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Annals of the Entomological Society of America, 2017, 1–9 doi: 10.1093/aesa/sax048 Ecology and Population Biology Research article

Tachinid (Diptera: ) Parasitoids of Danaus plexippus (Lepidoptera: Nymphalidae)

Karen Oberhauser,1,2 Dane Elmquist,3 Juan Manuel Perilla-Lopez, 4 Ilse Gebhard,5 Laura Lukens,1 and John Stireman4

1Department of Fisheries, Wildlife and Conservation Biology, University of , St. Paul, MN 55108 ([email protected]; [email protected]), 2Corresponding author, e-mail: [email protected], 3USDA-ARS, Yakima, WA 98902 ([email protected]. gov), 4Department of Biological Sciences, Wright State University, Dayton, OH 45435 ([email protected]; john.stireman@ wright.edu), and 56450 N. 2nd St. Kalamazoo, MI 49009 ([email protected]) Subject Editor: Ann Fraser Received 31 January 2017; Editorial decision 15 May 2017

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 , evaluate their relative frequency, and examine variation in their specificity, ovi- position 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 archippivora (Riley), Hyphantrophaga virilis (Aldrich & Webber), concinnata (Meigen), n. sp., saundersii (Williston), Lespesia sp., and 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 ( and Iowa) for C. concinnata. 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 re- port of multiparasitism of monarchs, in which more than one parasitoid species emerged from a host. The biol- ogy of the tachinid parasitoids we identified and their relationship with monarchs is examined.

Resumen La crıa extensiva de orugas de mariposas monarca (Danaus plexippus L.) con el apoyo del pro- grama de ciencia ciudadana Monarch Larva Monitoring Project (MLMP), revelo 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 tachınidos que atacan mariposas monarca en los Estados Unidos, se evalua su frecuencia relativa y examina su variacion especifica, estrategia de oviposicion y uso de los estadios del hospedante. Basados en los resultados de la crıa de mas de 20.000 mariposas monarca por parte de voluntarios del MLMP, se encontro un total de 9.8% de parasitoidismo durante los diferentes estadios (17% durante el quinto instar). Se identificaron tachınidos emergidos de 466 orugas, encontrandose siete especies de Tachinidae. En orden decreciente de frecuencia se encontro: Lespesia archippivora (Riley), Hyphantrophaga virilis (Aldrich & Webber), (Meigen), Leschenaultia n. sp., (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. presento un rango amplio de distribucion. Se presentan nuevos registros de C. concinnata para Texas y Iowa. Lespesia archippivora y C. conninnata fueron las especies que mas se encon- traron parasitando instares tardıos 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 recol- ectadas 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 ocurrio en las tres especies de parasitoides mas abundantes en

VC The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: [email protected] 1 2 Annals of the Entomological Society of America, 2017, Vol. 0, No. 0

frecuencias bajas. Se examina la biologıa de las especies de tachınidos encontradas y su relacion con las mar- iposas monarca.

Key words: parasitoid, Tachinidae, citizen science, multiparasitism

Monarch butterflies (Danaus plexippus L.) are cosmopolitan in dis- natural enemies causes significant mortality, and because under- tribution. Their natural history, migratory process, mimicry com- standing monarch mortality from natural enemies is important in plexes, and conservation have been the focus of research programs current efforts to set habitat restoration goals for this species, it is for decades (MonarchNet 2016), and monarchs have become a important to record the current assemblage of monarch fly parasit- model species for ecological and conservation research in oids. Here, we document and analyze the community of tachinid North America. Environmental factors that influence monarch pop- parasitoids reared from wild-collected monarchs from throughout ulation dynamics have been of particular interest to ecologists (e.g., their U.S. breeding range (but concentrated in the Upper Malcolm and Zalucki 1993, Oberhauser and Solensky 2004, Midwestern United States) and evaluate their associations with their Oberhauser et al. 2015), including abiotic factors such as weather monarch hosts. In the report that follows, we refer to all life stages (summarized by Nail and Oberhauser 2015) and biotic factors in- of monarchs (eggs, larvae, pupae, and adults) as monarchs, and all cluding predators, parasitoids, host plants, and diseases (summa- life stages of the as tachinid flies or flies. rized 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, in- cluding recent papers on the Pteromalid wasp, Pteromalus cassotis Materials and Methods (Walker) (Oberhauser et al. 2015, Stenoien et al. 2015a). Nail et al. Volunteer citizen scientists in the MLMP have documented temporal (2015) included tachinid fly parasitism in their analysis of immature and spatial variation in monarch egg and larval abundances since survival rates; McCoshum et al. (2016) used occurrence data to infer 1997. Monitoring sites include gardens, railroad right-of-ways, the ranges of several monarch natural enemies, including tachinid roadsides, abandoned fields, pastures, natural habitats, and restored flies, and Oberhauser et al. (2015), in their review of monarch natu- prairies (see Prysby and Oberhauser 2004 for details). Since 1999, a ral enemies, summarized studies in which parasitism by tachinid flies subset of MLMP volunteers have collected thousands of monarch was documented. Additionally, Prysby (2004), Oberhauser et al. larvae to measure tachinid parasitism frequencies, and since 2011, (2007), Oberhauser (2012), and Mueller and Baum (2014) used some volunteers have sent us samples of the flies that they rear from rearing studies to document tachinid fly parasitism frequencies in monarchs (Gebhard saved flies from 2005 and 2006, and these 78 the wild. With the exception of Mueller and Baum, all of the above flies are included in our analyses). Most volunteers collect monarchs studies were based on larvae collected from the wild and reared by to rear as fourth or fifth instars, but they have also contributed re- Monarch Larva Monitoring Project (MLMP) volunteers (a citizen cords and specimens from hundreds of monarchs collected as eggs science project; Prysby and Oberhauser 2004, MLMP 2016). While and younger larvae. They rear them in their homes, recording the results vary from year to year and location to location, on average, date, location, and larval stadium at collection, as well as the out- about 20% of monarchs collected as late instar larvae are parasit- come of each rearing (adult monarch, died of unknown cause, died ized by tachinid flies. Because of the relative ease with which mon- accidental death, parasitized by fly, parasitized by wasp). They re- arch eggs and larvae can be collected and reared to assess parasitism cord the number of parasitoids that emerge from each host, and a frequencies, tachinid flies are the only invertebrate natural enemies “notes” data field allows volunteers to record additional informa- for which we have a good understanding of population-level im- tion that they consider relevant. Volunteers have access to training pacts. Pteromalus cassotis attacks pupae, which are hard to find in videos, directions with photos, and field guides that provide infor- the wild, and studying attack frequencies of predators requires ob- mation on identifying larvae to stadium (MLMP 2016); while it is servations of wild individuals. possible that some of their identifications are inaccurate, the relative Based on identification of a subset of flies reared from wild- numbers observed in each stage suggest that errors are uncommon collected eggs and larvae, it was assumed that most of the tachinid (see, for example, Prysby and Oberhauser 2004). parasitoids of monarchs were Lespesia archippivora, which have We report here on two MLMP parasitoid data collection activi- also been reported as parasitoids of monarchs in Hawaii (Etchegary ties: 1) the outcomes of rearings that were reported to the MLMP and Nishida 1975a,b). However, in his summary of tachinid–host data entry portal, and 2) the identity of a subset of parasitoid speci- associations, Arnaud (1978; see also O’Hara 2016) reported several mens that we received from volunteers. It is important to note that additional tachinid parasitoids of monarchs in North America: we do not receive all of the tachinid flies from monarchs reared by obscura (Coquillett), Compsilura concinnata, Exorista our volunteers, and that some of the volunteers who send us flies do mella (Walker), Hyphantrophaga virilis, Lespesia schizurae not keep records of all of the monarchs that they rear. Thus, these (Townsend), Madremyia saundersii, pecosensis (Townsend), two data sets are somewhat independent. For the analyses of rearing and P. vulgaris (Falle´n). Chaetogaedia monticola (Bigot) is also listed outcomes reported to our data entry portal (the first activity listed as a parasitoid of monarchs by Arnaud; however, this record is de- above), we omitted data when monarch death was accidental (e.g., rived from artificial parasitism in the laboratory (Severin et al. dropping the larva or pupa, crushing it between the lid and rearing 1915). In addition, Sturmia convergens (Wiedemann) has been re- container, or losing track of it during feeding). We also omitted data ported as a parasitoid of monarchs in Australia and Southeast Asia when over 60% of the monarchs reared by a volunteer died of un- (Cantrell 1986). Because many of the reports in Arnaud (1978) date known causes; our rearing experience suggests that mortality rates back several decades (many from the 1800s) and represent inciden- this high are likely to be due to diseases transmitted as a result of tal or rare events as well as identification errors, because this suite of poor rearing techniques, and thus might not accurately reflect Annals of the Entomological Society of America, 2017, Vol. 0, No. 0 3 natural causes of mortality. Finally, we omitted data when all of the in the Upper Midwest (where most of our tachinid specimens origi- reared monarchs reported by a volunteer were parasitized, because nated) as a proxy for monarch abundance (for details, see Stenoien the volunteer might only be reporting cases of parasitism. However, et al. 2015b). We calculated Pearson’s correlation coefficients be- for the last criterion, deleting the small sample size cases with 100% tween the proportion of the parasitoid pool represented by our three parasitism frequencies could lead to an under-representation of par- most common species (L. archippivora, Compsilura concinnata, and asitized monarchs because 100% parasitism could be accurate with Hyphantrophaga virilis) and monarch density across the collection small sample sizes. We thus omitted all cases in which the total pro- years. As above, this comparison assumes that the specimens we re- portion of parasitized monarchs in a given sample size category was ceived represent the pool of parasitoids reared by all volunteers. It is significantly higher than the overall parasitism frequency (as deter- possible that volunteers are more likely to send unusual-looking par- mined by a chi-square association test). Using this criterion, we asitoids, but we have no reason to think that this pattern would vary omitted from our analysis all cases in which a volunteer reported re- from year to year. sults 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 un- til they send them to us (MLMP 2016). We pinned adult flies using Results standard techniques, recording the location, date, and age of the col- From 1999 to June 2016, MLMP volunteers collected and reared lected monarch, and the flies that emerged from each monarch. 20,837 monarch eggs and caterpillars (for records meeting the above Tachinidae specimens were identified to using the key pro- criteria). Of these, 20,386 were identified to stage (or larval sta- vided by Wood (1987), and to species using information provided dium) at collection; Fig. 1 (right-most column) illustrates the pro- by Sabrosky (1980), Aldrich and Webber (1924), and Toma and portions collected in each stage. The overall frequency of tachinid Guimaraes~ (2002), and with reference to identified museum speci- fly parasitism was 9.8%, but parasitism frequency increased nearly mens in the Stireman collection housed at Wright State University linearly with larval instar, reaching a maximum of 17% in fifth in- (JOSC, Dayton, ). Tachinidae names conform to the catalog of stars with a subsequent drop at the pupal stage (Fig. 2). O’Hara and Wood (2004). Specimens are deposited at the UMN Interestingly, a number of individuals collected as eggs (N ¼ 40) Monarch Lab, University of Minnesota, Saint Paul, MN, and the were parasitized by tachinids. Details on year to year and site to site JOSC collection, Wright State University, Dayton, OH. Note that variation and the probable timing of overall parasitoid attack during all data from specimens mailed to us for identification were included monarch development are analyzed in previous studies (Oberhauser when describing the tachinid community, regardless of sample size et al. 2007, Oberhauser 2012, Nail et al. 2015). from the sender, because we did not use these data to assess overall We received 1,146 fly specimens that we were able to at least parasitism frequencies (although they were used to assess the relative tentatively identify to species (Table 1; some individuals were dam- importance of the different parasitoid species). aged in transit, frozen as pharate adults, or sent as pupae, which we To glean information about the host stages attacked by the dif- were unable to identify). The flies came from 466 monarch hosts ferent parasitoid species, we calculated 95% binomial probability whose stages at collection, when known, are illustrated in the fifth confidence intervals for the proportions of monarchs collected as bar in Fig. 1. We identified seven species of Tachinidae from mon- eggs and fifth instars that produced the four most common parasit- archs collected in 16 states ranging from to , al- oid species (sample sizes of the other parasitoid species were too though most records are concentrated in the Upper Midwest (Table small for these analyses). We then compared these intervals to the 2). Most (75%) parasitism events were due to Lespesia archippivora total proportion of reared monarchs collected at these two stages. If and this species was found across the country (Tables 1 and 2), only the total proportion of reared monarchs did not fall within these in- being absent from three states from which we received few speci- tervals, we considered them significantly different. This comparison mens (FL, NC, ME). Compsilura concinnata and Hyphantrophaga assumes that volunteers who sent us specimens collected monarchs virilis were each responsible for 10% or more of the parasitized in the same age proportions as all volunteers who reared monarchs; monarchs (Table 1). we cannot test this assumption because not all of the volunteers re- There were significantly more L. archippivora larvae per mon- ported all of the monarchs they reared, but have no reason to think arch (mean: 3.14) than the three other species reared from nine or that it is not true. We did not make the same comparisons with the more monarchs (Table 1, C. concinnata, H. virilis or Leschenaultia proportions of monarchs collected as other larval instars to limit the n. sp; F3,348 ¼ 12.5, P < 0.0001). Of the 466 monarchs from which number of comparisons, but we do note a case in which this propor- tachinid larvae emerged, nine exhibited multiparasitism (they pro- tion was very different from the total proportions of reared mon- duced more than one fly species), containing all three pairwise com- archs. Eggs and fifth instars were most interesting to us because 1) binations of the three most common parasitoid species (Table 3). of the rarity of tachinid attacks of monarchs collected as eggs, and There was no relationship between the proportional representation 2) disproportionately high parasitism of monarchs collected as fifth of any of the top three species and monarch density in the Upper instars suggests attacks across multiple life history stages Midwest in any given year (Table 4). There was, however, signifi- (Oberhauser 2012). cant year to year variation in the representation of different species We compared the numbers of individual parasitoids that (v2 ¼ 178, df ¼ 14, P < 0.0001 for numbers of the three most com- emerged from host caterpillars across the four most abundant spe- mon species across years), with L. archippivora, C. concinnata, and cies using an ANOVA with post hoc LSD pairwise comparisons. H. virilis ranging from 13% in 2005 to 91% in 2012, 2.5% in 2012 This comparison provides information on the relative success of to 61% in 2005, and 1.3% in 2011 to 33% in 2015, respectively. these species in monarchs. While caterpillars collected as fifth instars are most likely to be Finally, we hypothesized that generalist parasitoids (species parasitized (Fig. 2), the four most common tachinid species showed known to attack many different hosts) would be more likely to at- substantial variation in which host stages they appear to attack (Fig. tack monarchs during years of high monarch abundance. We used 1). No monarchs collected as eggs produced C. concinnata or L. mean peak egg densities (eggs per observed plants) from MLMP sites archippivora; these two zero proportions are significantly lower 4 Annals of the Entomological Society of America, 2017, Vol. 0, No. 0

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

0% Cc (n = 21) Hv (n = 46) Lesch ns (n = 9) La (n = 233) Coll stage of All reared para monarchs monarchs (n = 1998) (n = 20386) egg L1 L2 L3 L4 L5 pupa

Fig. 1. 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.

0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 Proporon parasized Proporon 0.02 0.00 egg L1 L2 L3 L4 L5 pupa (n=5734) (n=1723) (n=1143) (n=1182) (n=2971) (n=7544) (n=64)

Fig. 2. 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.

than the overall proportion of reared monarchs collected as eggs illus- monarchs collected as fifth instars was significantly higher than the trated in Fig. 1 (95% binomial confidence intervals ¼ 0–0.161 and overall proportion of monarchs collected during this stadium 0–0.016, respectively, vs. 28% collected as eggs). Over half of the C. (CI ¼ 0.487–0.619, vs. 37% collected as fifth instars). concinnata emerged from monarchs collected as fourth instars (Fig. 1); Hyphantrophaga virilis representation in monarchs collected as both the 95% binomial confidence interval for this proportion (post hoc cal- eggs and fifth instars was as expected based on the overall proportions culation) does not overlap with the proportion of monarchs collected of monarchs collected in these stages. Sample sizes for the other three as fourth instars (CI ¼ 0.306–0.732 vs. 15% collected as fourth in- species preclude conclusive statistical tests, but all specimens of both stars). Leschenaultia n. sp. representation in monarchs collected as eggs M. saundersii and N. erecta were reared from caterpillars collected as was significantly higher than expected despite the relatively small sam- fifth instars. The stage at collection of the three caterpillars from which plesize(CI¼ 0.400–0.972). The frequency of L. archippivora in Lespesia sp. were reared was not recorded by volunteers. Annals of the Entomological Society of America, 2017, Vol. 0, No. 0 5

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 6 S.E.

Compsilura concinnata (Cc) 69 45 0.098 1.74b 6 0.195 Hyphantrophaga virilis (Hv) 114 63 0.137 1.99b 6 0.227 Leschenaultia n. sp. (Lesch) 10 9 0.020 1.22b 6 0.222 Lespesia archippivora (La) 943 347 0.748 3.14a 6 0.113 Lespesia sp. (Lsp) 3 3 0.007 1 6 0 Madremyia saundersii (Ms) 5 3 0.007 2.33 6 0.882 Nilea erecta (Ne) 2 2 0.004 1.5 6 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 para- sitism events only with number of hosts > 8, values followed by different superscripts are significantly different).

Table 2. 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 (N¼366) 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 (N¼17) ME 1 x NY 1 x OH 1 x PA 14 x x x Middle East (N¼20) MD 13 x NC 7 x x Southwest (N¼18) AZ 3 x x CA 15 x South Central (N¼35) OK 2 x x TX 33 x x Southeast (N¼1) FL 1 x

For species abbreviations, see Table 1.

Table 3. Summary of nine multiparasitism events (from a total of fifth instars (Oberhauser et al. 2007), so monarchs that survive 466 monarch hosts) to pupation are less likely to be parasitized. The 17% value is similar to frequencies calculated previously from MLMP data Species present Flies in each multispecies event (Oberhauser et al. 2007, Oberhauser 2012, Nail et al. 2015), but L. archippivora and H. virilis 2 La 2Hv 1 La 1Hv lower than that found by Mueller and Baum (2014) (36%). It L. archippivora and C. concinnata 1La3Cc should be noted that Mueller and Baum collected all specimens 1La1Cc from roadside and prairie sites within 25 km of Stillwater, OK, 1La1Cc in a single year, whereas MLMP data cover broad temporal and 3La1Cc geographic ranges; annual frequencies from the MLMP range C. concinnata and H. virilis 3Cc1Hv from 3% to 38% (Nail et al. 2015) and the highest value was in 1Cc2Hv 2012, when Mueller and Baum collected their data. 1Cc3Hv As assumed by the above authors, L. archippivora was the most common and widespread tachinid parasitoid of monarchs. It was The rows in the right column list all of the tachinid flies that were reared from each monarch host. 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 Discussion it was not reared are all in the eastern United States, but low sample sizes from these states preclude strong conclusions about this pat- Overview tern. However, 25% of the parasitized hosts contained other species, Our survey confirms previous claims that tachinid flies are signif- with H. virilis (14%) and C. concinnata (10%) being the other two icant parasitoids of monarchs (Prysby 2004; Oberhauser et al. most common monarch parasitoids. Given the extremely low num- 2007, 2015; Oberhauser 2012; Mueller and Baum 2014; Nail ber of records (<1% of the total), N. erecta, M. saundersii, and et al. 2015), with an overall parasitism level of 17% for mon- Lespesia sp. should be considered incidental parasitoids of mon- archs collected during the final larval stadium. The fact that a archs, but for Leschenaultia n. sp. the relationship is unclear, with lower proportion of monarchs collected as pupae are parasitized low prevalence (2%) but still a substantial number of rearings probably reflects the fact that tachinid flies often emerge from (N ¼ 9). 6 Annals of the Entomological Society of America, 2017, Vol. 0, No. 0

Table 4. 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 00000.133 Nilea erecta 0 0 0.027 00000 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).

Of the nine parasitoid species listed for monarchs by Arnaud D. plexippus are distinct from specimens reared from Erebidae and (1978), we found four: C. concinnata, H. virilis (listed by Arnaud as Lasiocampidae (JOS personal observation), and year to year varia- Eusisyropa virilis), L. archippivora, and M. saundersii. We found tion in parasitism frequency of monarchs suggest that L. archippi- three species previously unreported from monarchs: Lespesia sp., vora tracks monarch population densities with a one year time lag Leschenaultia n. sp., and Nilea erecta. The five species listed in (Oberhauser 2012). Further morphological and genetic analysis is Arnaud that were not represented in our collection include Buquetia needed to determine if L. archippivora indeed represents multiple, obscura, Exorista mella, Lespesia schizurae, Phryxe pecosensis, and monophagous species and how many. To our knowledge, P. vulgaris. Leschenaultia has never previously been reared from monarchs. The We are not certain of the identity of the “Lespesia sp.” speci- fact that the species we found has not been reported in other hosts mens. They are clearly not L. archippivora, but neither do they suggests that it may be a specialist on monarchs or on milkweed as- match L. schizurae (a species previously recorded from monarchs). sociated caterpillars (see below). Their bright yellow-gold parafrontal and parafacial regions are simi- Hyphantrophaga virilis, M. saundersii, N. erecta, and C. concin- lar to those of L. flavifrons, but they do not match this species in nata are all highly polyphagous, attacking a wide variety of other respects. Lespesia is a difficult genus taxonomically, with Lepidoptera spanning at least 10 families each (Arnaud 1978, Smith many morphologically similar species that themselves may consist of et al. 2007). These species are likely to use relatively general cues to cryptic species complexes (e.g., Smith et al. 2007). Further taxo- locate caterpillar hosts, and may take advantage of their ability to nomic investigation of these specimens is in progress. The attack monarchs when monarch densities are high, although we Leschenaultia specimens fail to completely match (or key to) any found no evidence of this (Table 4). It is also possible that they at- recognized North American species, and we suspect that they may tack monarchs when other hosts are rare, but we cannot test this hy- represent an undescribed species. pothesis with available data. Host associations of the Lespesia sp. are unclear, as the genus contains both highly specialized and highly polyphagous species (Arnaud 1978). Species Characteristics Compsilura concinnata has the broadest host range of any All of the tachinid species recorded in our survey belong to the sub- known tachinid, having been recorded from >180 host species in family , representing three tribes: Blondeliini three orders (Arnaud 1978, Boettner et al. 2000). Incredibly, it was (Compsilura), (Lespesia, Madremya, and Nilea), and intentionally introduced into North America from Europe over (Hyphantrophaga and Leschenaultia). These genera possess much of the 20th century to control the gypsy moth Lymantria dis- a variety of reproductive strategies, including deposition of tiny par dispar L. (Lepidoptera: Erebidae) and other caterpillar pests “microtype” eggs on leaves that are eaten by the host (H. virilis and (Sanchez and Carde 1998). This species is of substantial environ- Leschenaultia sp.; Wood 1987), laying incubated eggs directly on mental concern due to its frequent use of nontarget species; it has the host that immediately hatch and bore into the host (Lespesia been implicated in the decline of wild silk moths (Saturniidae) in archippivora, L. sp., and M. saundersii; Stapel et al. 1997), and in- Eastern U.S. forests (Boettner et al. 2000; Kellogg et al. 2003; jecting eggs into hosts with an abdominal piercer (C. concinnata; Elkinton and Boettner 2004, 2012). The regularity of attacks by this O’Hara 1985). Although O’Hara (2005) inferred the reproductive intentionally introduced biocontrol agent on monarchs is disturbing, strategy of N. erecta to be direct deposition of incubated eggs from given current concerns about declining monarch numbers (e.g., its systematic placement in the Eryciini, Wiman and Jones (2013) re- Semmens et al. 2016). port 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 mo- Host Stage nophagous parasitoids of D. plexippus are L. archippivora and We can use associations between parasitoid species and monarch Leschenaultia sp. According to Arnaud (1978), L. archippivora uses stage at collection to infer the stages at which monarchs are vulnera- a wide range of lepidopteran hosts belonging to 15 different families ble to the different parasitoids. The increase in parasitism with lar- (and one family of Hymenoptera). However, preliminary morpho- val stadium at collection (Fig. 2) suggests that monarch larvae logical examination suggests that specimens reared from continue to be vulnerable to fly parasitoids as they develop Annals of the Entomological Society of America, 2017, Vol. 0, No. 0 7

(Oberhauser 2012). The most common parasitoid in our study, L. than direct interference. This latter conclusion is also supported by archippivora, was never found in monarchs collected as eggs, in the observation that all of these species are at least facultatively gre- keeping with its direct deposition of eggs on host larvae, and was garious. Sample sizes of multiparastism are too low for robust statis- overrepresented in monarchs collected as fifth instars, supporting tical analysis, but the relatively frequent involvement of C. the previous conclusion that this parasitoid continues to attack mon- concinnata, particularly with H. virilis, supports our conjecture that arch larvae throughout their development (Etchegary and Nishida it may often attack relatively later than the other parasitoids (in- 1975a,b; Prysby 2004; Oberhauser 2012; also see Stapel et al. creasing the chance of multiparasitism) or that parasitized larvae 1997). Compsilura concinnata was never found in monarchs col- may be more vulnerable to attack by this species. lected as eggs or first instars; this species injects eggs into the host larva, and the eggs hatch immediately and migrate to the host mid- Variability Over Space and Time gut (Ichiki and Shima 2003, O’Hara 2005). Our results suggest that Tachinid fly parasitism is variable across years (Oberhauser 2012, it attacks monarchs older than first instars. Its overrepresentation in Nail et al. 2015), but the mechanisms driving this variability require hosts collected as fourth instars suggests that it often emerges from further study. Our finding of substantial parasitism by species other fourth instars or early fifth instars (so parasitized hosts are less likely than L. archippivora, the variable representation of L. archippivora to survive to be collected as fifth instars), although sample sizes are in the annual monarch parasitoid pool, and the fact that many of too small to draw strong conclusions. the parasitoids are generalists whose population dynamics will be Representation of H. virilis across monarch developmental driven by numbers of other species will complicate our ability to un- stages does not deviate from expectations based on the overall pro- derstand these mechanisms. portions of monarchs collected in these stages; H. virilis takes ad- Nearly all of the identified tachinid species reared from mon- vantage of its opportunistic parasitism to infest caterpillars of archs in this study have broad distributions, ranging over much of several Lepidoptera families (Arnaud 1978, O’Hara 2005). A sub- North America, coast to coast and from to . An ex- stantial fraction of parasitism events were from monarchs collected ception is C. concinnata, which was introduced and established as eggs. Because there is no evidence that any tachinid flies attack along both East and West coasts of North America and in the host eggs, this finding indicates that milkweed foliage “infected” Midwestern United States (Sanchez and Carde 1998), and has been with the microtype eggs of H. virilis was introduced by MLMP vol- slowly spreading from these introduction areas (Elkinton and unteers, either on the leaf on which the egg was collected or subse- Boettner 2004, 2012). It is largely absent from the (interior) south- quent leaves collected for feeding. This makes it difficult to ascertain ern half of the United States and the Rocky Mountain states and the host stage at which H. virilis typically attacks, as parasitoid eggs provinces. Our record from Texas represents a dramatic extension could be introduced at any time during rearing and larvae typically of its known range. Iowa also appears to be a new state record. Our emerge from the pupal stage of the host (Sellers 1930). However, limited records of the apparently undescribed Leschenaultia sp. sug- the lack of deviation from overall proportions of monarchs collected gest it has a broad geographic range over much of the eastern United across developmental stages suggests that they are attacked early in States. Sample sizes are insufficient to assess whether parasitoid their development by this parasitoid; continued attack would result community composition and parasitism frequencies vary signifi- in increasing proportions for monarchs collected at later stages. cantly across geography. We only know the stage of collection for nine monarchs that pro- In conclusion, the large temporal and spatial scales of this rear- duced Leschenaultia n. sp., but seven of these were collected as eggs, ing study allowed us to document new monarch parasitoids. While a significantly higher proportion than the overall egg collection. we had previously used MLMP data to show year to year variation This is consistent with their deposition of microtype eggs on foliage in parasitism frequencies, obtaining the reared specimens allowed us with feeding larvae, which then ingest the eggs (Mondor and Roland to document variation in the relative importance of different species. 1998). Again, the only explanation for the high representation in Our findings further illustrate the value of engaging citizen scientists monarchs collected as eggs is that parasitoid eggs were introduced in natural history research; indeed, a large proportion of all pub- with milkweed leaves supplied to the developing caterpillars. The lished research on monarch ecology utilizes citizen science data low number emerging from monarchs collected at other stages sug- (Ries and Oberhauser 2015, MonarchNet 2016). gests that monarchs may not be a typical host for this parasitoid spe- cies, i.e., it may be targeting other milkweed feeding hosts such as Eucheates egle (Erebidae). Although capable of developing in mon- archs, the low numbers of hosts collected at other stages that pro- Acknowledgments duced this parasitoid could indicate premature mortality of the We thank the hundreds of MLMP volunteers who have contributed to this monarch host (as with C. concinnata, some infected hosts may not project, especially Diane Rock, Betsy Johnson, Linda Rippert, Brenda Keith, survive to be collected as larvae, or die in captivity before the fly lar- and Annie Letaw, who, in addition to I.G. sent in the vast majority of the flies vae emerge). However, our sample size for monarchs parasitized by that we identified; and Monarch Lab students and staff for their help in devel- Leschenaultia n. sp. is too low to draw strong conclusions. oping protocols and communicating with volunteers. This research was sup- ported 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 Multiparasitism Foundation DEB 1442134. An earlier version of the manuscript benefited Multiparasitism (in which more than one parasitoid species emerges from the comments of two anonymous reviewers. 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 References Cited generally lack the ability to discriminate among parasitized and Aldrich, J. M., and R. T. Webber. 1924. The North American species of para- unparasitized hosts (Feener and Brown 1997, Kan et al. 2003) and sitic two-winged flies belonging to the genus Phorocera and allied genera. that competition in this community is exploitative in nature, rather Proc. U. S. Natl. Mus. 63: 1–90. 8 Annals of the Entomological Society of America, 2017, Vol. 0, No. 0

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