JKES Journal of the Kansas Entomological Society (Central States Entomological Society)
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Protandry of Western Corn Rootworm (Coleoptera: Chrysomelidae) Beetle Emergence Partially Due to Earlier Egg Hatch of Males DALTON C. LUDWICK,1 ANTHONY ZUKOFF,1,2 MATT HIGDON,3
AND
BRUCE E. HIBBARD3,4
JOURNAL OF THE KANSAS ENTOMOLOGICAL SOCIETY 90(2), 2017, pp. 94–99
Protandry of Western Corn Rootworm (Coleoptera: Chrysomelidae) Beetle Emergence Partially Due to Earlier Egg Hatch of Males DALTON C. LUDWICK,1 ANTHONY ZUKOFF,1,2 MATT HIGDON,3
AND
BRUCE E. HIBBARD3,4
ABSTRACT: The western corn rootworm, Diabrotica virgifera virgifera LeConte, exhibits protandry. The contribution of pre-hatch development to protandry in western corn rootworm was previously investigated with a small set of data from one population. To verify the contribution of pre-hatch development to protandry, more than 10,000 larvae from seven wild-type populations collected from across the Corn Belt were evaluated. Larvae were placed into containers on each day of egg hatch for each population and allowed to develop into adults. Duration of hatch for these populations ranged from 7 to 19 days with the percent of insects surviving to adulthood varying between 13 and 52%. For the first 25% of egg hatch, significantly more males emerge. Pre-hatch development does contribute to protandry, but overall the contribution is likely less than post-hatch development. KEY WORDS: Diabrotica virgifera virgifera, protandry, post-hatch development
Western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a pest of corn native to North America. Since its discovery, the range of this species has expanded across the United States of America and into Europe. In the USA alone, this pest is estimated to cause more than one billion dollars in economic losses annually (Metcalf 1986). Management strategies are becoming increasingly limited as WCR had developed resistance to many management tactics (Ball and Weekman, 1962; Meinke et al., 1998; Pereira et al., 2015; Levine et al., 2002; Gassmann et al., 2011; Zukoff et al., 2016). Several recent reviews have been written (Gray et al., 2009; Spencer et al., 2009; Meinke et al., 2009; Miller et al., 2009), but as always, understanding more about the biology and confirming the basic assumptions about this species may lead to better management strategies in the future. WCR males have evolved a strategy to increase access to females (Branson, 1987; Wiklund and Fagerstrom, 1977). Adult males emerge earlier than females, a phenomenon known as protandry, thereby increasing access to the number of virgin females (Wiklund and Fagerstrom, 1977). Peak male emergence is always prior to peak female emergence, but the extent of this has varied from several days to more than a week. There are two modes of development by which protandry can occur; sometimes both modes occur concurrently and create a larger difference in emergence between males and females than if just one mode was utilized. The first mode, pre-hatch development, is the developmental rate of embryos prior to eclosion. The second mode, post-hatch development, has been thoroughly documented for WCR (Jackson and Elliot, 1988). Posthatch development leads to protandry through the quicker development of males compared to females during the larval stages. Research by Branson (1987) documented a contribution
1
Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211 Southwest Research Extension Center, Kansas State University, Garden City, Kansas 3 USDA-ARS, 205 Curtis Hall, University of Missouri, Columbia, Missouri 65211 4 Corresponding author:
[email protected] This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or recommendation for its use by the USDA or the University of Missouri USDA and University of Missouri are equal opportunity providers and employers. 2
Received 31 March 2017; Accepted 2 October 2017 C 2017 Kansas Entomological Society
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of pre-hatch development to protandry in WCR by placing 76 larvae from one population on seedling corn. Here, multiple populations from across the Corn Belt were evaluated for the contribution of pre-hatch development to protandry. Materials and Methods Insect Rearing This study was conducted 2004–2006. Eggs used in the study were obtained from wild WCR collected from seven different Corn Belt locations by French Agricultural Research, Lamberton, Minnesota. After oviposition, French Agricultural Research stored the eggs at room temperature for 2 weeks, cooled to 15◦ C for two weeks, and then in a refrigerator at 7–8◦ C for several months to synchronize hatch (Fisher, 1989). Eggs were then removed from the refrigerator, shipped to Missouri, and resulting larvae were reared using methods similar to Meihls et al. (2011). Briefly, containers of soil and eggs were watched closely for initial hatch and once egg hatch began, up to 100 neonates per day from each location were transferred to 15 × 10 cm plastic containers (708 ml; The Glad Products Company, Oakland, CA). Present in each plastic container was sterilized growth medium and ∼15 grams of three to five day old germinated corn. When fewer than 100 neonates were available for a population on a given day of hatch, all available neonate larvae were transferred to a container with soil and corn. The daily transfer of neonates to plastic containers continued until hatching had ceased for each population. Infested corn developed in growth chambers (I-36LL, Percival Scientific, Perry, IA) under 14:10 (L:D) h at a temperature of 25◦ C and was watered as needed. After 14 days, corn plants were cut off at the soil surface, and the remaining contents inverted into a 33 × 19 cm container (5.7 liters; Sterilite Corporation, Clinton, SC) filled with new growth medium and germinated corn to allow larvae to complete development and pupate. Due to limited growth chamber space, the secondary containers were held in the greenhouse. Temperatures targeted 25◦ C with a standard error around this mean of ∼2 o C, with occasional temperature spikes or cooler temperatures, depending on outside temperatures at the time. Lighting targeted 14:10 light dark cycle and was subsidized with growth lights (Catalog No. 63599-1, RUUD Lighting, Racine, WI) as needed. Ten days after the smaller container was transferred, a fine mesh screen was placed over the larger container to prevent escape of adults. Adults were collected from their containers on a daily basis and stored in ethanol until no adults emerged for a period of two weeks. The number of males and females which emerged from each container was recorded.
Statistical Analysis The days on which neonate larvae hatched were divided into categories of hatch duration by increments of 6.25% due to differences in overall hatch duration between locations. These increments are hereafter referred to as hatch categories (Table 2). For example, if a larva hatched on day 1 of 15, then the first day of hatch would fall below 6.25% of hatch duration and would be assigned to category 1, whereas an insect which hatched on day 1 of 7 would be assigned to category 3 (i.e., 12.51–18.75%). This division of hatch duration created 16 categories. Analysis was done by comparing the sex ratio data for each hatch category against a 50:50 sex ratio in a one-tailed t-test at an alpha of 0.05. All replications for each hatch category were given equal weight regardless of the number of adults. If the number of
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Table 1. Hatch duration, number of larvae used, and total number of adults obtained from eggs collected from 7 locations. Hatch
Neonate
Containers
Total
Survival to
Location
duration (d)
larvae used
used
adults
adults (%)
MN Dodge City, KS Concordia, KS Columbia, MO* Caledonia, MN Janesville, WI Seneca, KS Summary
18 8 15 19 7 12 18
1,435 875 1,080 900 1,800 2,050 1,935 10,075
18 8 14 18 18 21 22 119
612 153 214 120 951 407 361 2818
42.65 17.49 19.81 13.33 52.83 19.85 18.66 27.97
* Insects originated from Dodge City, KS, wild-type insects. The progeny of these wild-type insects were reared in a field experiment in Columbia, MO. Due to rearing in Columbia, MO, fields before being brought into the greenhouse, these insects were considered to be a separate location.
replications for a hatch category was three or fewer, the lesser replicated categories were combined with the next category in order to obtain greater statistical power. Results A total of 10,075 neonate larvae were used in the study and 2,818 adults (1,466 males and 1,352 females) were collected from the containers. The duration of eclosion varied by location, lasting between 7 and 19 days (Table 1). Egg to adult survival was between 13.3 and 52.8% (Table 1). Significantly more males than females emerged during the first to fourth hatch categories (Table 2, Fig. 1). Male emergence was significantly less than female emergence during the tenth and twelfth hatch categories. The sex ratio was not significantly different for the other 12 hatch categories (Fig. 1). Table 2. Average number of males and females ± standard error of the mean and total number of adults emerging within each hatch category. Percent of
Hatch
Avg. male
Avg. female
Total
No. of
hatch duration
category
( ± SEM)
( ± SEM)
no. of adults
replications
00.00–6.25 06.26–12.50 12.51–18.75 18.76–25.00 25.01–31.25 31.26–37.50 37.51–43.75 43.76–50.00 50.01–56.25 56.26–62.50 62.51–68.75 68.67–75.00 75.01–87.50* 87.51–100*
1 2 3 4 5 6 7 8 9 10 11 12 13 15
33 121 198 144 261 124 443 167 49 231 185 267 269 326
4 6 7 7 8 6 11 10 4 10 6 9 9 13
*
6.3 15.6 23.6 14.2 19.9 13.0 19.1 5.8 5.3 7.3 12.8 11.3 17.1 4.1
± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.2 8.0 7.0 4.4 6.3 6.1 4.2 2.0 2.3 2.9 9.3 5.0 7.3 4.1
2.0 2.7 4.7 6.9 14.9 7.7 22.6 10.9 7.0 15.8 18.0 18.3 12.8 11.5
± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.0 1.3 2.7 3.2 3.7 2.5 8.1 3.3 2.5 6.3 6.1 5.2 4.5 3.3
Sections of hatch duration combined due to low replication within sections.
Fig. 1. Percentage of male emergence for each hatch category. Number of males were divided by the total number of adults to emerge within each hatch category for each replication of each population and multiplied by 100 to calculate a percentage. Then, these percentages were added together and divided by the number of replications to achieve an average male emergence for each hatch category. Bars represent standard error of the mean. Values with an asterisk above are significantly greater than 50%while values with an asterisk below are significantly less than 50%. A one-tailed t-test was conducted to determine statistical significance with an alpha of 0.05. The dashed line represents an even sex ratio.
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Discussion Prior studies of protandry by WCR reported that males emerged earlier than females by several days (Branson, 1987; Quiring and Timmins, 1990; Jackson and Elliot, 1988). Most of the previous experiments considered only post-hatch development’s contribution to protandry or the combined effects of both post-hatch and pre-hatch effects. This experiment focused solely on the contribution of pre-hatch development to protandry. Because each replication was given equal weight regardless of the number of adults, Fig. 1 and Table 2 do not always correlate. For example, while the first hatch category had an average of 6.3 males and 2.0 females (Table 2), three of the four replications had zero females and the fourth replication was 33% female, resulting 8.33% female (91.67% male) when averaged over the four replications (Fig. 1). Branson (1987), reported larvae from the first day of hatch was 75% male, but only evaluated 1 population. Here, males were significantly more likely to emerge than females during the first to fourth hatch categories (Fig. 1). Females were only significantly more likely to emerge when larvae from the tenth and twelfth hatch categories were placed on corn (Fig. 1). Male emergence was significantly greater than 50% for the first four categories. Pre-hatch development clearly contributes to protandry. However, when emerging WCR adults are observed in the field, males tend to dominate the landscape for at least three days (Quiring and Timmins, 1990). Both pre-hatch and post-hatch development each contribute to protandry, but post-hatch development likely contributes to a greater extent given final emergence curves in the field. Acknowledgments We thank Dr. Isaac Oyediran for his confirmation of beetle sex. We also thank French Agricultural Research, Lamberton, Minnesota for sending us various wild-type populations. Lastly, we thank two anonymous reviewers for suggestions that ultimately improved this manuscript. Literature Cited Ball, H. J., and G. T. Weekman. 1962. Insecticide resistance in the adult western corn rootworm in Nebraska. Journal of Economic Entomology 55:439–441. Branson, T. F. 1987. The contribution of prehatch and posthatch development to protandry in the chrysomelid, Diabrotica virgifera virgifera. Entomologia Experimentalis et Applicata 43:205–208. Fisher, J. R. 1989. Hatch of Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) eggs exposed to two different overwintering and hatch regimes. J. Kans. Entomol. Soc. 62:607–610. Gassmann, A. J., J. L. Petzold-Maxwell, R. S., Keweshan, and M. W. Dunbar. 2011. Field-evolved resistance to Bt maize by western corn rootworm. PLoS ONE. https://doi.org/10.1371/journal.pone.0022629. Gray, M. E., T. W. Sappington, N. J. Miller, J. Moeser, and M. O. Bohn. 2009. Adaptation and invasiveness of western corn rootworm: Intensifying research on a worsening pest. Annu. Rev. Entomol. 2009. 54:303–321. Jackson, J.J., and N.C. Elliot. 1988. Temperature-dependent development of immature stages of the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera:Chrysomelidae). Environmental Entomology 17:166–171. Levine, E., J.L. Spencer, S.A. Isard, D.W. Onstad, and M.E. Gray. 2002. Adaptation of the western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), to crop rotation: evolution of a new strain in response to a cultural management practice. American Entomologist 48:94–107. Meihls L.N., Higdon M.L., Ellersieck M.R., and B.E. Hibbard. 2011. Selection for resistance to mCry3Aexpressing transgenic corn in western corn rootworm. Journal of Economic Entomology 104:1045–1054. Meinke, L.J., B.D. Siegfried, R.J. Wright, and L.D. Chandler. 1998. Adult susceptibility of Nebraska western corn rootworm (Coleoptera: Chrysomelidae) populations to selected insecticides. Journal of Economic Entomology 91:594–600.
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Meinke, L. J., T. W. Sappington, D. W. Onstad, T. Guillemaud, N. J. Miller, J. Kom´aromi, N. Levay, L. Furlan, J. Kiss, and F. Toth. 2009. Recent research on the western corn rootworm: Western corn rootworm (Diabrotica virgifera virgifera LeConte) population dynamics. Ag. and Forest Entomol. 11:29–46. Metcalf, R. 1986. Foreword, pp. vii–xv. In Methods for the study of pest Diabrotica. Springer, New York, NY. Miller, N. J., T. Guillemaud, R. Giordano, B. D. Siegfried, M. E. Gray, L. J. Meinke, and T. W. Sappington. 2009. Recent research on the western corn rootworm: Genes, gene flow and adaptation of Diabrotica virgifera virgifera. Ag. and Forest Entomol. 11:47–60. Pereira, A. E., H. Wang, S. N. Zukoff, L. J. Meinke, B. W. French, and B. D. Siegfried. 2015. Evidence of field-evolved resistance to bifenthrin in western corn rootworm (Diabrotica virgifera virgifera LeConte) populations in western Nebraska and Kansas. PLoS ONE. 10, 1–16; doi: 10.1371/journalpone.0142299 (2015). Quiring, D. T., and P. R. Timmins. 1990. Influence of reproductive ecology on feasibility of mass trapping Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). Journal of Applied Ecology 27:956–982. Spencer, J. L., B. E. Hibbard, J. Moeser, and D. W. Onstad. 2009. Behavior and ecology of the western corn rootworm (Diabrotica virgifera virgifera LeConte) (Coleoptera: Chrysomelidae). Ag. and Forest Entomol. 11: 9–27. Wiklund, C., and T. Fagerstrom. 1977. Why do males emerge before females? A hypothesis to explain the incidence of protandry in butterflies. Oecologia 31: 153–158. Zukoff, S. N., K. R. Ostlie, B. Potter, L. N. Meihls, A. L. Zukoff, L. French, M. R. Ellersieck, B. W. French, and B. E. Hibbard. 2016. Multiple assays indicate varying levels of cross resistance of Cry3Bb1-selected field populations of the western corn rootworm to mCry3A, eCry3.1Ab, and Cry34/35Ab1. J. Econ. Entomol. 109: 1387–1398.
