to consider the fitness of populations surveilled during ecological studies. Several traits have been identified as indicative of fitness in mosquitoes and thus may be used in comparative ecological studies. Numerous studies have shown wing length to be predictive of both body mass and fecundity^{14,28,29,30,31}. Other traits, such as leg length, proboscis length, and body length, have been shown to correlate with fitness as well^32,33,34,35. Historically, these methods have involved the use of analogue grid slides and measuring lenses. However, high-detail image capture and analysis software offer the opportunity to obtain higher resolution measurements of traits with greater precision and reproducibility. Here, we outline the methodology for dissecting and measuring key morphological traits used in our lab for research on wild mosquitoes collected as part of ecological studies using image analysis software to obtain high-precision measurements.

Protocol

NOTE: Insects are exempt from legal or institutional review board considerations. As such, no approval is necessary for this kind of research.

1. Mosquito dissection and imaging

1. Transfer individual wild-collected female mosquitoes into a 90 mm Petri dish and place them under a dissecting microscope equipped with a digital camera.
   NOTE: This protocol can be used for both lab-reared and wild-collected mosquitoes. Any known mosquito collection methods can be used with this dissection methodology.
2. Adjust the magnification based on the species and individual insect, and focus so that the entire body of the mosquito is visible and occupies the majority of the camera’s field of view.
3. Using micro-scissors, dissect the following tissues/appendages (Figure 1): hind left leg at the coxa, left wing at the base of the jugum, left antenna at the antennifer, and proboscis at the clypeus
4. Transfer the dissected tissues onto a slide mounted with double-sided tape.
5. With the appendages removed, adjust the rest of the mosquito body so that its left side is visible. If necessary, additional limbs may be removed to allow the mosquito to sit flush with the Petri dish. Using the microscope’s camera, capture an image of the mosquito’s left side.
6. Without adjusting magnification or focus, remove the petri dish containing the mosquito and replace it with the slide mounted with the dissected tissues (antennae, proboscis, wing, and hind leg). Using the microscope’s camera, capture an image of the dissected tissues.
7. Without adjusting magnification or focus, remove the slide mounted with mosquito tissues, and replace it with the calibration slide (stage micrometer). Using the microscope’s camera, capture an image of the calibration slide.
   NOTE: Analysis of captured images can occur at a separate time from dissection.

2. Mosquito trait measurement

1. Open the images collected during mosquito dissection in the camera’s image analysis software. If the microscope camera does not include image analysis software, a standalone image analysis software such as ImageJ may be used.
2. Using the software’s measurement tool set to pixels, measure a 1 mm section of the calibration scale on the calibration slide image taken. Record the number of pixels per millimeter (px/mm) for the magnification and focus of this image.
3. Measurements of the mosquito can now be performed. Start by measuring and recording the length (in pixels) of the mosquito thorax and abdomen in the side-view image. Thoracic length is measured as the distance between the anterior of the thorax to the posterior of the scutellum as viewed from the side. Abdominal length is the distance from the anterior of the scutellum to the posterior of the abdomen³⁵. It may be necessary to use a multi-point measuring tool or multiple separate measurements on traits with bends or joints.
4. Then, perform measurements of the mosquito appendages. Start by measuring the lengths (in pixels) of the dissected antenna (distance between the terminal segment of antennae and the base of the antennifer), wing (distance between the axial incision and the apical margin), hind leg (sum of the lengths of the tibia and femur), and proboscis (distance between the base of clypeus and the termination of labium)^32,36.
5. Divide the pixel measurements of the collected traits by the px/mm measurement obtained from the calibration slide image to obtain the trait lengths in mm.

Results

Our laboratory utilizes the described methodology in two ongoing ecological studies examining the impacts of habitat fragmentation on mosquito fitness and the impacts of socio-economic and socio-demographic factors on mosquito abundance, diversity, and fitness. At present, several hundred mosquitoes have been analyzed across both studies. Below are representative data from one of the habitat fragmentation studies, including measurements and analyses.

Following dissection, the side of the mosqu...

Discussion

The methodology described here provides a means to assess mosquito morphological traits as a proxy for fitness. Such assessments are commonly used in ecological research on insects, birds, reptiles, and plants^{1,37,38,39,40,41}. Measuring key morphological traits allows researchers to conduct comparative ecology studies in the ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100mm petri dish	Fischer Scientific	08-748C
Dissecting microscope with mounted camera	AmScope or similar	SM-4T-144S
Double-sided tape	3M	NC0879005
Fine tip forceps	Aven inc.	18059USA	Size 4 or 5, depending on preference
Image analysis software	AmScope or similar	SM-4T-144S	Many microscpe mounted cameras come with image analysis software. If none is available, free programs such as ImageJ can be used.
Micro scissors	Wexler Surgical	SL2000.1
Microscope slides	Fischer Scientific	12-544-4
Microscope Stage Calibration Slide	AmScope or similar	A36CALM1