Home | MITC | MonarchLab | MLMP | SEE | Monarch Store | Links | About Us | Contact Us

Tachinid Fly (Lespesia archippivora) Parasitism of Monarch Butterfly Larvae (Danaus plexippus) 2006-2007

Kaitlyn Creasey
Zoology
Breck School
123 Ottawa Avenue North
Minneapolis, MN 55422

Introduction | Methods | Results | Conclusion | Acknowledgements | Sources Cited | Bibliography | Appendix 1: Monarch Instar Stages | Appendix 2: Tachinid Stages | Appendix 3: Map of Collection Sites

Introduction

Lespesia archippivora, a species in the family of tachinid flies (Figure 1), has been used for biological control. (The tachinid flies referred to throughout this paper are Lespesia archippivora.) Specifically, tachinid flies were introduced to the Hawaiian islands to control invasive armyworms (1). If introduced as biological controls in areas where they are not native, tachinid flies may overpopulate and become an invasive species, adversely affecting non-target species such as monarch butterflies (Danaus plexippus). Tachinid flies are known to parasitize monarch larvae, which are part of a delicate ecosystem, so understanding the effects of tachinid introduction on the monarch population is important. The purpose of my study was to determine effects of tachinid fly parasitism on the monarch population and expand knowledge of specific biology of tachinid flies with respect to monarch larvae.

Monarch larvae develop through five instar stages. (See Appendix 1 for instar stages.) First-instar is the stage of monarch larvae immediately after hatching. After a few days, monarch larvae molt to become second-instars. Monarch larvae molt three more times, going through third-, fourth-, and fifth-instar stages. The pupa stage is the end of the fifth-instar stage and occurs where the larvae "J" (Figure 2) and pupate. After approximately twelve days as pupae, adult monarch butterflies emerge (2).

Previous studies have looked at basic biology of tachinid flies. It is known that this species parasitizes larvae in a variety of species, among them monarch butterfly larvae. To reproduce, tachinid flies deposit their eggs on host monarch larvae. Once parasitic tachinid larvae hatch, they burrow into host larvae. (See Appendix 2 for development stages of tachinid flies.) Tachinid larvae develop through three instar stages while feeding off the host. Because tachinid flies need constant air supply, they attach spiracles to host monarch larvae trachea (1). Tachinid larvae synchronize developmental time to that of the host. As a result, either when monarch larvae "J" or pupate, monarchs die from effects of tachinid parasitism, at which point, full-grown tachinid larvae string down from dead monarch larvae (Figure 3) and pupate on the ground until they emerge as adult flies (3).

Oberhauser et al. studied percent of parasitized monarch larvae in the wild from 1999-2005. Data on monarch larvae collected over six years in Wisconsin, Michigan, and North Carolina show that an average of 12% of monarch larvae died from parasitism by tachinid flies. These data, however, do not reflect the variation in the percent of wild parasitized monarch larvae at a given collection site. Data from individual sites and years suggest that tachinid parasitism is independent of site and year. The Oberhauser study also concluded that monarch larvae collected at more advanced developmental stages had a higher chance of being parasitized (3).

Cardoza et al. studied the life cycle of Lespesia aletae, a species of tachinid flies using two species of moth larvae as hosts: Spodoptera frugiperda and Syntomeida epilais. The Cardoza study concluded that length of pupation for female tachinid pupae is one day longer than for male pupae when hosted by S. frugiperda and pupa width for males was greater from host S. epilais. Cardoza also found that pupa mass of tachinids decreased when more tachinid larvae were laid in hosts (4).

A study by Etchegaray et al. (1975) showed tachinid flies prefer to lay eggs on late second-, third-, or early fourth-instar monarch larvae and are usually unable to lay eggs on fifth-instars because these instars are able to shake off fly eggs. Although unusual, Etchegaray found that tachinid flies were able to lay eggs on first-instar monarch larvae. Etchegaray also observed superparasitism when 20 or more tachinid eggs were laid on a single monarch larva; the monarch larva and tachinid larvae died as a result (1).

My study had four goals. The first goal was to continue the Oberhauser study into 2006 to determine percentage of tachinid parasitized monarch larvae in the wild. My second goal was to investigate effects of number of eggs laid on a single monarch larva versus tachinid pupa mass. Based on the study by Cardoza, my hypothesis for this goal was that number of tachinid larvae laid on a monarch larva would affect the mass of tachinid larvae. My third goal was to find relationships, if any, between tachinid sex, mass, and duration of the pupal stage. Based on the Cardoza study, my hypothesis for this goal was that female tachinid pupae would be larger than male tachinid pupae and duration of pupal stage would be longer for females. My final goal was to determine which monarch instar stage tachinid flies prefer as hosts and average number of eggs laid on each monarch instar stage. Based on the Etchegaray study, my hypothesis for this goal was that tachinid flies would not be able to lay eggs on fifth-instar monarch larvae and it would be unusual for them to lay eggs on first-instar monarch larvae.

Methods

Collecting and Raising Monarchs

First-, second-, third-, fourth-, and fifth-instar stages of monarch larvae were collected between June 5th and August 11th, 2006, from locations in Falcon Heights Park in Minnesota; a restored prairie in Amery, Wisconsin; land in Cumberland, Wisconsin; the Minneapolis-St. Paul Air Reserve Station; Brainerd, Minnesota; and the University of Minnesota Monarch Lab Garden. (See Appendix 3 for locations of collection sites.) These monarch larvae were raised in individual deli containers at the University of Minnesota and given fresh milkweed and water daily. Each monarch larva was numbered; and collection date, site location, and instar at collection were recorded. The number of monarch larvae that lived to be adult butterflies, died from tachinid parasitism, or died from a different cause were recorded.

Raising Tachinid Flies

After tachinid larvae emerged from monarch larvae and pupated, they were placed into separate Petri dishes. The monarch number; date monarch collected; instar at collection; date of fly emergence; instar of monarch at emergence; number of flies per monarch larva; and fly pupa length, width, and mass were recorded. Once flies emerged from the pupa stage, they were anesthetized in a freezer for five minutes, and sex was determined under a microscope. Flies were placed in cages and given powdered milk, water, and sugar.

Choice Study

Two second-, two third-, and two fourth-monarch instars were placed together in cages with tachinid flies for two hours. After exposure, the monarch larvae were placed in individual deli containers, assigned a number, and given milkweed and water daily. The number of tachinid eggs laid on each monarch larvae were counted, and the sex of the flies was recorded.

No Choice Study

First- and fifth-monarch larvae instars were placed in tachinid cages and observed to determine if tachinid flies parasitized first- or fifth-instars and how many eggs were laid on each monarch larvae, if any.

Statistical Analysis

Two-sample t-tests, linear-regression tests, and chi-square tests were performed on the data using the program Statistix to determine significance of percent of wild parasitized monarch larvae from different collection sites; correlations between sex, pupa mass, duration of pupation of tachinid flies, and number of tachinid larvae per monarch larvae; and significance of monarch instar at collection versus percentage of wild parasitized monarch larvae. A p-value of 0.05 was to indicate statistical significance.

Results

Wild-Collected Monarchs

A total of 7% of monarch larvae in the wild were parasitized by tachinid flies in 2006 at the collection sites (Table 1). Sample size, however, varied with both collection site and instar stage at which monarch larvae were collected (Figure 4). Falcon Heights Park and Cumberland had no parasitism, the Air Reserve Station had 6% parasitism, and the Amery had 18% parasitism (Figure 4). Also, there was a tendency for monarch larvae collected during later instars to have higher parasitism rates (0.1>p>0.05), although this was not significant at the 0.05 level of confidence (Figure 5).

Table 1: Wild Monarch Larvae Survey

Percent healthy adults	Percent parasitized by tachinid flies	Percent died from other causes
70.9 (n=61)	7.0 (n=6)	22.1 (n=19)

Tachinid Fly Pupae

Male tachinid pupae were heavier than females (p=0.0001) (Figure 6) and were in the pupal stage longer (p=0.0001) (Figure 7). The mean mass of female tachinid pupae was 0.0346 and the mean of female pupae was 0.0436. The mean duration of pupal stage for females was 11.85 days, while the mean duration of pupal stage for males was 13.31 days. The number of fly larvae laid per monarch varied from one to eight tachinid larvae (Figure 8). When one to four flies emerged from monarchs, the mean mass of these fly pupae was greater than those that emerged in groups of five to eight (Figure 9). The mean mass for fly pupae that emerged in groups of one to four was 0.0368 g and the mean mass for fly pupae that emerged in groups of five to eight was 0.0214 g (t = 4.218, df = 14, p = 4.29 x 10^-4).

Experimental Parasitism

Of the 167 monarch larvae exposed to tachinid flies in the laboratory, eight parasitized monarch larvae died before the fly larvae emerged and were dissected. These monarch larvae contained from 12 to 31 immature fly larvae (Figure 10). An additional seven died before being dissected; it is not known whether these monarch larvae were parasitized. A total of 95 monarch larvae survived to adulthood, while 27 died of causes other than tachinid fly parasitism.

Results from the experimental parasitism study showed fly larvae emerged successfully from two monarch larvae that were exposed as first- and fourth-instars (Table 2). One of these monarch larvae was seen writhing in defense. Three tachinid flies emerged from the pupa of this monarch. The second parasitized monarch larva was parasitized as a first-instar, and two tachinids emerged. No fifth-instars monarch larvae were parasitized. Monarch larvae exposed to flies less than four days old were not parasitized.

Table 2: Successful Monarch Parasitisms by Tachinid Flies

Monarch Instar	Number of Tachinid Larvae
First	2
Fourth	3

Conclusion

Overall in 2006, there was a 7% tachinid parasitism rate for wild monarch larvae collected from the six sites (Appendix 3). However, percent of parasitism varied at each site. There were no parasitized monarch larvae found at Falcon Heights Park or Cumberland. The 2006 results suggest Amery may have a larger population of tachinid flies than the Air Reserve Station; the Oberhauser study reported similar results of variation in percent of parasitism (2). Factors that could have affected counts at each site may have resulted from isolated patches of monarch larvae populations. In my study, 22.1% of monarch larvae died before I could determine if they were parasitized.

Monarch larvae collected during later instars did not have a significantly higher chance of being parasitized than earlier instars (0.1>p>0.05). It was unexpected that fourth-instars were not parasitized since third- and fifth-instars were. This occurred even with a large sample size (n=25).

When a larger number of tachinid larvae emerged from host monarch larvae, the average mass of the tachinid pupae was significantly less (p=4.29 x 10^-4), which supported my hypothesis that the number of tachinid larvae laid on a monarch larvae would affect mass of tachinid larvae. Male tachinid pupae were statistically heavier than females (p=0.0001) and remained in the pupal stage longer than females (p=0.0001), which did not support my hypotheses that female tachinid pupae would be larger than male tachinid pupae and the duration of pupal stage would be longer for females.

I was not successful in determining which monarch instar stages were preferred by tachinid flies. There were only two successful parasitisms on first- and fourth-instar monarch larvae; however, overparasitism was common in my laboratory study. This suggests that female flies cannot tell if a host has been previously parasitized. Flies less than four days old did not parasitize monarch larvae, which suggests that the flies are not reproductively mature until this age.

The study of percent of parasitized monarch larvae will be continued. Also, further steps for this study will require maintaining a population of tachinid flies in the laboratory. Correlations between number of tachinid larvae in a host and tachinid larvae mass should be determined. This on-going study of relationships between tachinid flies will help to protect species that exist in delicate ecosystems.

Acknowledgements

I have received help from many people, most importantly Dr. Karen Oberhauser. She helped me decide on a topic, provided me with papers on previous tachinid fly research, helped me perfect my methods, and showed me how to analyze my data. I also received help sexing flies from Roger Moon at the University of Minnesota. Thanks also go to Grant Bowers, Kate Plowman, Matt Kaiser, Reba Batalden, De Cansler, Lis Young Isebrand, and Sarah Kempke, who helped me raise monarch larvae needed for my experiments. My research class at Breck School and Lois Fruen helped me with my presenting skills and my paper. The University of Minnesota and Breck School provided funding for my research.

Sources Cited

1. Etchegaray, Julia B., and Toshiyuki Nishida. "Biology of Lespesia archippivora (Diptera: T." Hawaiian Entomological Society 22(41-49): 1975.
2. Monarch Larvae Monitoring Project. 2006. University of Minnesota. 29 Sept. 2006 <http://www.mlmp.org/>.
3. Oberhauser, Karen. Personal interview. 14 July 2006.
4. Cardoza, Yasmin J., Nancy D. Epsky, and Robert R. Heath. "Biology and Development of Lespesia aletiae (Diptera: Tachinidae) in Two Lepidopteran Species in the Laboratory." Florida Entomologist 80(1997): 289-300.

Bibliography

Feener Jr., Donald H., and Brian V. Brown. "Diptera as Parasitoids." Annual Review: Entomology 42(1997): 73-96.

Marshall, Stephen A. Insects: Their Natural History and Diversity. Buffalo, New York: Firefly Books, 2006.

O'Hara, James E.. "Oviposition Strategies in the Tachinidae, a Family of Beneficial Parasitic Flies." Agriculture and Forestry Bulletin 8(1985): 31-34.

Parmesan, Camille, Nils Ryrholm, Constanti Stefanescu, and Jane K. Hill. "Poleward shifts in geographical ranges of butterfly species associated with regional warming." Nature 10 June 1999: 579-583.

Reitz, Stuart. "Superparasitism and Intraspecific Competition by the Solitary Larval- Pupal Parasitoid Archytas marmoratus (Diptera: Tachinidae)." Florida Entomologist 78(1995): 578-585.

Stapel, J. Oscar, John R. Ruberson, Harry R. Gross, Jr. , and W. Joe Lewis. "Progeny Allocation by the Parasitoid Lespesia archippivora (Diptera: Tachinidae) in Larvae of Spodoptera exigua (Lepidoptera: Noctuidae)." Environmental Entomology. 26(1997): 265-271.

Stireman III, John O. "Host location and selection cues in a generalist tachinid parasitoid." Entomologia Experimentalis et Applicata 103(2002): 23-34.

Appendix 1: Monarch Instar Stages

(Photos used with permission of Dr. Karen Oberhauser)

Egg: A monarch egg is about 1.2 mm in height and is usually attached to the bottom side of a milkweed leaf.
First-instar: The front tentacles of first-instar larvae are small bumps, and back tentacles are barely visible. The first-instar monarch larva is about 2-6 mm in length and does not have stripes.
Second-instar: The front tentacles of second-instar larvae are 0.3 mm in length, and back tentacles are small bumps. The second-instar has black, yellow, and white stripes and is about 6-9 mm in length.
Third-instar: The front tentacles of third-instar larvae are 1.7 mm long, and back tentacles are 0.9 mm. A third-instar is between 2 mm and 3.5 mm in length, and the stripes have become more distinct.
Fourth-instar: The front tentacles of fourth-instar larvae are 5 mm long, and back tentacles are 2 mm long. This instar is between 13 and 25 mm in length.
Fifth-instar: The front tentacles of fifth-monarch instar are 11 mm long, and back tentacles are 4 mm long. Fifth-instars are between 25 and 45 mm in length.

Appendix 2: Tachinid Stages

(Photos by author)

First-instar: First-instar tachinid larvae are 0-5 mm in length.	(Not Pictured)
Second-instar: Second-instar tachinid larvae are 5-10 mm in length.
Third-instar: Third-instar tachinid larvae are 10-12 mm in length.
Tachinid pupae: Tachinid pupae are 6-8 mm in length.

Appendix 3: Map of Collection Sites

MonarchLab Home | Research | Biology | Monarch Rearing | Conservation | Ask the Expert