transfer and rearing of butterfly eggs on agar-based diet until adult emergence

白蝴蝶的人工饲养和重金属耐受性

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.

作者聚焦：调查卷心菜蝴蝶对城市污染物的耐受性 

在受控条件下饲养卷心菜白蝴蝶（Pieris rapae）：具有重金属耐受性的案例研究

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 Transfer and Rearing of Butterfly Eggs on Agar-Based Diet Until Adult Emergence at JoVE.com

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

以琼脂为主的饮食转移和饲养蝴蝶卵，直到成虫出现

打印出包含人工饮食制备的称重成分的配方。将所有干成分（琼脂除外）称重到一个容器中。将干配料混合物放入搅拌机中，并在配料混合物中加入五毫升亚麻籽油。将每个日粮批次的15克细网琼脂与400毫升蒸馏水在一升烧杯中混合。在微波炉中加热混合物，直到琼脂接近沸腾，整个混合物中都有细小的气泡。煮沸后，加入400毫升水龙头温度的蒸馏水，使温度降低，与干配料混合。接下来，将 74 盎司的减肥杯放在柜台上，边缘接触。将琼脂混合物加入搅拌机中并充分混合。混合后，将混合物倒入减肥杯中，确保饮食覆盖每个杯子的底部。饮食冷却后，盖上杯子。用饮食类型标记杯子并将它们堆叠在托盘上。在 24 摄氏度的气候室内，将寄主植物叶子和蝴蝶卵装在 30 盎司熟食杯中，并带有网罩。一周后，检查杯子是否有晚期第一龄或早期第二龄幼虫。使用画笔将幼虫转移到人工饮食中。将三只幼虫放入每个四盎司的杯子中。将杯子侧放在塑料箱中，让碎屑落到底部并远离饮食，从而降低霉菌和疾病的风险。将减肥杯放在低至中等光照水平的受控温度条件下。通过透明杯盖检查，每隔一到两天监测杯子是否有霉菌或疾病。让幼虫化蛹并在饮食杯中出现。用干净的手小心地将成年蝴蝶从杯子中取出，将所有四个翅膀紧贴身体以稳定地握住。要标记蝴蝶，请抓住干燥的个体的头部和胸部。然后，使用细尖锐器，在后翅上标记一个数字。通过结合翅膀标记和生殖器检查来确定蝴蝶的性别。接下来，用一只手抓住成年人的头部和胸部，同时用另一只手打开蜡玻璃信封。然后，将成虫转移到信封中，并通过信封固定翅膀。在实验前将蝴蝶保持在五到六摄氏度长达一周。在本案例研究中，蝴蝶存活率不受镍浓度增加的影响，但对铜和锌都有显着影响。蝴蝶发育速度受铜和锌的影响。随着铜浓度的增加，显影时间增加，从50 ppm开始与对照有明显偏差。随着锌浓度的增加，开发时间增加，从100 ppm开始与对照有显着偏差。

transfer-rearing-butterfly-eggs-on-agar-based-diet-until-adult

Research

JoVE Journal

Biology

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

466 次观看.  University of Minnesota. 打印出包含人工饮食制备的称重成分的配方。将所有干成分（琼脂除外）称重到一个容器中。将干配料混合物放入搅拌机中，并在配料混合物中加入五毫升亚麻籽油。将每个日粮批次的15克细网琼脂与400毫升蒸馏水在一升烧杯中混合。在微波炉中加热混合物，直到琼脂接近沸腾，整个混合物中都有细小的气泡。煮沸后，加入400毫升水龙头温度的蒸馏水，使温度降低，与?...

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

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

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 ...

科研

生物学

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.

在受控条件下收集和饲养成年雌蝶以进行卵采集

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.

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.