Abstract
Extensive rearing of monarch larvae (Danaus plexippus L.) through the citizen science Monarch Larva Monitoring Project (MLMP) revealed that monarchs’ primary parasitoids are flies in the family Tachinidae and that these parasitoids result in appreciable larval mortality. We document the tachinid community that attacks monarchs in the United States, evaluate their relative frequency, and examine variation in their specificity, oviposition strategy, and use of host stages. Based on results of rearing >20,000 monarchs by MLMP volunteers, overall parasitism by tachinids across life stages was 9.8% (17% for monarchs collected as fifth instars). We identified the flies that emerged from 466 monarch hosts, and found seven Tachinidae species. In decreasing order of frequency, these included Lespesia archippivora (Riley), Hyphantrophaga virilis (Aldrich & Webber), Compsilura concinnata (Meigen), Leschenaultia n. sp., Madremyia saundersii (Williston), Lespesia sp., and Nilea erecta (Coquillett). Lespesia sp., Leschenaultia n. sp., and N. erecta had not been previously reported as monarch parasitoids, and Leschenaultia n. sp. is apparently undescribed. We include new state records (Texas and Iowa) for C. concinnata. Lespesia archippivora and C. concinnata were overrepresented as parasitoids of later instars and were absent from monarchs collected as eggs, but H. virilis and Leschenaultia sp., which lay their eggs on foliage, were reared from caterpillars collected as eggs. To our knowledge, we include the first report of multiparasitism of monarchs, in which more than one parasitoid species emerged from a host. The biology of the tachinid parasitoids we identified and their relationship with monarchs is examined.
Resumen La cría extensiva de orugas de mariposas monarca (Danaus plexippus L.) con el apoyo del programa de ciencia ciudadana Monarch Larva Monitoring Project (MLMP), reveló que los parasitoides primarios de las mariposas monarca son moscas de la familia Tachinidae y que estos parasitoides son responsables de una considerable mortalidad de orugas. Se documenta la comunidad de tachínidos que atacan mariposas monarca en los Estados Unidos, se evalúa su frecuencia relativa y examina su variación especifica, estrategia de oviposición y uso de los estadios del hospedante. Basados en los resultados de la cría de más de 20.000 mariposas monarca por parte de voluntarios del MLMP, se encontró un total de 9.8% de parasitoidismo durante los diferentes estadios (17% durante el quinto instar). Se identificaron tachínidos emergidos de 466 orugas, encontrándose siete especies de Tachinidae. En orden decreciente de frecuencia se encontró: Lespesia archippivora (Riley), Hyphantrophaga virilis (Aldrich & Webber), Compsilura concinnata (Meigen), Leschenaultia n. sp., Madremyia saundersii (Williston), Lespesia sp., y Nilea erecta (Coquillett). Lespesia sp., Leschenaultia n. sp. y N. erecta son reportadas por primera vez como parasitoides de larvas de mariposas monarca, Leschenaultia n. sp. presentó un rango amplio de distribución. Se presentan nuevos registros de C. concinnata para Texas y Iowa. Lespesia archippivora y C. conninnata fueron las especies que más se encontraron parasitando instares tardíos y no se encontraron en monarcas recolectadas desde huevo, en cambio, las que depositan micro-huevos H. virilis y Leschenaultia sp. sobre el follaje, fueron criadas de orugas recolectadas en estado de huevo, en las frecuencias altas esperadas. Hasta donde sabemos se presenta el primer reporte de multiparasitismo en mariposas monarca, con mas de una especie de parasitoide emergiendo de un hospedante. El multiparasitismo ocurrió en las tres especies de parasitoides más abundantes en frecuencias bajas. Se examina la biología de las especies de tachínidos encontradas y su relación con las mariposas monarca.
Monarch butterflies (Danaus plexippus L.) are cosmopolitan in distribution. Their natural history, migratory process, mimicry complexes, and conservation have been the focus of research programs for decades (MonarchNet 2016), and monarchs have become a model insect species for ecological and conservation research in North America. Environmental factors that influence monarch population dynamics have been of particular interest to ecologists (e.g., Malcolm and Zalucki 1993, Oberhauser and Solensky 2004, Oberhauser et al. 2015), including abiotic factors such as weather (summarized by Nail and Oberhauser 2015) and biotic factors including predators, parasitoids, host plants, and diseases (summarized by de Roode 2015, Oberhauser et al. 2015).
With regard to parasitoids, tachinid flies (Diptera: Tachinidae) are best-studied, although there are reports of other parasitoids, including recent papers on the Pteromalid wasp, Pteromalus cassotis (Walker) (Oberhauser et al. 2015, Stenoien et al. 2015a). Nail et al. (2015) included tachinid fly parasitism in their analysis of immature survival rates; McCoshum et al. (2016) used occurrence data to infer the ranges of several monarch natural enemies, including tachinid flies, and Oberhauser et al. (2015), in their review of monarch natural enemies, summarized studies in which parasitism by tachinid flies was documented. Additionally, Prysby (2004), Oberhauser et al. (2007), Oberhauser (2012), and Mueller and Baum (2014) used rearing studies to document tachinid fly parasitism frequencies in the wild. With the exception of Mueller and Baum, all of the above studies were based on larvae collected from the wild and reared by Monarch Larva Monitoring Project (MLMP) volunteers (a citizen science project; Prysby and Oberhauser 2004, MLMP 2016). While results vary from year to year and location to location, on average, about 20% of monarchs collected as late instar larvae are parasitized by tachinid flies. Because of the relative ease with which monarch eggs and larvae can be collected and reared to assess parasitism frequencies, tachinid flies are the only invertebrate natural enemies for which we have a good understanding of population-level impacts. Pteromalus cassotis attacks pupae, which are hard to find in the wild, and studying attack frequencies of predators requires observations of wild individuals.
Based on identification of a subset of flies reared from wild-collected eggs and larvae, it was assumed that most of the tachinid parasitoids of monarchs were Lespesia archippivora, which have also been reported as parasitoids of monarchs in Hawaii (Etchegary and Nishida 1975a,b). However, in his summary of tachinid–host associations, Arnaud (1978; see also O'Hara 2016) reported several additional tachinid parasitoids of monarchs in North America: Buquetia obscura (Coquillett), Compsilura concinnata, Exorista mella (Walker), Hyphantrophaga virilis, Lespesia schizurae (Townsend), Madremyia saundersii, Phryxe pecosensis (Townsend), and P. vulgaris (Fallén). Chaetogaedia monticola (Bigot) is also listed as a parasitoid of monarchs by Arnaud; however, this record is derived from artificial parasitism in the laboratory (Severin et al. 1915). In addition, Sturmia convergens (Wiedemann) has been reported as a parasitoid of monarchs in Australia and Southeast Asia (Cantrell 1986). Because many of the reports in Arnaud (1978) date back several decades (many from the 1800s) and represent incidental or rare events as well as identification errors, because this suite of natural enemies causes significant mortality, and because understanding monarch mortality from natural enemies is important in current efforts to set habitat restoration goals for this species, it is important to record the current assemblage of monarch fly parasitoids. Here, we document and analyze the community of tachinid parasitoids reared from wild-collected monarchs from throughout their U.S. breeding range (but concentrated in the Upper Midwestern United States) and evaluate their associations with their monarch hosts. In the report that follows, we refer to all life stages of monarchs (eggs, larvae, pupae, and adults) as monarchs, and all life stages of the flies as tachinid flies or flies.
Materials and Methods
Volunteer citizen scientists in the MLMP have documented temporal and spatial variation in monarch egg and larval abundances since 1997. Monitoring sites include gardens, railroad right-of-ways, roadsides, abandoned fields, pastures, natural habitats, and restored prairies (see Prysby and Oberhauser 2004 for details). Since 1999, a subset of MLMP volunteers have collected thousands of monarch larvae to measure tachinid parasitism frequencies, and since 2011, some volunteers have sent us samples of the flies that they rear from monarchs (Gebhard saved flies from 2005 and 2006, and these 78 flies are included in our analyses). Most volunteers collect monarchs to rear as fourth or fifth instars, but they have also contributed records and specimens from hundreds of monarchs collected as eggs and younger larvae. They rear them in their homes, recording the date, location, and larval stadium at collection, as well as the outcome of each rearing (adult monarch, died of unknown cause, died accidental death, parasitized by fly, parasitized by wasp). They record the number of parasitoids that emerge from each host, and a “notes” data field allows volunteers to record additional information that they consider relevant. Volunteers have access to training videos, directions with photos, and field guides that provide information on identifying larvae to stadium (MLMP 2016); while it is possible that some of their identifications are inaccurate, the relative numbers observed in each stage suggest that errors are uncommon (see, for example, Prysby and Oberhauser 2004).
We report here on two MLMP parasitoid data collection activities: 1) the outcomes of rearings that were reported to the MLMP data entry portal, and 2) the identity of a subset of parasitoid specimens that we received from volunteers. It is important to note that we do not receive all of the tachinid flies from monarchs reared by our volunteers, and that some of the volunteers who send us flies do not keep records of all of the monarchs that they rear. Thus, these two data sets are somewhat independent. For the analyses of rearing outcomes reported to our data entry portal (the first activity listed above), we omitted data when monarch death was accidental (e.g., dropping the larva or pupa, crushing it between the lid and rearing container, or losing track of it during feeding). We also omitted data when over 60% of the monarchs reared by a volunteer died of unknown causes; our rearing experience suggests that mortality rates this high are likely to be due to diseases transmitted as a result of poor rearing techniques, and thus might not accurately reflect natural causes of mortality. Finally, we omitted data when all of the reared monarchs reported by a volunteer were parasitized, because the volunteer might only be reporting cases of parasitism. However, for the last criterion, deleting the small sample size cases with 100% parasitism frequencies could lead to an under-representation of parasitized monarchs because 100% parasitism could be accurate with small sample sizes. We thus omitted all cases in which the total proportion of parasitized monarchs in a given sample size category was significantly higher than the overall parasitism frequency (as determined by a chi-square association test). Using this criterion, we omitted from our analysis all cases in which a volunteer reported results from only one or two monarchs.
Volunteers who send us flies (the second activity listed above) are asked to wait until the adults eclose, and to freeze specimens until they send them to us (MLMP 2016). We pinned adult flies using standard techniques, recording the location, date, and age of the collected monarch, and the flies that emerged from each monarch. Tachinidae specimens were identified to genus using the key provided by Wood (1987), and to species using information provided by Sabrosky (1980), Aldrich and Webber (1924), and Toma and Guimarães (2002), and with reference to identified museum specimens in the Stireman collection housed at Wright State University (JOSC, Dayton, Ohio). Tachinidae names conform to the catalog of O'Hara and Wood (2004). Specimens are deposited at the UMN Monarch Lab, University of Minnesota, Saint Paul, MN, and the JOSC collection, Wright State University, Dayton, OH. Note that all data from specimens mailed to us for identification were included when describing the tachinid community, regardless of sample size from the sender, because we did not use these data to assess overall parasitism frequencies (although they were used to assess the relative importance of the different parasitoid species).
To glean information about the host stages attacked by the different parasitoid species, we calculated 95% binomial probability confidence intervals for the proportions of monarchs collected as eggs and fifth instars that produced the four most common parasitoid species (sample sizes of the other parasitoid species were too small for these analyses). We then compared these intervals to the total proportion of reared monarchs collected at these two stages. If the total proportion of reared monarchs did not fall within these intervals, we considered them significantly different. This comparison assumes that volunteers who sent us specimens collected monarchs in the same age proportions as all volunteers who reared monarchs; we cannot test this assumption because not all of the volunteers reported all of the monarchs they reared, but have no reason to think that it is not true. We did not make the same comparisons with the proportions of monarchs collected as other larval instars to limit the number of comparisons, but we do note a case in which this proportion was very different from the total proportions of reared monarchs. Eggs and fifth instars were most interesting to us because 1) of the rarity of tachinid attacks of monarchs collected as eggs, and 2) disproportionately high parasitism of monarchs collected as fifth instars suggests attacks across multiple life history stages (Oberhauser 2012).
We compared the numbers of individual parasitoids that emerged from host caterpillars across the four most abundant species using an ANOVA with post hoc LSD pairwise comparisons. This comparison provides information on the relative success of these species in monarchs.
Finally, we hypothesized that generalist parasitoids (species known to attack many different hosts) would be more likely to attack monarchs during years of high monarch abundance. We used mean peak egg densities (eggs per observed plants) from MLMP sites in the Upper Midwest (where most of our tachinid specimens originated) as a proxy for monarch abundance (for details, see Stenoien et al. 2015b). We calculated Pearson’s correlation coefficients between the proportion of the parasitoid pool represented by our three most common species (L. archippivora, Compsilura concinnata, and Hyphantrophaga virilis) and monarch density across the collection years. As above, this comparison assumes that the specimens we received represent the pool of parasitoids reared by all volunteers. It is possible that volunteers are more likely to send unusual-looking parasitoids, but we have no reason to think that this pattern would vary from year to year.
Results
From 1999 to June 2016, MLMP volunteers collected and reared 20,837 monarch eggs and caterpillars (for records meeting the above criteria). Of these, 20,386 were identified to stage (or larval stadium) at collection; Fig. 1 (right-most column) illustrates the proportions collected in each stage. The overall frequency of tachinid fly parasitism was 9.8%, but parasitism frequency increased nearly linearly with larval instar, reaching a maximum of 17% in fifth instars with a subsequent drop at the pupal stage (Fig. 2). Interestingly, a number of individuals collected as eggs (N = 40) were parasitized by tachinids. Details on year to year and site to site variation and the probable timing of overall parasitoid attack during monarch development are analyzed in previous studies (Oberhauser et al. 2007, Oberhauser 2012, Nail et al. 2015).
Collection stage of monarchs that produced the four parasitoid species (C. concinnata (Cc), H. virilis (Hv), Leschenaultia n. sp. (Lesch ns), L. archippivora (La)) that emerged from ≥10 monarchs. For comparison, collection ages for all parasitized hosts and the total number of monarchs reared by MLMP volunteers are also shown. Not all volunteers identified the monarch age at collection, so sample sizes are lower than those illustrated in Tables 2 and 3.
Collection stage of monarchs that produced the four parasitoid species (C. concinnata (Cc), H. virilis (Hv), Leschenaultia n. sp. (Lesch ns), L. archippivora (La)) that emerged from ≥10 monarchs. For comparison, collection ages for all parasitized hosts and the total number of monarchs reared by MLMP volunteers are also shown. Not all volunteers identified the monarch age at collection, so sample sizes are lower than those illustrated in Tables 2 and 3.
Proportions of monarchs collected and reared by MLMP volunteers from 1999–2016 that were parasitized by tachinid flies. Columns represent the stage at collection, with the total number of monarchs collected at each stage given under each column.
Proportions of monarchs collected and reared by MLMP volunteers from 1999–2016 that were parasitized by tachinid flies. Columns represent the stage at collection, with the total number of monarchs collected at each stage given under each column.
We received 1,146 fly specimens that we were able to at least tentatively identify to species (Table 1; some individuals were damaged in transit, frozen as pharate adults, or sent as pupae, which we were unable to identify). The flies came from 466 monarch hosts whose stages at collection, when known, are illustrated in the fifth bar in Fig. 1. We identified seven species of Tachinidae from monarchs collected in 16 states ranging from California to Maine, although most records are concentrated in the Upper Midwest (Table 2). Most (75%) parasitism events were due to Lespesia archippivora and this species was found across the country (Tables 1 and 2), only being absent from three states from which we received few specimens (FL, NC, ME). Compsilura concinnata and Hyphantrophaga virilis were each responsible for 10% or more of the parasitized monarchs (Table 1).
Summary of tachinid fly parasitism of monarchs collected by MLMP volunteers from 2005–2016
| Tachinid species | No. | Total hosts (w/multi) | Proportion | Mean flies/monarch ± S.E. |
|---|---|---|---|---|
| Compsilura concinnata (Cc) | 69 | 45 | 0.098 | 1.74b ± 0.195 |
| Hyphantrophaga virilis (Hv) | 114 | 63 | 0.137 | 1.99b ± 0.227 |
| Leschenaultia n. sp. (Lesch) | 10 | 9 | 0.020 | 1.22b ± 0.222 |
| Lespesia archippivora (La) | 943 | 347 | 0.748 | 3.14a ± 0.113 |
| Lespesia sp. (Lsp) | 3 | 3 | 0.007 | 1 ± 0 |
| Madremyia saundersii (Ms) | 5 | 3 | 0.007 | 2.33 ± 0.882 |
| Nilea erecta (Ne) | 2 | 2 | 0.004 | 1.5 ± 0.500 |
| Tachinid species | No. | Total hosts (w/multi) | Proportion | Mean flies/monarch ± S.E. |
|---|---|---|---|---|
| Compsilura concinnata (Cc) | 69 | 45 | 0.098 | 1.74b ± 0.195 |
| Hyphantrophaga virilis (Hv) | 114 | 63 | 0.137 | 1.99b ± 0.227 |
| Leschenaultia n. sp. (Lesch) | 10 | 9 | 0.020 | 1.22b ± 0.222 |
| Lespesia archippivora (La) | 943 | 347 | 0.748 | 3.14a ± 0.113 |
| Lespesia sp. (Lsp) | 3 | 3 | 0.007 | 1 ± 0 |
| Madremyia saundersii (Ms) | 5 | 3 | 0.007 | 2.33 ± 0.882 |
| Nilea erecta (Ne) | 2 | 2 | 0.004 | 1.5 ± 0.500 |
No.—total number of each tachinid species that emerged from 466 parasitized monarch specimens; Total hosts (w/multi) —total number of specimens from which each fly species emerged, including those from which more than one species emerged; Proportion—the proportion of the parasitized specimens from which each fly species emerged (including multispecies events); Mean flies/monarch—the mean number of flies that emerged from each monarch (for single species parasitism events only with number of hosts > 8, values followed by different superscripts are significantly different).
U.S. states (divided by regions) in which monarchs parasitized by each tachinid fly species were collected
| Region | State | No. (monarchs) | Cc | Hv | Lesch | La | Lsp | Ms | Ne |
|---|---|---|---|---|---|---|---|---|---|
| Upper Midwest (N=366) | IA | 9 | x | x | |||||
| MI | 250 | x | x | x | x | x | x | ||
| MN | 21 | x | x | ||||||
| ND | 1 | x | |||||||
| WI | 85 | x | x | x | |||||
| Northeast (N=17) | ME | 1 | x | ||||||
| NY | 1 | x | |||||||
| OH | 1 | x | |||||||
| PA | 14 | x | x | x | |||||
| Middle East (N=20) | MD | 13 | x | ||||||
| NC | 7 | x | x | ||||||
| Southwest (N=18) | AZ | 3 | x | x | |||||
| CA | 15 | x | |||||||
| South Central (N=35) | OK | 2 | x | x | |||||
| TX | 33 | x | x | ||||||
| Southeast (N=1) | FL | 1 | x |
| Region | State | No. (monarchs) | Cc | Hv | Lesch | La | Lsp | Ms | Ne |
|---|---|---|---|---|---|---|---|---|---|
| Upper Midwest (N=366) | IA | 9 | x | x | |||||
| MI | 250 | x | x | x | x | x | x | ||
| MN | 21 | x | x | ||||||
| ND | 1 | x | |||||||
| WI | 85 | x | x | x | |||||
| Northeast (N=17) | ME | 1 | x | ||||||
| NY | 1 | x | |||||||
| OH | 1 | x | |||||||
| PA | 14 | x | x | x | |||||
| Middle East (N=20) | MD | 13 | x | ||||||
| NC | 7 | x | x | ||||||
| Southwest (N=18) | AZ | 3 | x | x | |||||
| CA | 15 | x | |||||||
| South Central (N=35) | OK | 2 | x | x | |||||
| TX | 33 | x | x | ||||||
| Southeast (N=1) | FL | 1 | x |
For species abbreviations, see Table 1.
There were significantly more L. archippivora larvae per monarch (mean: 3.14) than the three other species reared from nine or more monarchs (Table 1, C. concinnata, H. virilis or Leschenaultia n. sp; F3,348 = 12.5, P < 0.0001). Of the 466 monarchs from which tachinid larvae emerged, nine exhibited multiparasitism (they produced more than one fly species), containing all three pairwise combinations of the three most common parasitoid species (Table 3). There was no relationship between the proportional representation of any of the top three species and monarch density in the Upper Midwest in any given year (Table 4). There was, however, significant year to year variation in the representation of different species (χ2 = 178, df = 14, P < 0.0001 for numbers of the three most common species across years), with L. archippivora, C. concinnata, and H. virilis ranging from 13% in 2005 to 91% in 2012, 2.5% in 2012 to 61% in 2005, and 1.3% in 2011 to 33% in 2015, respectively.
Summary of nine multiparasitism events (from a total of 466 monarch hosts)
| Species present | Flies in each multispecies event |
| L. archippivora and H. virilis | 2 La 2Hv |
| 1 La 1Hv | |
| L. archippivora and C. concinnata | 1 La 3 Cc |
| 1 La 1 Cc | |
| 1 La 1 Cc | |
| 3 La 1 Cc | |
| C. concinnata and H. virilis | 3 Cc 1 Hv |
| 1 Cc 2 Hv | |
| 1 Cc 3 Hv |
| Species present | Flies in each multispecies event |
| L. archippivora and H. virilis | 2 La 2Hv |
| 1 La 1Hv | |
| L. archippivora and C. concinnata | 1 La 3 Cc |
| 1 La 1 Cc | |
| 1 La 1 Cc | |
| 3 La 1 Cc | |
| C. concinnata and H. virilis | 3 Cc 1 Hv |
| 1 Cc 2 Hv | |
| 1 Cc 3 Hv |
The rows in the right column list all of the tachinid flies that were reared from each monarch host.
Tachinid fly species representation in the parasitized monarchs that were collected each year
| Year | 2005 | 2006 | 2011 | 2012 | 2013 | 2014 | 2015 | 2016 | Corr |
|---|---|---|---|---|---|---|---|---|---|
| Host no. | 23 | 51 | 78 | 174 | 10 | 35 | 80 | 15 | |
| Compsilura concinnata | 0.609 | 0 | 0.080 | 0.025 | 0.100 | 0.032 | 0.230 | 0.133 | −0.42 |
| Hyphantrophaga virilis | 0.261 | 0.275 | 0.013 | 0.037 | 0.400 | 0.258 | 0.328 | 0 | −0.18 |
| Leschenaultia sp. | 0 | 0 | 0 | 0.012 | 0 | 0 | 0.115 | 0 | |
| Lespesia archippivora | 0.130 | 0.725 | 0.867 | 0.913 | 0.400 | 0.710 | 0.328 | 0.733 | 0.25 |
| Lespesia sp. | 0 | 0 | 0 | 0.012 | 0.100 | 0 | 0 | 0 | |
| Madremyia saundersii | 0 | 0 | 0.013 | 0 | 0 | 0 | 0 | 0.133 | |
| Nilea erecta | 0 | 0 | 0.027 | 0 | 0 | 0 | 0 | 0 | |
| MLMP host density | 0.36 | 0.52 | 0.24 | 0.50 | 0.10 | 0.14 | 0.36 | 0.13 |
| Year | 2005 | 2006 | 2011 | 2012 | 2013 | 2014 | 2015 | 2016 | Corr |
|---|---|---|---|---|---|---|---|---|---|
| Host no. | 23 | 51 | 78 | 174 | 10 | 35 | 80 | 15 | |
| Compsilura concinnata | 0.609 | 0 | 0.080 | 0.025 | 0.100 | 0.032 | 0.230 | 0.133 | −0.42 |
| Hyphantrophaga virilis | 0.261 | 0.275 | 0.013 | 0.037 | 0.400 | 0.258 | 0.328 | 0 | −0.18 |
| Leschenaultia sp. | 0 | 0 | 0 | 0.012 | 0 | 0 | 0.115 | 0 | |
| Lespesia archippivora | 0.130 | 0.725 | 0.867 | 0.913 | 0.400 | 0.710 | 0.328 | 0.733 | 0.25 |
| Lespesia sp. | 0 | 0 | 0 | 0.012 | 0.100 | 0 | 0 | 0 | |
| Madremyia saundersii | 0 | 0 | 0.013 | 0 | 0 | 0 | 0 | 0.133 | |
| Nilea erecta | 0 | 0 | 0.027 | 0 | 0 | 0 | 0 | 0 | |
| MLMP host density | 0.36 | 0.52 | 0.24 | 0.50 | 0.10 | 0.14 | 0.36 | 0.13 |
Host no. —the total number of hosts from which specimens we received were reared; values after each parasitoid species are the proportion of the monarchs that contained that species in each year; host density—mean peak density at Upper Midwestern MLMP sites, a proxy for overall monarch abundance (see text and Stenoien et al. (2015b) for details). Corr—Pearson correlation coefficient for correlations between proportions and host density for species reared in seven or more years. None of these correlations are significant at the 0.05 level of confidence (all P > 0.30). Calculation of annual MLMP host density (eggs per plant) explained in text and Stenoien et al. (2015b).
While caterpillars collected as fifth instars are most likely to be parasitized (Fig. 2), the four most common tachinid species showed substantial variation in which host stages they appear to attack (Fig. 1). No monarchs collected as eggs produced C. concinnata or L. archippivora; these two zero proportions are significantly lower than the overall proportion of reared monarchs collected as eggs illustrated in Fig. 1 (95% binomial confidence intervals = 0–0.161 and 0–0.016, respectively, vs. 28% collected as eggs). Over half of the C. concinnata emerged from monarchs collected as fourth instars (Fig. 1); the 95% binomial confidence interval for this proportion (post hoc calculation) does not overlap with the proportion of monarchs collected as fourth instars (CI = 0.306–0.732 vs. 15% collected as fourth instars). Leschenaultia n. sp. representation in monarchs collected as eggs was significantly higher than expected despite the relatively small sample size (CI = 0.400–0.972). The frequency of L. archippivora in monarchs collected as fifth instars was significantly higher than the overall proportion of monarchs collected during this stadium (CI = 0.487–0.619, vs. 37% collected as fifth instars). Hyphantrophaga virilis representation in monarchs collected as both eggs and fifth instars was as expected based on the overall proportions of monarchs collected in these stages. Sample sizes for the other three species preclude conclusive statistical tests, but all specimens of both M. saundersii and N. erecta were reared from caterpillars collected as fifth instars. The stage at collection of the three caterpillars from which Lespesia sp. were reared was not recorded by volunteers.
Discussion
Overview
Our survey confirms previous claims that tachinid flies are significant parasitoids of monarchs (Prysby 2004; Oberhauser et al. 2007, 2015; Oberhauser 2012; Mueller and Baum 2014; Nail et al. 2015), with an overall parasitism level of ∼17% for monarchs collected during the final larval stadium. The fact that a lower proportion of monarchs collected as pupae are parasitized probably reflects the fact that tachinid flies often emerge from fifth instars (Oberhauser et al. 2007), so monarchs that survive to pupation are less likely to be parasitized. The 17% value is similar to frequencies calculated previously from MLMP data (Oberhauser et al. 2007, Oberhauser 2012, Nail et al. 2015), but lower than that found by Mueller and Baum (2014) (36%). It should be noted that Mueller and Baum collected all specimens from roadside and prairie sites within 25 km of Stillwater, OK, in a single year, whereas MLMP data cover broad temporal and geographic ranges; annual frequencies from the MLMP range from 3% to 38% (Nail et al. 2015) and the highest value was in 2012, when Mueller and Baum collected their data.
As assumed by the above authors, L. archippivora was the most common and widespread tachinid parasitoid of monarchs. It was found in 13 of the 16 states from which we received specimens, and in every year for which we have records. The three states from which it was not reared are all in the eastern United States, but low sample sizes from these states preclude strong conclusions about this pattern. However, 25% of the parasitized hosts contained other species, with H. virilis (14%) and C. concinnata (10%) being the other two most common monarch parasitoids. Given the extremely low number of records (<1% of the total), N. erecta, M. saundersii, and Lespesia sp. should be considered incidental parasitoids of monarchs, but for Leschenaultia n. sp. the relationship is unclear, with low prevalence (2%) but still a substantial number of rearings (N = 9).
Of the nine parasitoid species listed for monarchs by Arnaud (1978), we found four: C. concinnata, H. virilis (listed by Arnaud as Eusisyropa virilis), L. archippivora, and M. saundersii. We found three species previously unreported from monarchs: Lespesia sp., Leschenaultia n. sp., and Nilea erecta. The five species listed in Arnaud that were not represented in our collection include Buquetia obscura, Exorista mella, Lespesia schizurae, Phryxe pecosensis, and P. vulgaris.
We are not certain of the identity of the “Lespesia sp.” specimens. They are clearly not L. archippivora, but neither do they match L. schizurae (a species previously recorded from monarchs). Their bright yellow-gold parafrontal and parafacial regions are similar to those of L. flavifrons, but they do not match this species in other respects. Lespesia is a difficult genus taxonomically, with many morphologically similar species that themselves may consist of cryptic species complexes (e.g., Smith et al. 2007). Further taxonomic investigation of these specimens is in progress. The Leschenaultia specimens fail to completely match (or key to) any recognized North American species, and we suspect that they may represent an undescribed species.
Species Characteristics
All of the tachinid species recorded in our survey belong to the subfamily Exoristinae, representing three tribes: Blondeliini (Compsilura), Eryciini (Lespesia, Madremya, and Nilea), and Goniini (Hyphantrophaga and Leschenaultia). These genera possess a variety of reproductive strategies, including deposition of tiny “microtype” eggs on leaves that are eaten by the host (H. virilis and Leschenaultia sp.; Wood 1987), laying incubated eggs directly on the host that immediately hatch and bore into the host (Lespesia archippivora, L. sp., and M. saundersii; Stapel et al. 1997), and injecting eggs into hosts with an abdominal piercer (C. concinnata; O'Hara 1985). Although O'Hara (2005) inferred the reproductive strategy of N. erecta to be direct deposition of incubated eggs from its systematic placement in the Eryciini, Wiman and Jones (2013) report that N. erecta females lay their eggs on foliage, and the larvae burrow into the host.
The only tachinid species reported here that are potentially monophagous parasitoids of D. plexippus are L. archippivora and Leschenaultia sp. According to Arnaud (1978), L. archippivora uses a wide range of lepidopteran hosts belonging to 15 different families (and one family of Hymenoptera). However, preliminary morphological examination suggests that specimens reared from D. plexippus are distinct from specimens reared from Erebidae and Lasiocampidae (JOS personal observation), and year to year variation in parasitism frequency of monarchs suggest that L. archippivora tracks monarch population densities with a one year time lag (Oberhauser 2012). Further morphological and genetic analysis is needed to determine if L. archippivora indeed represents multiple, monophagous species and how many. To our knowledge, Leschenaultia has never previously been reared from monarchs. The fact that the species we found has not been reported in other hosts suggests that it may be a specialist on monarchs or on milkweed associated caterpillars (see below).
Hyphantrophaga virilis, M. saundersii, N. erecta, and C. concinnata are all highly polyphagous, attacking a wide variety of Lepidoptera spanning at least 10 families each (Arnaud 1978, Smith et al. 2007). These species are likely to use relatively general cues to locate caterpillar hosts, and may take advantage of their ability to attack monarchs when monarch densities are high, although we found no evidence of this (Table 4). It is also possible that they attack monarchs when other hosts are rare, but we cannot test this hypothesis with available data. Host associations of the Lespesia sp. are unclear, as the genus contains both highly specialized and highly polyphagous species (Arnaud 1978).
Compsilura concinnata has the broadest host range of any known tachinid, having been recorded from >180 host species in three orders (Arnaud 1978, Boettner et al. 2000). Incredibly, it was intentionally introduced into North America from Europe over much of the 20th century to control the gypsy moth Lymantria dispar dispar L. (Lepidoptera: Erebidae) and other caterpillar pests (Sanchez and Carde 1998). This species is of substantial environmental concern due to its frequent use of nontarget species; it has been implicated in the decline of wild silk moths (Saturniidae) in Eastern U.S. forests (Boettner et al. 2000; Kellogg et al. 2003; Elkinton and Boettner 2004, 2012). The regularity of attacks by this intentionally introduced biocontrol agent on monarchs is disturbing, given current concerns about declining monarch numbers (e.g., Semmens et al. 2016).
Host Stage
We can use associations between parasitoid species and monarch stage at collection to infer the stages at which monarchs are vulnerable to the different parasitoids. The increase in parasitism with larval stadium at collection (Fig. 2) suggests that monarch larvae continue to be vulnerable to fly parasitoids as they develop (Oberhauser 2012). The most common parasitoid in our study, L. archippivora, was never found in monarchs collected as eggs, in keeping with its direct deposition of eggs on host larvae, and was overrepresented in monarchs collected as fifth instars, supporting the previous conclusion that this parasitoid continues to attack monarch larvae throughout their development (Etchegary and Nishida 1975a,b; Prysby 2004; Oberhauser 2012; also see Stapel et al. 1997). Compsilura concinnata was never found in monarchs collected as eggs or first instars; this species injects eggs into the host larva, and the eggs hatch immediately and migrate to the host midgut (Ichiki and Shima 2003, O'Hara 2005). Our results suggest that it attacks monarchs older than first instars. Its overrepresentation in hosts collected as fourth instars suggests that it often emerges from fourth instars or early fifth instars (so parasitized hosts are less likely to survive to be collected as fifth instars), although sample sizes are too small to draw strong conclusions.
Representation of H. virilis across monarch developmental stages does not deviate from expectations based on the overall proportions of monarchs collected in these stages; H. virilis takes advantage of its opportunistic parasitism to infest caterpillars of several Lepidoptera families (Arnaud 1978, O'Hara 2005). A substantial fraction of parasitism events were from monarchs collected as eggs. Because there is no evidence that any tachinid flies attack host eggs, this finding indicates that milkweed foliage “infected” with the microtype eggs of H. virilis was introduced by MLMP volunteers, either on the leaf on which the egg was collected or subsequent leaves collected for feeding. This makes it difficult to ascertain the host stage at which H. virilis typically attacks, as parasitoid eggs could be introduced at any time during rearing and larvae typically emerge from the pupal stage of the host (Sellers 1930). However, the lack of deviation from overall proportions of monarchs collected across developmental stages suggests that they are attacked early in their development by this parasitoid; continued attack would result in increasing proportions for monarchs collected at later stages.
We only know the stage of collection for nine monarchs that produced Leschenaultia n. sp., but seven of these were collected as eggs, a significantly higher proportion than the overall egg collection. This is consistent with their deposition of microtype eggs on foliage with feeding larvae, which then ingest the eggs (Mondor and Roland 1998). Again, the only explanation for the high representation in monarchs collected as eggs is that parasitoid eggs were introduced with milkweed leaves supplied to the developing caterpillars. The low number emerging from monarchs collected at other stages suggests that monarchs may not be a typical host for this parasitoid species, i.e., it may be targeting other milkweed feeding hosts such as Eucheates egle (Erebidae). Although capable of developing in monarchs, the low numbers of hosts collected at other stages that produced this parasitoid could indicate premature mortality of the monarch host (as with C. concinnata, some infected hosts may not survive to be collected as larvae, or die in captivity before the fly larvae emerge). However, our sample size for monarchs parasitized by Leschenaultia n. sp. is too low to draw strong conclusions.
Multiparasitism
Multiparasitism (in which more than one parasitoid species emerges from the host) of monarchs has not been reported previously, and the low frequency of such events suggest that it is a rare and random occurrence. That it occurs at all supports the view that tachinids generally lack the ability to discriminate among parasitized and unparasitized hosts (Feener and Brown 1997, Kan et al. 2003) and that competition in this community is exploitative in nature, rather than direct interference. This latter conclusion is also supported by the observation that all of these species are at least facultatively gregarious. Sample sizes of multiparastism are too low for robust statistical analysis, but the relatively frequent involvement of C. concinnata, particularly with H. virilis, supports our conjecture that it may often attack relatively later than the other parasitoids (increasing the chance of multiparasitism) or that parasitized larvae may be more vulnerable to attack by this species.
Variability Over Space and Time
Tachinid fly parasitism is variable across years (Oberhauser 2012, Nail et al. 2015), but the mechanisms driving this variability require further study. Our finding of substantial parasitism by species other than L. archippivora, the variable representation of L. archippivora in the annual monarch parasitoid pool, and the fact that many of the parasitoids are generalists whose population dynamics will be driven by numbers of other species will complicate our ability to understand these mechanisms.
Nearly all of the identified tachinid species reared from monarchs in this study have broad distributions, ranging over much of North America, coast to coast and from Canada to Mexico. An exception is C. concinnata, which was introduced and established along both East and West coasts of North America and in the Midwestern United States (Sanchez and Carde 1998), and has been slowly spreading from these introduction areas (Elkinton and Boettner 2004, 2012). It is largely absent from the (interior) southern half of the United States and the Rocky Mountain states and provinces. Our record from Texas represents a dramatic extension of its known range. Iowa also appears to be a new state record. Our limited records of the apparently undescribed Leschenaultia sp. suggest it has a broad geographic range over much of the eastern United States. Sample sizes are insufficient to assess whether parasitoid community composition and parasitism frequencies vary significantly across geography.
In conclusion, the large temporal and spatial scales of this rearing study allowed us to document new monarch parasitoids. While we had previously used MLMP data to show year to year variation in parasitism frequencies, obtaining the reared specimens allowed us to document variation in the relative importance of different species. Our findings further illustrate the value of engaging citizen scientists in natural history research; indeed, a large proportion of all published research on monarch ecology utilizes citizen science data (Ries and Oberhauser 2015, MonarchNet 2016).
Acknowledgments
We thank the hundreds of MLMP volunteers who have contributed to this project, especially Diane Rock, Betsy Johnson, Linda Rippert, Brenda Keith, and Annie Letaw, who, in addition to I.G. sent in the vast majority of the flies that we identified; and Monarch Lab students and staff for their help in developing protocols and communicating with volunteers. This research was supported by the University of Minnesota Monarch Lab, and the development of the MLMP was supported by National Science Foundation ESI 9731429. Contributions of J.M.P.L. and J.O.S. were supported by National Science Foundation DEB 1442134. An earlier version of the manuscript benefited from the comments of two anonymous reviewers.
References Cited
Author notes
Subject Editor: Ann Fraser
