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California’s Butterflies |
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Captive Breeding Manual |
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Murrieta Captive Rearing Facility
Quino Checkerspot (Euphydryas editha quino) Captive Breeding Manual
Prepared by:
Gordon F. Pratt Entomology Department University of California Riverside, CA 92521 permit #TE004939-6
1. Introduction
The Quino Checkerspot Butterfly (QCB) was at one time considered to be one of the most abundant southern California butterflies. There were times when there were so many QCB that they could be seen even in downtown San Diego (Murphy pers. comm.). The species has rapidly decreased in numbers, so much so that when the species was finally petitioned for the endangered species list in the late 1980s, it was already thought to be extinct. Later in the mid 1990s populations were found in the Temecula/Murrieta area of Riverside County and in the eastern San Diego and Jacumba areas of San Diego County (Mattoni et al. 1997).
Despite the past abundance of QCB and the extensive work on other populations of the Edith’s Checkerspot, very little was actually known about the biology of QCB. As an example, QCB was believed to be restricted to below 3,000 feet elevation. Recent studies have shown it to occur up to over 5,000 feet elevation (Pratt et al. 2001). Also QCB was previously thought to be restricted to one larval food plant. It is now found to use as many as 5 larval food plants (Pratt et al. 2001).
Most North American butterflies need adaptive behaviors to get through periods when there are little to no available larval food plants. Generally, butterflies go through what is called diapause (similar to hibernation). Butterflies can diapause as eggs, larvae, pupae or adults. QCB diapause as larvae and can diapause in a number of different instars, except perhaps as mature larvae. This diapause period is generally quite long running from May through January or February and particularly long (more than one year) when quality of food plants are not sufficient to support larval development. These poor plant conditions are usually due to low precipitation. Under these conditions it is believed larvae will respond by returning to diapause and waiting out another year.
Since QCB spend most of there life in diapause, where larvae diapause is extremely important. Despite the importance to larval survival, very little is known of the sites selected by larvae to diapause. In captivity larvae have been observed to diapause on the soil surface, under leaf litter, and up in a plant several inches above the soil surface. The choices made in captivity could be due to the reduced number of potential choices. There is some controversy as to the number of larvae that will return to diapause in nature.
This program is to captive breed populations of the Federally Endangered QCB from 4 regions of southern California, for potential release and experiments. The regions are 1) Lake Skinner and west in Riverside County, 2) east of Wilson Valley above 4,000 feet to western San Jacinto Mountains, 3) southwestern San Diego County, and 4) southeastern San Diego County. The 4 regions were selected to represent areas of likely genetic differences amongst populations of QCB, caused by elevation and food plant differences. An additional region (east of Lake Skinner and west of region 2 below 4,000 feet elevation [Wilson Valley north to Brown Canyon and south to northern San Diego County]), was at one time included in the above regions. It was lost several years ago and has not been replaced due to not being able to find females in the area despite several attempts during years of high rainfall. The Riverside County populations were at least one time interconnected. At present no intermediate populations are known between the southern San Diego and Riverside County populations. There may be a connection between southwestern and southeastern San Diego through such areas as through Campo.
2. Propagation of host plants used for rearing the QCB
Plantago erecta seeds were obtained from S & S Seeds and collected from the Temecula area and grown in Riverside and Murrieta. Plantago patagonica, Collinsia concolor, and Antirrhinum coulterianum seeds were collected from the Anza area. Castilleja exserta seed were obtained from Theodore Payne. Several species of Penstemons have been used to rear Quino larvae. These species include Penstemon gloxinoides, Penstemon pinifolius, and Penstemon eatoni. Penstemon gloxinoides is not a real species but a nursery name used for a number of crosses of Penstemon species (Nold 1999). There is no clear species representing this name. Some plants that are under the name Penstemon gloxinoides are not as good as others. Other Penstemons do not produce high survivals.
Taxonomy
All of the plants that QCB uses as larval food plants in captivity are closely related plants. All of the plants, other than Plantago, at one time belonged to the family Scrophulariaceae. More recently Plantago has been found by DNA analyses to be more closely related to some members of the Scrophulariaceae than some members of the Scrophulariaceae are to others (Olmstead et al. 2001).
The Scrophulariaceae was recently split into a number of closely related families. Most of the plants, Plantago, Collinsia, Penstemon, and Antirrhinum are now placed in the Veronicaceae. The food plants in the Castilleja and Cordylanthus genera are placed in the closely related family Orobanchaceae (Olmstead et al. 2001).
Seed collection
Seeds are easily collected from Collinsia, Cordylanthus, and Antirrhinum since they are formed in capsules that open and allow the seeds to pop out and blow away. The branches of these plants are collected late in development just as they are senescing. Plantago seeds are purchased from S &S Seeds or collected from the field late in the season. They are allowed to dry in paper bags. In the fall the seed capsules of the Plantago erecta are broken open by light grinding and the seed chaff is removed by winnowing. The Plantago patagonica are not separated from the branches, since they are more difficult to separate.
Penstemons
Penstemons were purchased from various Nurseries: Parkview, Home Depot, Lowes, and Theodore Payne. These plants are fertilized once a month and watered 2 to 3 times a week. New plants are obtained from cuttings that are used for rearing larvae. After the leaves have been eaten in the aquariums, the branches are put into yogurt containers with water. After about a week the branches are put into pots with soil. Many of these branches form new plants.
Seed Germination
Plantago erecta seeds are placed out in flats that fit neatly into the bottom of the aquariums used for rearing QCB (Figure 1). These flats are watered once daily at around 5:40 PM for 40 minutes with an automatic watering system. Normally the seeds germinate within about 10 days.
Figure 1. Plantago seedlings in flats at the Murrieta Captive Breeding Facility
3.1 QCB Life History
QCB has a complicated life history partly because it is adapted to annual food plants in an unpredictable environment. The larvae usually break diapause in winter sometime after the first fall or winter rains. These larvae were in diapause throughout the summer from the past spring. They come out and feed, and if conditions are good they will continue to grow, pupate, and develop into adults, while if the conditions are not good they may return to diapause. Some larvae are likely to return to diapause even under good conditions. Those adults that eclose that spring need to mate and oviposit quickly several weeks before the annual food plants dies and dries up. The females oviposit egg clusters from about 40 to 150 eggs. The eggs hatch within a couple of weeks. The young prediapause larvae feed until the food plant dies and dries up. It is thought that if the plant dies too early the larvae will not have enough nutrition to enter diapause. The larvae that do eat enough enter diapause until the following rains in late fall or winter.
3.2 Diapausing Larvae
Figure 2. Diapausing larvae kept in yogurt containers
Larvae that enter diapause are counted and placed into labeled yogurt containers with the stud line (Figure 2). They are kept in the diapause room at room temperature throughout the remaining spring, summer, and fall. Plantago erecta is planted in mid October. When the seedlings have sprouted and filled out the flats, the larvae are broken from diapause. This is done by pulling out the diapausing larvae from the yogurt containers and placing them on wet paper towel with cuttings of Penstemon branches within Gladlock-brand plastic containers (Figure 3). These containers are opened and exposed to the air for a minute or so twice daily, preferably 12 hours apart. The larvae will begin to feed upon the Penstemon leaves when they break diapause. The larvae are kept inside these containers for at least a week.
Figure 3. Breaking diapause with Gladlock-brand containers
3.3 Developing Larvae
Aquariums are set up with the Plantago flats set on the bottom (Figure 4). Additional Penstemon branches (placed through holes in lids of yogurt containers filled with water) are added in some cases to provide feeding choices for the larvae. For each aquarium a lamp is run with a timer set on a 10 hour light/14 hour dark schedule. The Plantago flats are changed every 5 days, since the quality of the Plantago will rapidly decrease after about 5 days. The Plantago flats are returned to the outdoors where they continue to grow and may be reused in a week or so. Each time the Plantago flats are changed the larvae are counted.
Larvae that return to diapause are found by forming shelters and curling up within them. These larvae are put back into yogurt containers, labeled as to the stud line, and put back into the diapause room where they are stored until the following season.
Figure 4. Aquarium with Plantago and additional Penstemon cuttings
4 Pupae
Larvae pupate on the netting placed over the aquarium, in shelters with the Plantago, in the corners of the aquarium, as well as a number of other locations within the aquarium. The pupae after the exoskeleton has hardened are collected from the aquariums and placed in yogurt containers (labeled as to stud line) with paper toweling (Figure 5). Shortly after collecting they will be weighed to the nearest milligram. This can be used to help sex the pupae since QCB females average about twice the weight of males.
The paper toweling placed inside the yogurt containers allows the freshly emerged adults a way to crawl up the side of the container to a place to expand their wings. These yogurt containers will be placed inside netted cages (labeled as to stud line). Usually within about 2 weeks, dependent on temperatures, the adults hatch from these pupae. The cages are exposed to natural light. It has been found that butterflies that have been allowed to eclose with light seem to be healthier than adults that eclose in the dark. Males that eclose are put into a cage labeled with the stud line at which they came from. Flying adults can affect the ability of freshly emerged adults to expand their wings. These males are then aged for three days. Females are immediately put into the fridge.
Figure 5. Yogurt container with pupae and fully expanded QCB
5 Adults
5.1 Male aging
Males need to be aged for three days. It seems that there is also a learning process as these males age, so the more males exposed together the more capable they seem to mate. After about three days of aging, males can often be found chasing each other in the cage exhibiting behaviors of trying to mate with one another. While males are aging they need to be fed. A honey/water mixture of one part honey to three parts water is used to feed them. This same mixture is used to feed females (Figure 6).
When the adult QCB does not immediately feed, the proboscis will be unfurled with an insect pin and placed into the paper toweling with the honey/water mixture. This has been a good method for getting individuals that are not use to feeding to learn the process. If females are going to be stored in the fridge for longer than a day they will be fed until satiated.
Figure 6. Feeding female on paper towel soaked with the honey/water mixture
5.2 Mating
To mate QCB, males will be exposed to strong light. Preferably the number of males in the cage to be used for mating will be 10 or more. Often they will be taken outdoors and put on a chair in the fenced enclosure (Figure 7). Sometimes because of excessive wind outdoors the males will be exposed to the light at the front window. When the males start to show a lot of activity, such as flying around and/or chasing each other the female to be mated will be taken from the fridge for mating. The tip of the abdomen of the female will touch the antennae of the male chosen to be mated. Generally if the male is interested he will exhibit a behavior of wing fanning and twisting of the abdomen. At this point the female is placed beside the male and left until he successfully copulates with her.
At initial attachment the male will be facing in the same direction as the female (Figure 8). When the male has become comfortable with the mating connection, he will turn around and face away from the female (Figure 9). It is at this point the pair can be removed from the cage and placed inside a stud line labeled a yogurt container. These containers are then placed indoors and watched for the next hour or more. If the male breaks from the female much before 40 minutes the mating usually has not been successful. If the mating takes longer than a couple of hours the mating usually has not been successful and the process is repeated with another male.
Figure 7. Cage of males used for mating
Figure 8. Freshly copulated QCB with male facing the same direction as the female
Figure 9. Successful copulation with male facing in the direction away from the female
Field Mating
In order to improve the genetic diversity of the lab population, females will be brought into the field for mating with field caught males. The advantage of this technique is that males can mate multiple times, so that once they have mated with the caged female, he can be released back in the same location as to where he was collected and presumably mate with additional field females. I have had lab males mate successfully 4 times in captivity. The same process of determining the success of the copulation is used here. The coupled pair will be placed inside a yogurt container and put into the shade. Generally these males mate rapidly if they are going to be successful. Once he breaks from the copulation he is released back into the field. If the mating is too short, like just a few minutes a new male is collected and the process is continued.
Only females from the same location as the field collected males will be used. In other words if the females from the colony are from the Lake Skinner site, then this field mating can only be done at this or a neighboring site. For instance these females can not be crossed with males from Marron Valley, Jacumba, or Anza.
Adult Collection and Transport
Another method of improving the genetic diversity of the lab colony, or establish a new lab colony, is to field collect females. Generally field collected females are already mated. To improve the likelihood of the female already being mated and having less of an affect on the field population only females that exhibit significant wear will be chosen. If the field population is not doing well females can be caught late in the season when they will not be producing many more successful progeny because the food plants are dying and drying up.
Field females will be searched for on native nectar sources. These females are the most likely to survive transport since they will already have fed. In some cases females will be fed the honey/water mixture in the field. The females will be placed in individual yogurt containers or large vials. A cooler without ice will be used for transporting the adults. It has been speculated by past experience that near freezing can kill stored sperm. The cooler will insulate the butterfly and provide darkness so that they do not fly around and use up energy. Upon arrival at the Murrieta facility the females will be fed and set up in oviposition containers.
Ovipositing females
Mated females will be immediately fed and put into oviposition containers on the surrogate Penstemon food plant (Figure 10). Each female will be fed the honey/water mixture saturated in a piece of paper toweling daily. This process will be done late in the day after she has completed ovipositing her daily egg mass. In some cases when the females do not unfurl their proboscises they will be unfurled for them by an insect pin.
The females will be left inside these containers for a period of time dependent on when her first egg batch begins hatching. Once they start hatching she will be moved to a new oviposition container. If she is not removed as she flies around inside the container she will get caught in the silken shelters created by the young larvae. These affects will be two fold since she will damage herself as well as the young larvae. This process of switching the female to a new oviposition container is repeated if she lives beyond the first egg batch hatching in the second container. Usually the female QCB dies before this happens.
Figure 10. The oviposition containers used for egg laying
6. Egg and Prediapause Larval Maintenance
Egg clusters can vary in size. Generally the first batch oviposited is the largest often 100 to 200 eggs. As the female ages her egg batches get smaller and smaller and can drop down to about 20 eggs per batch. As the female ages the fertility of her eggs seems to droop as well. This could be due to the quality of stored sperm decreasing with age.
When the eggs are first oviposited they are generally a yellow green. They turn to a bright yellow within a day or so and turn a brick brown a few days later (Figure11). This color change is important since it is useful at determining whether the copulation was successful. If the eggs remain a dingy yellow green for several days the female is generally not producing fertilized eggs. If the female’s progeny is needed she will be mated with another male.
The prediapause larvae (Figure 12) are maintained in the oviposition container until they have eaten most of the Penstemon leaves inside the container. Once that happens they are moved to yogurt containers lined with paper toweling and with fresh Penstemon leaves. Each container is labeled as to the stud line. The larvae are left in the containers for some time since the first instar is rather delicate to being moved. Other plants, such as Collinsia concolor and Antirrhinum coulterianum, may be substituted for Penstemon leaves to help save some of the Penstemon plants for future QCB. Plantago erecta is not used since this species easily molds and makes poor food for prediapause larvae. The QCB larvae are reared to diapause inside these yogurt containers
Figure 11. Fertile QCB egg batch
The containers are opened twice daily, plants molding or loosing their fresh green look are removed daily, and fresh food plant is added when needed. Once larvae have reached diapause stage they are put into stud line labeled yogurt containers inside the diapause room where they are maintained through the remaining spring and summer. Larvae that have entered diapause can be told from non-diapause larvae by their not feeding and forming a shelter often in the paper toweling.
Figure 12. First instar prediapause QCB larvae
7. Maintaining Diapause Larvae
Diapausing larvae are maintained in yogurt containers in the diapause room. Some larvae adapted to high elevation conditions may be put into a refrigerator or environmental chamber to simulate winter conditions. These larvae may be taken out of these refrigerators or environmental chambers, or the conditions of the environmental chambers may be changed to simulate the conditions that are occurring in the field. Populations found at high elevations such as south of Anza have proved difficult to rear. It is thought that the cooler conditions this population occurs may be the reason.
These yogurt containers have lids that do not form tight couplings and allow some air flow. For this reason the containers do not need to be opened frequently to air out. I have found that they can be left for months with the lids on without larval mortality. What mortality occurs is generally before entering diapause or shortly after breaking diapause.
Mattoni, R., G. F. Pratt, T. R. Longcore, J. F. Emmel, and J. N. George. 1997. The endangered quino checkerspot butterfly, Euphydryas editha quino (Lepidoptera: Nymphalidae). J. Res. Lep. 34: 99-118.
Nold, R. 1999. Penstemons. Timber Press, Portland Oregon, 259 pp.
Olmstead, R. G., C. W. Depamphilis, A. D. Wolfe, N. D. Young, W. J. Elisons, and P. A Reeves. 2001. Disintegration of the Scrophulariaceae. American Journal of Botany 88 (2): 348-361.
Pratt, G. F., E. W. Hein, & D. M. Krofta. 2001. Newly discovered populations and food plants extend the range of the endangered quino checkerspot butterfly, Euphydryas editha quino (Nymphalidae) in Southern California. J. Lep. Soc. 55: 176-178.
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