Name: Measuring Wing Traits and Estimating Fecundity in Adult Female Butterflies
Uploaded: 2023-08-18T13:00:00
Duration: 1 min 32 s
Description: Watch this Scientific Journal Video about Measuring Wing Traits and Estimating Fecundity in Adult Female Butterflies at JoVE.com

measuring wing traits and estimating fecundity in adult female butterflies

Artificial Rearing and Heavy Metal Tolerance in Cabbage White Butterflies

The small cabbage white butterfly (Pieris rapae, hereafter &#34;cabbage white&#34;) is a cosmopolitan pest species of mustard crops, such as cabbage, broccoli, and canola1,2,3. At the same time, the cabbage white is a powerful system for research in biology and a commonly used butterfly model, as they can be easily reared and manipulated in controlled lab experiments4,5. Research on cabbage white butterflies has provided critical insights with respect to host searching6,7,8, nectar resource use9,10,11, mate choice and sexual selection12,13,14, wing pattern development and evolution15,16,17, and responses to novel and changing environments18,19. Many of these insights rely on the fact that cabbage whites can be reared on artificial diets4,20,21, which can be precisely manipulated to reflect poor nutritional conditions22,23, ecologically relevant pollutant levels24,25,26,27, or transitions to novel host plants28,29. The present study uses an experiment on exposure to heavy metals to illustrate basic methods for rearing cabbage white butterflies on an artificial diet in the laboratory and key performance measures of larvae and adults. Many aspects of these methods apply to other butterflies30,31 and moths32,33,34 that can be reared on an artificial diet.
In this paper, an experiment on metal tolerance is used to illustrate the general methods of rearing cabbage white butterflies. Heavy metals are a common anthropogenic pollutant stemming from the degradation of human products, industrial processes, and legacy contamination from historical use in pesticides, paints, and other products35,36,37,38. Many heavy metals, including lead, copper, zinc, and nickel, can move from soil and water into plant tissue39,40,41,42, and metals in dust can be deposited on plant leaves43,44,45, resulting in multiple routes of exposure to phytophagous insect larvae. Heavy metal exposure early in life can have negative effects on animal development, especially on neural tissue, and high levels can be lethal35,36,46,47,48. A number of studies have shown the negative effects of metal exposure on developing insects, including both pests and beneficial insects49,50,51. The large number of heavy metal pollutants, and the fact that they often co-occur in human environments52, means that precise lab methods are needed in which researchers can expose developing insects to different levels and combinations of diverse metals to understand and mitigate their environmental effects.
The present work contrasts the impacts of common metals on cabbage white survival and development, focusing on copper (Cu), zinc (Zn), and nickel (Ni), three common pollutants in human environments. For instance, forbs from rural Minnesota roadsides contain up to 71 ppm Zn, 28 ppm Cu, and 5 ppm Ni53. This experiment manipulates the levels of these metals in artificial diets of cabbage white butterflies at levels corresponding to, and exceeding, the levels seen in the environment. An artificial diet is used to contrast the relative toxicity of these metals, predicting that cabbage whites would be more sensitive to metal pollutants that are not an integral part of their physiology (nickel) relative to those that occur, albeit at small levels, in enzymes and tissue (copper and zinc; Figure 1). Throughout, this text provides methodological details and accompanying video visualizations to illustrate the rearing and research methods of this important butterfly model system.

Author Spotlight: Investigating the Tolerance of Cabbage Butterflies to Urban Pollutants 

Rearing the Cabbage White Butterfly (Pieris rapae) in Controlled Conditions: A Case Study with Heavy Metal Tolerance

In this study, we're interested in understanding how organisms deal with anthropogenic environments like cities, roadsides, and crop fields. We use cabbage white butterflies as a system because they're easy to manipulate experimentally. For instance, we're studying genetic variation intolerance to urban pollutants.We collect samples from different locations and populations to study variation over species or space. Nets, coolers, and envelopes are our main tools in the field. Butterfly traits are measured by microscopy and ICP-MS is the best method to measure heavy metal concentrations in small samples.One of the current experimental challenges is studying the variation across species and their developmental adjustments to human environments. The main difficulty lies in rearing diverse species under controlled experimental conditions. While cabbage white butterflies are easy to raise, finding suitable analogous diets for 10 or 20 different species that feed on various plants presents a significant challenge from an experimental perspective.We're interested in mitigating exposure to urban toxins, the ecological interventions that could decrease the toxicity of heavy metals and other urban pollutants, such as microplastics, are being explored. Additionally, we aim to investigate how to design pockets of habitat within city spaces to encourage the health of plants and animals living there, including humans.

Watch this Scientific Journal Video about Measuring Wing Traits and Estimating Fecundity in Adult Female Butterflies at JoVE.com

Measuring Wing Traits and Estimating Fecundity in Adult Female Butterflies  

To measure wing traits on dead butterflies, hold the thorax in one hand and using forceps, remove each wing at its base. Place the wings in a light box and take photographs for measurements. To measure aspects of reproduction, house the adult butterflies in mating cages, allowing one day for male reproductive maturation and one day for mating.To estimate fecundity, either sacrifice females for egg counts through dissection at set time points or collect eggs daily on host plants. To estimate egg loads, remove the abdomen, place it in PBS, and make a cut along the ventral side. Using forceps, separate the innards from the cuticle and pull the ovaries from the gut, trachea, and other abdomen contents.Carefully uncurl the four ovarioles within each ovary, and observe the transition from mature, yolked, and shelled eggs to immature follicles. Then, use a counter to tally the total number of mature eggs. To assess the mating status of a dissected female, open the bursa copulatrix and separate the spermatophores.

measuring-wing-traits-estimating-fecundity-adult-female

Research

JoVE Journal

Biology

The cabbage white butterfly (Pieris rapae) is an important system for applied pest control research and basic research in behavioral and nutritional ecology. Cabbage whites can be easily reared in controlled conditions on an artificial diet, making them a model organism of the butterfly world. In this paper, a manipulation of heavy metal exposure is used to illustrate basic methods for rearing this species. The general protocol illustrates how butterflies can be caught in the field, induced to lay eggs in greenhouse cages, and transferred as larvae to artificial diets. The methods show how butterflies can be marked, measured, and studied for a variety of research questions. The representative results give an idea of how artificial diets that vary in components can be used to assess butterfly performance relative to a control diet. More specifically, butterflies were most tolerant to nickel and least tolerant to copper, with a tolerance of zinc somewhere in the middle. Possible explanations for these results are discussed, including nickel hyper-accumulation in some mustard host plants and recent evidence in insects that copper may be more toxic than previously appreciated. Finally, the discussion first reviews variations to the protocol and directions for troubleshooting these methods, before considering how future research might further optimize the artificial diet used in this study. Overall, by providing a detailed video overview of the rearing and measurement of cabbage whites on artificial diets, this protocol provides a resource for using this system across a wide range of studies.

This paper presents a detailed protocol for rearing the cabbage white butterfly in controlled lab conditions with an artificial diet, which allows precise manipulations of early-life nutrition and toxin exposure. The representative results show how heavy metal toxicity can be assayed with this protocol.

The cabbage white butterfly (Pieris rapae) is an important system for applied pest control research and basic research in behavioral and nutritional ...

Collection and Rearing of Adult Female Butterflies in Controlled Conditions for Egg Harvesting

To begin, collect adult female butterflies with an aerial insect net and place them in the cage to harvest eggs. Place the host plants in pots or in containers with water to maintain the leaf pressure. In case of direct egg collection and transfer to diet, attach a host plant leaf to the top of a plastic cup of water using a rubber band.Then, stretch a piece of Parafilm around the cup's edge, so that females touching the leaf will oviposit onto the Parafilm. Spray the cage with water to maintain the relative humidity high in the cage. If potted plants are watered in the cage, place a towel under the pot to prevent water pooling and to protect butterflies from becoming trapped.Prepare 10%honey water for butterfly feeding. Then, to set up the feeder, rinse the yellow or orange sponges thoroughly with tap water and cut them into small squares. Place the sponges in 60 millimeter plastic Petri dishes and add honey water to them.Change the feeders daily and clean the sponges in a mild bleach solution, followed by thorough rinsing with tap water to prevent mold growth.

Transfer and Rearing of Butterfly Eggs on Agar-Based Diet Until Adult Emergence

Print out a recipe containing weighing ingredients for artificial diet preparation. Weigh all the dry ingredients, except agar, into one container. Place the dry ingredients mixture into a blender and add five milliliters of flaxseed oil to the ingredient mixture.Mix 15 grams of fine mesh agar for each diet batch with 400 milliliters of distilled water in a one-liter beaker. Heat the mixture in a microwave until the agar is close to boiling, with fine bubbles throughout the mixture. After boiling, add 400 milliliters of faucet-temperature distilled water to bring the temperature down to mix with the dry ingredients.Next, place 74-ounce diet cups on the counter with the edges touching. Add the agar mixture to the blender and thoroughly mix. After mixing, pour the mixtures into diet cups, ensuring the diet covers the bottom of each cup.After the diet cools, place lids on the cups. Label the cups with diet type and stack them on trays. House host plant leaves with butterfly eggs in 30-ounce deli cups with a mesh cover inside a 24-degrees Celsius climate chamber.After one week, inspect the cups for late first or early second instar larvae. Using a paintbrush, transfer the larvae to the artificial diet. Place three larvae into each four-ounce cup.Place the cups on their sides in a plastic bin, allowing frass to fall to the bottom and away from the diet, reducing mold and disease risk. House the diet cups in controlled temperature conditions with low to moderate light levels. Monitor the cups for mold or disease every one to two days by inspecting through the clear cup lids.Allow the larvae to pupate and emerge in the diet cups. Carefully remove adult butterflies from the cups with clean hands, gripping all four wings close to their body for a stable hold. To mark the butterflies, hold the dry individuals by the head and thorax.Then, using a fine-tip sharpie, mark a number on the hind wing. Determine the sex of the butterflies by combining wing markings and genitalia examination. Next, grasp the adult by its head and thorax using one hand while opening a wax glassine envelope with the other hand.Then, transfer the adult into the envelope and secure the wings through the envelope. Maintain the butterflies at five to six degrees Celsius for up to one week before experimentation. In this case study, the butterfly survival rate was not affected by increasing the concentration of nickel, but there was a significant effect for both copper and zinc.Butterfly developmental rate was impacted by copper and zinc. As copper concentration increased, there was an increase in development time, with a significant deviation from the control starting at 50 ppm. As zinc concentration increased, there was an increase in development time, with a significant deviation from the control starting at 100 ppm.