Dodd, Luke E.

Fulton, Shelby A.

Rieske, Lynne K.

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Western Kentucky UniversityTopSCHOLAR®Mammoth Cave Research Symposia 11th Research Symposium 2016Apr 19th, 3:15 PMEvaluating the Energetic Value of LepidopteraUsing Bomb CalorimetryShelby A. FultonDepartment of Biological Sciences, Eastern Kentucky University, shelby_fulton5@mymail.eku.eduLuke E. DoddDepartment of Biological Sciences, Eastern Kentucky University, luke.dodd@eku.eduLynne K. RieskeDepartment of Entomology, University of KentuckyFollow this and additional works at: http://digitalcommons.wku.edu/mc_reserch_sympPart of the Animal Sciences Commons, Forest Sciences Commons, Geology Commons,Hydrology Commons, Other Earth Sciences Commons, and the Plant Sciences CommonsThis is brought to you for free and open access by TopSCHOLAR®. It has been accepted for inclusion in Mammoth Cave Research Symposia by anauthorized administrator of TopSCHOLAR®. For more information, please contact todd.seguin@wku.edu.Recommended CitationShelby A. Fulton, Luke E. Dodd, and Lynne K. Rieske, "Evaluating the Energetic Value of Lepidoptera Using Bomb Calorimetry"(April 19, 2016). Mammoth Cave Research Symposia. Paper 12.http://digitalcommons.wku.edu/mc_reserch_symp/11th_Research_Symposium_2016/Day_two/12Proceedings for Celebrating the Diversity of Research in the Mammoth Cave Region: 11th Research Symposium at Mammoth Cave National Park. Editors: Shannon R. Trimboli, Luke E. Dodd, and De’Etra Young.85Evaluating the Energetic Value of Lepidoptera Using Bomb CalorimetryShelby A. Fulton1, Luke E. Dodd1, and Lynne K. Rieske21 Department of Biological Sciences, Eastern Kentucky University2 Department of Entomology, University of KentuckyAbstractLepidopterans are a core resource for many of North America’s insectivorous bats. These predators consume Lepidoptera of varying sizes, and some bat species remove the wings of lepidopteran prey prior to consumption. Selection of larger prey and subsequent wing removal may allow bats to optimize the energetic value afforded by lepidopteran prey. To explore the relationships between caloric yield, body size, and wing presence, laboratory-reared Trichoplusia ni moths were grouped into large and small size classes. Wings were removed from half of the moths in each size class. Bomb calorimetry was used to determine the gross heat (cal/g) of moths in each treatment. To account for potential differences in energetic value among species, specimens of Malacosoma americanum, Halysidota tessellaris, and Iridopsis sp. moths were also combusted. Larvae of M. americanum were field-collected in April 2012 and reared in the laboratory. Adult H. tessellaris and Iridopsis sp. moths were wild-caught using an illuminated substrate at Mammoth Cave National Park in June - July 2015. No differences were detected for size class or wing condition of T. ni (P ≥ 0.05). Additionally, no differences were detected in the caloric yields of the various lepidopteran species, except between Ma. americanum and Iridopsis sp. (P = 0.03). These results suggest that lepidopteran prey of various species and sizes may be of similar prey quality, and that the removal of wings by bats may be unrelated to caloric yield. Even so, we believe the lack of differences detected in this study indicate that our approach was likely too coarse of a method to capture subtle energetic differences among lepidopteran prey. Future studies including additional insect orders will clarify the potential limitations of conducting prey quality studies by bomb calorimetry.IntroductionLepidoptera are a core resource for many of North America’s insectivorous bats, and have been detected in the diets of all Kentucky bat species tested (Lacki et al. 2007). The gleaning species Myotis septentrionalis and Corynorhinus rafinesquii are lepidopteran specialists, with this prey taxon representing nearly 50% of the diet of M. septentrionalis (Dodd et al. 2012) and more than 80% of the diet of C. rafinesquii (Lacki and Dodd 2011). Lepidoptera are also common in the diets of more generalist predators, including M. lucifugus, M. sodalis, and Perimyotis subflavus. Although M. lucifugus and M. sodalis may consume diverse diets, these species often rely on lepidopteran prey (Brack and LaVal 1985, Whitaker 2004, Feldhamer et al. 2009, Clare et al. 2014). The generalist predator P. subflavus opportunistically consumes soft-bodied arthropods, including lepidopterans (Whitaker 2004, Lacki et al. 2007, Dodd et al. 2014).The ubiquity of Lepidoptera as a prey resource for insectivorous bats is thought to be a consequence of high digestive efficiency. The carbohydrate chitin, which Proceedings for Celebrating the Diversity of Research in the Mammoth Cave Region: 11th Research Symposium at Mammoth Cave National Park. Editors: Shannon R. Trimboli, Luke E. Dodd, and De’Etra Young.86forms arthropods’ hard exoskeletons, is difficult for most mammals to digest (Strobel et al. 2013). However, some bat species have the ability to optimize digestion of arthropod prey due to specialized gastrointestinal microflora (Strobel et al. 2013, Whitaker et al. 2004). These bats, including M. septentrionalis, M. lucifugus, M. sodalis, and P. subflavus, host chitinase-producing bacteria in the digestive tract (Whitaker et al. 2004). The enzyme chitinase promotes the breakdown of chitin, but does not allow it to be completely digested. As a result, insects with high chitin levels have low digestive efficiency (Barclay et al. 1991).Some bats (e.g., Corynorhinus species) reject lepidopteran body parts such as the legs and wings (Lacki and Dodd 2011). This behavior may be a result of low palatability, but is thought to be due to low digestibility of these chitin-rich structures (Barclay et al. 1991). Smaller moths have lower digestive efficiency, likely due to the increased difficulty of removing indigestible or unpalatable structures from small prey (Barclay et al. 1991). Although larger moths are more digestible, it is not yet clear whether selection of larger moths affords a caloric benefit. The relationships between caloric yield, body size, and wing presence are poorly understood. Thus, our objectives were: (1) explore the relationships between caloric yield, body size, and wing presence by determining the mean gross heat (cal/g) generated across large, small, winged, and wingless representatives of a model lepidopteran species (Trichoplusia ni), (2) investigate potential differences in energetic value among species by using bomb calorimetry to combust Malacosoma americanum, Halysidota tessellaris, and Iridopsis sp. moths, and (3) evaluate the overall viability of bomb calorimetry as a method of conducting prey quality studies.Methods Malacosoma americanum tents and larvae were field-collected in April 2012 at Mammoth Cave National Park (N 37° 11.83’, W 86°04.50’). Tents (n = 1-3) were placed in plastic housing (32 cm × 26 cm × 9 cm) lined with paper towels to absorb moisture and provide substrate. The developing insects were supplied ad libitum with fresh, field-collected Prunus sp. foliage. Throughout the three-week rearing process, some tents were disposed of to maintain hygienic conditions. Pupae were subsequently removed from plastic housing and placed individually in plastic diet cups (30 ml) until emergence. Adult moths were flash-frozen within 24 hr of emergence; adult moths (in diet cups) were submerged in liquid nitrogen for 5-10 seconds, and immediately stored in a -80°C freezer.Larvae of T. ni were reared communally from 25 eggs on 110 g of a pinto bean-based diet in a 240 ml Styrofoam cup kept at ambient conditions (Evenden and Haynes 2001). Other details of the rearing methods are described by Shorey and Hale (1965). Pupae were separated, sexed, placed individually in diet cups (30 ml), and flash-frozen in liquid nitrogen within 24 hr of adult emergence. Specimens were then stored in a -20°C freezer. Adult T. ni were divided into large and small size classes (individual masses of 118 ± 0.80 and 87 ± 0.69 mg, respectively), and wings were removed from half of the moths in each size class. Wild-caught moths were collected from June - July 2015 at the Mammoth Cave International Center for Science and Learning (N 37° 12.44’, W 86° 7.93’). A cotton sheet was hung vertically and stretched taut at ground level; the sheet was Proceedings for Celebrating the Diversity of Research in the Mammoth Cave Region: 11th Research Symposium at Mammoth Cave National Park. Editors: Shannon R. Trimboli, Luke E. Dodd, and De’Etra Young.87illuminated between approximately 2000 and 2300 hours with a 10 w black light and electrical harness (Universal Light Trap, Bioquip Products, Rancho Dominguez, CA, USA) (Figure 1). Lepidoptera attracted to the sheet were collected in plastic diet cups and immediately placed on ice. Specimens were temporarily stored at -18°C and transferred to -80°C within 7 d. Although numerous taxa were collected, H. tessellaris and Iridopsis sp. were selected for combustion due to their ready abundance and conspicuous appearance (Covell 2005).To prepare for combustion, all frozen Lepidoptera were transferred to open, heat-resistant vials and dried in a 55°C oven for approximately 24 hr. Specimens were consolidated by treatment (Table 1) and ground with a mortar and pestle for 30-60 seconds until a coarse powder was attained. A Parr 1281 Oxygen Bomb Calorimeter (Parr Instrument Company, Moline, IL, USA) was calibrated daily using a 1.0 g benzoic acid pellet (Parr Instrument Company, Moline, IL, USA). To determine whether sample weight affects gross heat generated by bomb calorimetry, we combusted Ma. americanum samples weighing 200-250 mg, 400-450 mg, 600-650 mg, and 800-850 mg. Following this assessment of methods, a standard sample weight of 250 mg was used for T. ni, H. tessellaris, and Iridopsis sp. treatments. The number of bomb calorimetry samples combusted was dependent upon the volume of processed lepidopteran material available for each treatment. All treatments were combusted according to instructions provided by the bomb calorimeter manufacturer.We determined the mean gross heat ± SE (cal/g) generated by the combustion of each treatment. A one-way analysis of variance (ANOVA) was used to test for differences between Ma. americanum sample weight Figure 1: Cotton sheet deployed at Mammoth Cave National Park to sample Lepidoptera, illuminated by 10 w black lights with electrical harnesses.Species Size ClassWings Sample Weight (mg)NT. ni Large Yes 250 6T. ni Large No 250 6T. ni Small Yes 250 6T. ni Small No 250 6T. ni* - Yes 250 2Ma. americanum - Yes 200-250 5Ma. americanum - Yes 400-450 5Ma. americanum - Yes 600-650 5Ma. americanum - Yes 800-850 3H. tessellaris - Yes 250 7Iridopsis sp. - Yes 250 4Table 1: Summary of Trichoplusia ni, Malacosoma americanum, Halysidota tessellaris, and Iridopsis sp. treatments. The treatment marked with an asterisk was not included in the initial comparison of small vs. large-bodied and winged vs. wingless T. ni, but was included in the comparison of species. N = number of samples combusted per treatment.Proceedings for Celebrating the Diversity of Research in the Mammoth Cave Region: 11th Research Symposium at Mammoth Cave National Park. Editors: Shannon R. Trimboli, Luke E. Dodd, and De’Etra Young.88classes, and a 2×2 ANOVA was used to test for differences between T. ni treatments. To test for potential differences in energetic value among species, Wilcoxon Rank-Sum tests were used to make pairwise comparisons (using 250 mg samples).ResultsMalacosoma americanum was found to have a significantly greater caloric yield than Iridopsis sp. (W5,4 = 19, P = 0.03), although no additional differences were detected between pairwise comparisons across species (Figure 2).  No differences were detected between any Ma. americanum weight classes (F3,14 = 1.6, P > 0.05) (Figure 3) or T. ni treatments (F3,23 = 0.86, P > 0.05) (Figure 4). DiscussionThe lack of differences detected between Ma. americanum weight classes suggests the gross heat generated by combustion is likely not affected by sample weight. These data indicate that any sample weight (adhering to manufacturer’s specifications for safe calorimeter usage) could be combusted effectively. Based on these findings, we recommend that future studies reduce sample weights to conserve raw material and maximize the number of combustion reactions possible.We found no differences in energetic value between any T. ni treatment, suggesting that the removal of lepidopteran wings by bats may be unrelated to caloric yield. These results support the commonly accepted hypothesis that bats reject lepidopteran wings due to indigestibility (Barclay et al. 1991, Lacki and Dodd 2011). The lack of any significant differences between large and small T. ni indicates that caloric yield is independent of body size. However, Ma. americanum appears to have a significantly greater caloric yield than Iridopsis sp., likely due to the larger body size of Ma. americanum. This explanation is supported by previously published literature regarding the energy density of fish; Glover et al. (2010) found Figure 2: Mean gross heat ± SE (cal/g) generated by combustion of coarsely ground samples of Malacosoma americanum, Trichoplusia ni, Halysidota tessellaris, and Iridopsis sp. using bomb calorimetry. We combusted five samples of Ma. americanum, twenty-six of T. ni, seven of H. tessellaris, and four of Iridopsis sp.Figure 3: Mean gross heat ± SE (cal/g) generated by combustion of coarsely ground Malacosoma americanum samples using bomb calorimetry. Five samples weighing 200-250 mg, 400-450 mg, and 600-650 mg, and three samples weighing 800-850 mg were combusted.Proceedings for Celebrating the Diversity of Research in the Mammoth Cave Region: 11th Research Symposium at Mammoth Cave National Park. Editors: Shannon R. Trimboli, Luke E. Dodd, and De’Etra Young.89that the caloric yield of largemouth bass is directly related to body mass, with larger bass generally possessing greater energetic density.Given that Lepidoptera are relatively soft-bodied (Freeman 1981), we suspect these prey may have comparatively less chitin than many insect orders, thus allowing predators to maximize digestive efficiency. Although it is likely that consuming Lepidoptera affords a digestive advantage, the similarity in energetic value among study species may suggest that lepidopteran prey of various species and sizes is of similar prey quality. However, based on the inconsistency of our results regarding caloric yield and body size, we believe the lack of differences detected in this study indicates our technique is likely too coarse of a method to capture subtle energetic differences among Lepidoptera. Future studies including additional insect orders will clarify the potential limitations of conducting prey quality studies by bomb calorimetry.AcknowledgementsThis project was funded by the Joint Fire Science Program (JFSP #14-1-05-22). The authors thank Eastern Kentucky University and the University of Kentucky for making this work possible. In particular, we thank the laboratory of Dr. Ken Haynes for providing materials related to rearing Trichoplusia ni, as well as Adam Bohannon and Dr. Winston Lin for assistance when using the bomb calorimeter. We thank Rachael Griffitts, Adrienne Kinney, and Abe Nielson for technical assistance. We are indebted to Dr. Joe Johnson for his intellectual and logistic contributions during the genesis of this project. Finally, we are grateful to the staff of Mammoth Cave National Park for continuing support.Literature CitedBarclay, R.M.R., M.A. Dolan, and A. Dyck. 1991. The digestive efficiency of insectivorous bats. Canadian Journal of Zoology 69:1853-1856.Brack, V., and R.K. Laval. 1985. Food habits of the Indiana Bat in Missouri. Journal of Mammalogy 66:308–315.Clare, E.L., W.O.C. Symondson, H. Broders, F. Fabianek, E.E. Fraser, A. Mackenzie, A. Boughen, R. Hamilton, C.K.R. Willis, F. Martinez-Nuñez, A.K. Menzies, K.J.O. Norquay, M. Brigham, J. Poissant, J. Rintoul, R.M.R. Barclay, and J.P. Reimer. 2014. The diet of Myotis lucifugus across Canada: assessing foraging quality and diet variability. Molecular Ecology 23:3618–3632.Covell, C.V. 2005. A field guide to moths of eastern North America: special publication number 12. Virginia Museum of Natural History, Martinsville,Virginia. p. 496.Figure 4: Mean gross heat ± SE (cal/g) generated by combustion of coarsely ground Trichoplusia ni samples using bomb calorimetry. Six samples were combusted per treatment (small with wings present, small with wings removed, large with wings present, large with wings removed).Proceedings for Celebrating the Diversity of Research in the Mammoth Cave Region: 11th Research Symposium at Mammoth Cave National Park. Editors: Shannon R. Trimboli, Luke E. Dodd, and De’Etra Young.90Dodd, L.E., E.G. Chapman, J.D. Harwood, M.J. Lacki, and L.K. Rieske. 2012. Identification of prey of Myotis septentrionalis using DNA-based techniques. Journal of Mammalogy 93:1119–1128. Dodd, L.E., M.J. Lacki, D.C. Cox, and L.K. Rieske. 2014. Prey consumed by bats across central Appalachia prior to the detection of White-nose Syndrome. Journal of the Kentucky Academy of Science 75:85-93.Evenden, M.L., and K.F. Haynes. 2001. Potential for the evolution of resistance to pheromone-based mating disruption tested using two pheromone strains of the cabbage looper, Trichoplusia ni. Entomologia Experimentalis et Applicata 100:131-134.Feldhamer, G.A., T.C. Carter, and J.O. Whitaker. 2009. Prey consumed by eight species of insectivorous bats from southern Illinois. American Midland Naturalist 162:43-51.Freeman, P. W. 1981. Correspondence of food habits and morphology in insectivorous bats. Journal of Mammalogy 62:154-159.Glover, D.C., D.R. DeVries, R.A. Wright, and D.A. Davis. 2010. Sample preparation techniques for determination of fish energy density via bomb calorimetry: an evaluation using Largemouth Bass. Transactions of the American Fisheries Society 139:671-675.Lacki, M.J., S.K. Amelon, and M.D. Baker. 2007. Foraging ecology of forest bats. Pp. 83–128, In M.J. Lacki, J. P. Hayes, and A. Kurta (Eds.). Bats in forests: conservation and management. Johns Hopkins University Press, Baltimore, Maryland. Lacki, M.J., and L.E. Dodd. 2011. Diet and foraging behavior of Corynorhinus in eastern North America. Pp. 39–52, In S.C. Loeb, M.J. Lacki, and D.A. Miller (Eds.). Conservation and management of big-eared bats: a symposium. US Department of Agriculture, Forest Service, Southern Research Station, Asheville, North Carolina.Shorey, H.H., and R.L. Hale. 1965. Mass-rearing of the larvae of nine noctuid species on a simple artificial medium. Journal of Economic Entomology 58:522-524.Strobel, S., A. Roswag, N.I. Becker, T.E. Trenczek, and J.A. Encarnação. 2013. Insectivorous bats digest chitin in the stomach using acidic mammalian chitinase. PloS ONE 8:e72770.Whitaker, J.O. 2004. Prey selection in a temperate zone insectivorous bat community. Journal of Mammalogy 85:460–469.Whitaker, J.O., H.K. Dannelly, and D.A. Prentice. 2004. Chitinase in insectivorous bats. Journal of Mammalogy 85:15-18. 

Evaluating the Energetic Value of Lepidoptera Using Bomb Calorimetry

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Evaluating the Energetic Value of Lepidoptera Using Bomb Calorimetry

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