Lespesia archippivora (Diptera: Tachinidae) Survival and Sex Ratios within Monarch Butterfly (Lepidoptera: Nymphalidae) Hosts.
The tachinid fly Lespesia archippivora (Riley) (Diptera: Tachinidae) parasitizes a wide range of lepidopteran species (Arnaud, 1978). Monarch butterflies (Danaus plexippus) (L.) (Lepidoptera: Nymphalidae) are preferred hosts of L. archippivora and parasitism is an important source of mortality for monarch larvae and pupae (Schaffner and Griswold, 1934; Oberhauser et al., 2007; Oberhauser, 2012; Oberhauser et al., 2017). Female L. archippivora lay eggs oil the outer integument of caterpillars (Belshaw, 1994). After hatching the fly maggots penetrate the host larva's integument, where they grow and develop (Etchegaray and Nishida, 1975). Once developed, maggots sever the nervous system, paralyzing and killing the larva, and exit the carcass on silken strands to the leaf litter below to pupate (Etchegaray and Nishida, 1975).
Female flies may produce dozens or even hundreds of eggs within a lifetime, but only lay 1-10 eggs per monarch larva (Etchegaray and Nishida, 1975). Parasitism by multiple females can result in up to 20 eggs within a single monarch, but this number typically results in maggots dying within the host (Etchegaray and Nishida, 1975; Belshaw, 1994). Parasitism rates are known to vary among monarch host populations, yet the factors influencing the success of the parasitoid itself have been studied in only a few populations. To better understand what factors are associated with variation in parasitoid success, we examined whether tachinid survival, growth, or sex ratio changed as the number of larvae parasitizing caterpillars increased. We also documented the seasonal pattern of parasitism on monarchs and asked whether fly growth and survival changed over the course of a season. Determining how these factors are associated with the success of L. archippivora is an important step in understanding the mechanisms driving variation in parasitism of monarchs.
We collected 419 late instar monarch larvae from milkweed gardens and prairies within the Omaha, Nebraska metropolitan area (41[degrees]15'N 96[degrees]0'W) in 2016 and 2017 (Geest et al., 2019). Larvae were reared in the lab and checked daily for signs of parasitism. Parasitoids that emerged were allowed to pupate ill the rearing container. If a monarch larva died, we waited 24 h to see if parasitoids emerged; if none emerged, we dissected the larva to determine if parasitoids were present (Oberhauser et al., 2007). Fly larvae found upon dissection were allowed to pupate normally (three cases). We defined the number of larvae emerging from a caterpillar as the brood size, although this may underestimate the number eggs laid by female tachinids given some eggs may fail to hatch and some larvae may die at early stages within the caterpillar. Newly emerged adult flies were collected and identified using existing morphological keys for Nearctic tachinids (Wood, 1987; O'Hara, 2013). We measured the fresh mass of each adult to 1 nig, and both and wing length in mm, immediately upon specimen collection. Sex of adult flies was determined by the presence of two proclinate orbital setae on each side of the head between the frontal setae and eye in females (McAlpine, 1981; J. E. O'Hara, pers. comm.). Summary values are listed as mean [+ or -] se.
RESULTS AND DISCUSSION
In total 424 L. archippivora flies were collected from 124 parasitized monarch larvae. The majority of flies emerged during the monarch's 5th instar (93 broods consisting of 358 flies). The remainder emerged during either the monarch 4th instar (eight flies from four broods) or chrysalis (56 flies from 27 broods). The number of flies emerging from parasitized monarch larvae, hereafter brood size, ranged from one to 10 flies, with most caterpillars producing one to four flies (Table 1). Brood size was largest for 5th instar monarch larvae compared to 4th instar or chrysalis stage (ANOVA [F.sub.2,121] = 6.774, P = 0.002; mean brood size from instar 4 = 2.00 [+ or -] 1.13, instar 5 = 3.85 [+ or -] 0.24, chrysalis = 2.15 [+ or -] 0.43). Survival, measured as the proportion of each brood that successfully pupated, was significantly higher for flies emerging during the monarch's 5th instar (0.845 [+ or -] 0.036) compared to those emerging earlier (0.250 [+ or -] 0.174) or later (0.664 [+ or -] 0.670) in the monarch life-cycle (ANOVA [F.sub.2,121] = 7.717, P < 0.001). We analyzed the proportion of each brood that successfully pupated to emerge as adults as a function of brood size with ANCOVA, including year and day as covariates (overall [F.sub.3,120] = 4.449, P = 0.005). Survival increased with increasing brood size ([beta] = 0.033 [+ or -] 0.014, P = 0.016). There was also a significant year effect with survival being higher in 2016 than 2017 ([beta] = 0.092 [+ or -] 0.039, P = 0.020). A pattern of decreasing survival across days in the season was not significant ([beta] = -0.002 [+ or -] 0.001, P = 0.051).
We analyzed variation in the mass and size of adult flies (Table 1) relative to sex, brood size, date, and year using ANCOVA. The overall model for mass was significant ([F.sub.4,346] = 13.38. P < 0.001) with females being heavier than males ([beta] = 0.714 [+ or -] 0.245, P = 0.004) and flies from larger broods being lighter ([beta] = -0.335 [+ or -] 0.091, P < 0.001). Mass increased across the season ([beta] = 0.0.029 [+ or -] 0.007, P < 0.001) and was greater in 2017 than 2016 ([beta] =-1.046 [+ or -] 0.285, P < 0.001). Similar effects of sex, brood size, and day were seen for wing length (overall [F.sub.4,346] = 7.59, P < 0.001; sex [beta] = 0.179 [+ or -] 0.071, P = 0.012; brood size [beta] =-0.110 [+ or -] 0.026, P < 0.001; day [beta] =-0.007 [+ or -] 0.002, P < 0.001), although there was not a significant year effect (P = 0.251). For body length, only brood size had a significant effect (overall [F.sub.4,346] = 10.21, P < 0.001; brood size [beta]=-0.151 [+ or -] 0.026, P < 0.001; day P = 0.222, year P = 0.840), although the effect of sex approached significance ([beta] = 0.138 [+ or -] 0.071, P = 0.053).
The smaller size of each fly from larger broods did not entirely compensate for the greater total number of flies, as the total mass of adult flies produced by a caterpillar (brood mass) increased with brood size (Table 1). This positive relationship between brood size and mass was significant when controlling for year, day, and the proportion of females in each brood (overall [F.sub.4,101] = 19.48, P < 0.001; brood size ([beta] = 4.508 [+ or -] 0.571, P < 0.001; day ([beta] = 0.106 [+ or -] 0.040, P = 0.009, year ([beta] = - 3.997 [+ or -] 1.704, P = 0.021; proportion female ([beta] = 7.965 [+ or -] 3.830, P = 0.040).
The sex ratio of L. archippivora was biased towards males, with 222 males and 129 females emerging as adults (six adults were damaged before they could be sexed). Our measured 63.2% male to 36.8% female ratio differs significantly from 50:50 (binomial test, Z = 4.911, P < 0.001). Variation in the proportion of females in a brood was not explained by the combination of brood size, year, and day (overall [F.sub.3,102] = 0.397, P = 0.756).
In our study we found peak time periods for I., archippivora in early and late summer (Fig. 1). Overall, 25.4 % of monarchs collected in 2016 were parasitized and 32.8 % were parasitized in 2017 (Geest et al., 2019). We collected parasitized monarch larvae between 16 May and 14 June and again from 15 July to 3 September. During the 15 June to 14 July period, 37 monarch larvae were collected, but none was parasitized. This period corresponds to a low point in monarch reproduction between generations; however, based on the overall parasitism rates we observed (Geest et al., 2019), we would still expect eight to 12 of the monarchs to produce L. archippivora larvae. This pattern is consistent with a bivoltine life cycle with a gap between generations occurring from mid-June to mid-July, as suggested by historical records that show L. archippivora exhibiting two or three generations per year, with an early generation in May and a second generation in late summer to fall (Schaffner and Griswold, 1934).
All of the parasitoids that successfully pupated were identified as L. archippivora based on the current morphological keys we employed (Wood, 1987; O'Hara, 2013). Most previous studies of tachinid parasitism have similarly attributed parasitism to L. archippivora (e.g., Oberhauser et al., 2007). There are other tachinids known to parasitize monarchs, though L, archippivora is the most widespread and most common (Oberhauser et al. 2017). However, the other species of tachinids found by Oberhauser et al., (2017) were primarily from states south or east of our study area. While they did not collect from Nebraska, the only other tachinid noted from the neighboring stales was Compsilura concinnata (Meigen) (Diptera: Tachinidae). That said, we cannot rule out the possibility some of the parasitoids that died as pupae and were unidentified could represent other species, or that ciyptic species not distinguished by the keys we employed may have been present.
The range from 1 to 10 in brood size we found (Table I) is consistent with previous studies, although our mean brood size (3.42 [+ or -] 0.21. median = 3) was slightly higher than those reported previously (range 2.4 to 3.1, Etchegaray and Nishida, 1975; Mueller and Baum, 2014; Oberhauser et al., 2007; Oberhauser et al., 2017). Fly survival through the pupal stage did not suffer as a result of larger brood sizes; in fact, the proportion of L. archippivora surviving to adulthood increased as more parasitoids shared a host, in contrast to the pattern observed in some other parasitoids (e.g., Charnov and Skinner, 1984). However, flies from larger broods were smaller and lighter, which likely has a negative impact on female fecundity and longevity of adults (Stapel et al, 1997; Sternberg et al, 2011). Why the survival of flies in larger broods does not decline, but actually increases, is unclear. Female tachinids may adjust the number of eggs they lay based on caterpillar size (Stapel et al, 1997). If fly larvae are sharing larger caterpillars, competition may be minimized, despite the larger brood mass associated with larger broods. Further complications might occur if parasitized monarchs respond by "self-medicating" through dietary (Singer et al. 2009; Smilanich et al, 2011) or other behavioral changes (e.g., Karban, 1998). For monarchs there is evidence that differences in cardenolide levels among Asclepias host plants might impact parasitism (Oberhauser et al, 2007). Finally, the presence of the protozoan parasite, Ophryocystis elektroscirrha, may increase the chances that monarch larvae resist L. archippivora parasitism, and highlights the fact that some flies die prior to emerging (Sternberg et al, 201 1).
Parasitism of monarchs by L. archippivora is widespread and may be especially important for the migratory generation of monarchs produced in late summer. Our results suggest that L. archippivora incur no survival cost by engaging in multiple parasitism of monarch larvae, which likely helps ensure the population of flies is large enough to parasitize the late-summer generation of monarchs. That said, multiply parasitized caterpillars still hosted a greater mass of fly larvae, which likely drives the decrease in size and mass of flies emerging as brood size increases. This may also play a role in the male-biases sex ratio observed, as life history theory assumes a male bias for parasites with limited host resources that result in smaller individuals (e.g. Charnov et al, 1981). Regardless of the impact on individual flies, the successful ability of L. archippivora to parasitize caterpillars multiply accentuates its potential to decrease the survival of monarch caterpillars critical for producing the generation of butterflies that overwinter and are responsible for initiating the subsequent year's population. This negative impact on future generations of butterflies underscores the importance for monarch conservation of understanding factors that contribute to variation in the ability of L. archippivora lo successfully parasitize their hosts.
Acknowledgments.--Funding was provided by the University of Nebraska Omaha Department of Biology, Office of Research and Creative Activity, and Office of Graduate Studies, and Prairie Biotic Research, Inc. Small Grants Program. Thanks to Ted Burk for comments on earlier drafts of this paper and to James O'Hara for his guidance on identifying and sexing L. archippivora. Additional thanks to Jeff Dietz, Lindsay Brown, Devin Christensen, Isabella Lombardo, Elizabeth Kamtz, Caylynn Cruse, and Mia Siebrasse for their assistance.
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EMILY A. GEEST (1), L. LAREESA WOLFENBARGER (2), and JOHN P. MCCARTY (3), Department of Biology, University of Nebraska Omaha, Omaha 68182. Submitted 7 March 2019; Accepted 21 June 2019
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TABLE 1.--The number of Lespesia archippivam larvae emerging from caterpillars or brood size ranged from 1 to 10. Brood survival is measured as the proportion of larvae emerging from each host that survive to adulthood. Mean ([+ or -] SE) body length, wing length, and fresh mass of each fly, as well as the sum of the masses of all flies from each brood vary with brood size. Numbers of males and females include all individuals of known sex. Mean % Female values are the mean percentage of females among broods and are based on subset of broods consisting of at least two individuals of known sex. Because the number of large broods is relatively small, the final row ([greater than or equal to] 5) summarizes the data for broods of 5 to 10 larvae to illustrate the differences between larger and smaller broods, however, analyses described in the text used all 10 brood sizes Body Brood N Brood length size broods survival (mm) 1 29 0.66 [+ or -] 0.09 7.17 [+ or -] 0.29 2 25 0.76 [+ or -] 0.08 6.46 [+ or -] 0.20 3 24 0.82 [+ or -] 0.07 6.41 [+ or -] 0.16 4 17 0.79 [+ or -] 0.09 6.74 [+ or -] 0.17 5 6 0.83 [+ or -] 0.13 6.06 [+ or -] 0.25 6 4 0.96 [+ or -] 0.04 6.28 [+ or -] 0.26 7 9 0.95 [+ or -] 0.02 5.75 [+ or -] 0.16 8 5 0.83 [+ or -] 0.11 5.62 [+ or -] 0.22 9 2 0.89 [+ or -] 0.11 5.05 [+ or -] 0.32 10 3 1.00 [+ or -] 0.00 5.94 [+ or -] 0.24 [greater than 29 0.91 [+ or -] 0.04 5.81 [+ or -] 0.09 or equal to] 5 Wing Fly Brood N length mass size broods (mm) (mg) 1 29 5.42 [+ or -] 0.30 10 [+ or -] 1 2 25 4.80 [+ or -] 0.21 9 [+ or -] 1 3 24 4.73 [+ or -] 0.17 8 [+ or -] 1 4 17 4.85 [+ or -] 0.18 7 [+ or -] 1 5 6 4.56 [+ or -] 0.27 6 [+ or -] 1 6 4 4.65 [+ or -] 0.27 9 [+ or -] I 7 9 4.39 [+ or -] 0.17 5 [+ or -] I 8 5 4.22 [+ or -] 0.23 5 [+ or -] 1 9 2 4.20 [+ or -] 0.33 4 [+ or -] 1 10 3 4.43 [+ or -] 0.24 7 [+ or -] 1 [greater than 29 4.41 [+ or -] 0.09 6 [+ or -] 1 or equal to] 5 Brood Brood N mass Number size broods (mg) of [female] 1 29 10 [+ or -] 3 7 2 25 16 [+ or -] 3 9 3 24 22 [+ or -] 3 24 4 17 24 [+ or -] 4 15 5 6 23 [+ or -] 6 12 6 4 51 [+ or -] 7 13 7 9 32 [+ or -] 5 23 8 5 34 [+ or -] 6 9 9 2 33 [+ or -] 10 6 10 3 72 [+ or -] 8 11 [greater than 29 37 [+ or -] 3 74 or equal to] 5 Brood N Number size broods of [male] Mean % [female] 1 29 12 2 25 29 23.5 [+ or -] 8.7 3 24 33 41.2 [+ or -] 8.7 4 17 39 26.8 [+ or -] 6.7 5 6 13 50.0 [+ or -] 14.8 6 4 10 56.7 [+ or -] 4.1 7 9 35 38.5 [+ or -] 5.7 8 5 22 25.8 [+ or -] 10.2 9 2 10 36.5 [+ or -] 7.9 10 3 19 36.7 [+ or -] 8.8 [greater than 29 109 40.5 [+ or -] 4.0 or equal to] 5 FIG. 1.--Changes in occurrence of Lespesia archippivora in monarch larvae (Danaus plexippus) over the course of a season. Over the course of two years, 418 monarch larvae were reared in the lab and screened for parasitism. The total bar height represents the number of monarch larvae collected during 5 d periods with the dark portion of the bars indicating the monarchs parasitized by L. archippivora, with the overall percentage of caterpillars parasitized during each period shown above each bar 12 May 0% 19 May 65% 26 May 50% 02 Jun 0% 09 Jun 56% 16 Jun 0% 23 Jun 0% 30 Jun 0% 07 Jul 0% 14 Jul 27% 21 Jul 11% 28 Jul 10% 04 Aug 29% 11 Aug 19% 18 Aug 46% 25 Aug 29% 01 Sep 50% Note: Table made from bar graph.
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|Title Annotation:||Notes and Discussion Piece|
|Author:||Geest, Emily A.; Wolfenbarger, L. Lareesa; McCarty, John P.|
|Publication:||The American Midland Naturalist|
|Date:||Oct 1, 2019|
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