Structure & Development 47 (2018) 457e464

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Arthropod Structure & Development

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Flight duration and flight muscle ultrastructure of unfed hawk

Bernard W.M. Wone a, b, 1, *, Jaika Pathak a, 1, Goggy Davidowitz a a Department of Entomology, University of Arizona, Tucson, AZ, USA b Department of Biology, University of South Dakota, Vermillion, SD, USA a r t i c l e i n f o a b s t r a c t

Article history: Flight muscle breakdown has been reported for many orders of , but the basis of this breakdown Received 28 January 2018 in insects with lifelong dependence on flight is less clear. Lepidopterans show such muscle changes Accepted 17 May 2018 across their lifespans, yet how this change affects the ability of these insects to complete their life cycles Available online 13 June 2018 is not well documented. We investigated the changes in muscle function and ultrastructure of unfed aging adult hawk moths (). Flight duration was examined in young, middle-aged, and Keywords: advanced-aged unfed moths. After measurement of flight duration, the main flight muscle (dorso- Aging longitudinal muscle) was collected and histologically prepared for transmission electron microscopy to Flight muscle breakdown Mitochondria compare several measurements of muscle ultrastructure among moths of different ages. Muscle function fi fl Muscle function assays revealed signi cant pos itive correlations between muscle ultrastructure and ight distance that Muscle ultrastructure were greatest in middle-aged moths and least in young moths. In addition, changes in flight muscle Flight capacity ultrastructure were detected across treatment groups. The number of mitochondria in muscle cells peaked in middle-aged moths. Many wild M. sexta do not feed as adults; thus, understanding the changes in flight capacity and muscle ultrastructure in unfed moths provides a more compl ete understanding of the ecophysiology and resource allocation strategies of this species. © 2018 Elsevier Ltd. All rights reserved.

1. Introduction However, in orders such as the (Ziegler, 1991; Stjernholm et al., 2005; Jervis et al., 20 05; Boggs, 2009) with Flying insects rely on flight for a multitude of essential functions, lifelong dependence on flight, it is not clear if or when flight muscle including finding food or mates, dispersing, or avoiding predators. breakdown occurs. However, flight is not a lifelong requirement in several orders of Studies of adult lepidopterans suggest that muscle breakdown insects such as the Diptera (Hocking, 1952), Coleoptera (Jackson, might occur in many of these species (Karlsson, 1994, 1998; 1933; Chapman, 1956), Heteroptera (Brinkhurst, 1959; Edwards, Stjernholm and Karlsson, 2000; Norberg and Leimar, 20 02; 1969; Andersen, 1973; Solbreck, 1986; Kaitala, 1988), Homoptera Stjernholm et al., 2005; Stjernholm and Karlsson, 2008; Åhman (Johnson, 1953, 1976; Kobayashi and Ishikawa, 1993), Hymenoptera and Karlsson, 2009), but whether there is degeneration of muscle (Janet, 1906, 1907; Jones et al., 1978), and (Readym and function and capacity over the entire lifespan of these insects is not ~ Josephson, 1982; Shiga et al., 1991; Tanaka, 1991, 1993; Tanaka and clear (Stjernholm and Karlsson, 2008). Recently, Niitepold et al. Suzuki, 1998). For such insects, flight muscle breakdown is associ- (2014) showed that body mass, flight, and peak metabolic rates ated with reproduction. In many of these species, adults emerge decrease with age in the lepidopterans Speyeria mormonia and with fully developed flight muscles, which subsequently break Colias eurytheme, suggesting that muscle mass and function down before or during the reproductive stage (Johnson, 1976; Zera decrease with age. Still, the exact ways in which aging might affect and Denno, 1997; Marden, 2000) and flight is only required for the flight capacity in flying insects are not known. Stjernholm et al. pre-reproductive migratory or dispersal phase in early adult life (2005) reported intriguing age-related reallocation of resources before initiating reproduction (Johnson, 1969; Harrison, 1980). from flight muscle to reproductive organs with no adverse effects on flight capacity under natural conditions in lepidopterans. In contrast, amino acids from nectar are allocated to flight muscles when moths * Corresponding author. Department of Biology, University of South Dakota, have the opportunity to feed (Levin et al. 2017 a, b), suggesting that Vermillion, SD, USA. fl E-mail address: [email protected] (B.W.M. Wone). maintenance of ight capability is prioritized when possible. Inter- 1 Co-first Author. estingly, the body masses of hawk moths continuously decrease https://doi.org/10.1016/j.asd.2018.05.003 1467-8039/© 2018 Elsevier Ltd. All rights reserved. 458 B.W.M. Wone et al. / Arthropod Structure & Development 47 (2018) 457e464 whether or not they are feeding (Ziegler, 1991). If lepidopterans do plate was then attached to the arm of a flight mill with a magnet. experience decreased muscle mass across their lifespan, how might Flight capacity, measured as distance flown, was measured only such muscle breakdown affect their ability to fly and complete their once on each on either Day 1, Day 3, or Day 6 depending on life cycle? their treatment group (n ¼ 30, 10 samples per age group). These We investigated the changes in muscle function and muscle nocturnal moths were flown during the dark cycle when their flight ultrastructure during the lifespan of unfed hawk moths. This moth activity was greatest. The moths were allowed to fly on the mill for species lives in variable environments with the potential for as long as they wanted, but if they stopped flying for more than  frequent episodes of starvation (Davidowitz, 2002; Alarcon et al., 0.5 h the flight trial was ended and distance flown was recorded. 2008; Contreras et al., 2013; Levin et al., 2016) and flight is an The computer-monitored flight mill setup was maintained at a essential component of their life history. Both male and female temperature of 27 ± 1  C and RH between 35% and 40%. moths hover when probing flowers for nectar, which can be a metabolically demanding mode of locomotion (Bartholomew and 2.3. Histological preparation Casey, 1978; Lehmann and Dickinson, 1997). In addition to requiring flight to feed, males need to fly to find females (Levin After measuring flight capacity, the main flight muscles (dorso- et al., 2016), and females need to fly and hover to oviposit. Our longitudinal muscle) were removed and histologically prepared for study asked whether age affects flight capacity in starved Manduca transmission electron microscopy (TEM) following Ribi (1987). In sexta, what changes might occur in flight muscle ultrastructure as brief, flight muscles were longitudinally sectioned from moths in M. sexta ages, and how such changes might affect flight capacity. each age treatment (n ¼ 30, five females and five males per age group) and fixed in 0.2 M piperazine-N,N0-bis(2-ethanesulfonic 2. Materials and methods acid) (PIPES), 25% glutaraldehyde, 16% paraformaldehyde, satu- rated picric acid, and distilled water for 1 h. Double-distilled water 2.1. Experimental was used in all washes and rinses. Each fixed muscle was then washed in 0.1 M PIPES twice for 20 min. Post-fixation of each sample Carolina sphinx moths (Lepidoptera: : M. sexta (hawk was performed in 0.2 M PIPES with 4% osmium tetroxide and moths)) from the colony at the University of Arizona, Tucson, AZ distilled water to preserve lipids in each sample. After 30 min, each were used for all experiments. Larvae were reared on an ad lib sample was rinsed and left overnight in 0.1 M PIPES. A solution of artificial diet (Davidowitz et al., 2003) and once eclosed, adults were 10% acetone and 2% aqueous uranyl acetate in distilled water was reared for the experiments described below. For the flight capacity used to stain the muscle tissues. Dehydration of the prepped tissues and muscle ultrastructure experiment, adults were reared individ- was carried out through a series of 25%, 50%, 70%, and 90% aqueous ually in a small 28 cm L  28 cm W  28 cm H, 299 cm3 cage ethanol solutions for 10 min each. Each specimen was then dehy- (BioQuip Products, Rancho Dominguez, CA, USA). These cages were drated again three times in 100% ethanol for 40 min per incubation. kept in a reversed light/dark cycle of 16:8 h maintained at 27 ± 2 C Each sample was infiltrated first in 75% propylene oxide with 25% and 40e45% RH. Moths were not fed to avoid any possible influence embedding resin overnight, then in 25% propylene oxide with 75% of adult-derived nutrients (Arrese and Soulages, 2010; Gondim et al., embedding resin for 6 h, and finally in 100% embedding resin 2013; Levin et al., 2017a; b) on any changes in flight capacity and overnight. The samples were embedded in longitudinal orientation muscle ultrastructure. Moreover, recent studies have shown that in 100% ACLAR® sheets (Ted Pella, Inc., Redding, CA, USA) and wild-caught adult M. sexta routinely experience limited nectar polymerized at 80  C for 36 h with care taken that all samples were resources: 15% did not feed at all and 73% did not feed on their placed in the same orientation. Thin sections (~200 mm) of preferred nectar resources (Levin et al., 2016). Similarly, laboratory- embedded samples were then cut longitudinally using an ultrami- reared M. sexta routinely do not feed (Ziegler, 1991), as indicated by crotome and were later stained with lead to enhance contrast their metabolic profiles across diel time and age (Wone et al., 2018). between different cellular structures. Three slides were prepared Because the average lifespan of unfed female M. sexta is 8.0 d and the from mid-longitudinal sections of each flight muscle for each moth. average lifespan of unfed male M. sexta is 4.9 d (Wone et al., 2018), We evaluated flight muscle ultrastructures including the number we divided the moths into three age groups: Day 1 (young), Day 3 of mitochondria, size of mitochondria, percent area of mitochondria, (middle-aged), or Day 6 (advanced-aged) moths to determine the number of sarcomeres, percent area of sarcomeres, and length of changes in flight muscle across their lifespan, with a total of n ¼ 5 sarcomeres using TEM imaging. Micrographs were recorded on a moths per sex per age group, for a total of n ¼ 30 moths. CM-12 TEM at 8800  magnification (Philips, Andover, MA, USA). TEM images were initially processed using Photoshop CS5 software 2.2. Flight capacity measurements (Adobe Systems Incorporated, San Jose, CA) and then muscle ul-  trastructures were quantified using Image J v1.45 (Abramoff et al., We used a custom-built computer-monitored flight mill to 2004). The value of each measurement for each sample was the evaluate the flight capacity of M. sexta, because flight mills provide average of 30 randomly chosen images at 8800  magnification exact measurements of flight distance, speed, and duration from three slides per moth (10 randomly chosen images at (Thomas and William, 1992; Cui et al., 2013; Hao et al., 2013; Chen 8800  magnification per muscle slide). All reported measurements et al., 2015). Flight mills do not provide good estimates of free flight from the TEM image of each sample are within the field of view in an performance (Riley et al. 1997), however, they are considered a area of 174 mm2 at 8800  magnification. standard approach for comparing the effects of treatments when investigating flight behavior in insects (Schumacher et al., 1997; 2.4. Statistical analysis Ishiguri and Shirai, 2004; Tu et al., 2010; Chen et al., 2015). To prepare moths for the flight capacity measurements, newly eclosed MANOVA was performed in RStudio (RStudio Team, 2015) to moths were gently handled and scales were removed from an area analyze any multivariate effects in the number of mitochondria, of less than 4 mm2 on the dorsal thoracic region of each moth's size of mitochondria, percent area of mitochondria, number of ® body. A drop of Loctite Super Glue Gel (Henkel Corporation, sarcomeres, percent area of sarcomeres, length of sarcomeres, and Westlake, OH, USA) was used to glue a small metal plate (approx- distance flown, with age and sex as factors. A significant MANOVA imately 3  2 mm) onto the bare pronotum of each moth. The small was followed by separate ANOVA performed in RStudio (RStudio B.W.M. Wone et al. / Arthropod Structure & Development 47 (2018) 457e464 459

Team, 2015) with the dependent variables number of mitochondria, percent area of mitochondria was much lower in Day 1 post- size of mitochondria, percent area of mitochondria, number of eclosion moths compared to Day 3 or Day 6 moths (Fig. 1D). No sarcomeres, percent area of sarcomeres, length of sarcomeres, or effect of sex on the number, size, or percent area of mitochondria in distance flown, and age and sex as factors. Adult body mass was not flight muscle was detected. included as a covariate to possibly account for body size effects on The number of sarcomeres in the flight muscle changed with age muscle ultrastructure (i.e., number of mitochondria), because the (F2, 27 ¼ 28.6, p < 0.0001). M. sexta collected on Day 1 post-eclosion microscopic area of muscle tissue examined was standardized had significantly fewer sarcomeres compared to those collected on across all individuals. At this microscopic level (i.e., cellular-level), Day 3 or Day 6 (Fig. 1E). Percent area of sarcomeres increased as the body size effects on the number of mitochondria are not expected moths aged (F2, 27 ¼ 27.3, p < 0.0001). Notably, percent area of because the number of mitochondria is well regulated in any given sarcomeres was much lower in Day 1 post-eclosion moths cell type (Robin and Wong, 1988). Individual univariate models compared to Day 3 or Day 6 moths (Fig. 1F). The length of the were evaluated post hoc by pairwise comparisons using Tukey's sarcomeres in the flight muscle also changed with age (F2, 27 ¼ 80.2, HSD (Honestly Significant Difference). To determine whether flight p < 0.0001). Sarcomere lengths were greatest in Day 3 post-eclosion capacity (i.e., flight distance) is related to number of mitochondria, moths compared to Day 1 or Day 6 moths, whereas sarcomere size of mitochondria, percent area of mitochondria, number of length was shortest in Day 1 moths compared to Day 3 or Day 6 sarcomeres, percent area of sarcomeres, or length of sarcomeres, moths (Fig. 1G). No effect of sex on the number, percent area, or these variables were regressed on distance flown. Effect size for the length of sarcomeres in the flight muscle was detected. individual linear regressions was calculated using Cohen's f2 (Cohen, 1988). 4. Discussion

3. Results Here, we provide the first detailed analysis of changes in flight capacity and flight muscle ultrastructure in unfed adult M. sexta over 3.1. Flight capacity their lifespan. The present study provides insights into how flight capacity and muscle ultrastructure change over time in M. sexta. MANOVA indicated significant multivariate effects for number Such changes might affect the ability of moths with a lifelong of mitochondria, size of mitochondria, percent area of mitochon- dependency on flight to find food, mates, and oviposition sites while dria, number of sarcomeres, percent area of sarcomeres, length of experiencing potentially long or frequent episodes of starvation. sarcomeres, and distance flown by age (F14, 20 ¼ 173.06, p < 0.0001, Wilks's lambda ¼ 0.00026), but not by sex in unfed adults. 4.1. Flight duration Flight capacity in M. sexta changed significantly with age (F2, 27 ¼ 491.4, p < 0.0001), with maximal flight capacity on Day 3 Muscle function and strength have been linked to age (Demontis post-eclosion compared to Days 1 and 6 (Fig. 1A). Interestingly, et al., 2013a; b), as we have also shown in M. sexta (Wone et al., 2018). moths collected on Day 1 and Day 6 post-eclosion could be grouped Flight duration in Day 1 post-emergence moths was minimal and together according to distance flown compared to moths collected on was maximal in Day 3 post-emergence moths. There was a signifi- Day 3. Moths collected on Day 1 and Day 6 post-eclosion all flew less cant increase in flight duration in Day 3 and this is positively corre- than 800 m, whereas those collected on Day 3 all flew farther than lated with flight muscle ultrastructure. These results are consistent 2500 m (Fig. 1A). No effect of sex on flight capacity was detected. with those from other flying insects, such as the honeybee (Herold, The regression analysis revealed highly significant positive cor- 1965), housefly (Sohal et al., 1972; Sohal, 1976), green-veined white relations between the number of mitochondria and distance of butterfly Pieris napi (Åhman and Karlsson, 2009), and Drosophila 2 2 flight (F1, 29 ¼ 503.6, p < 0.0001, R ¼ 0.95, f ¼ 19.0; Fig. 2A), the (Sohal, 1975; Ridgel and Ritzmann, 2005; Kim et al., 2011; Demontis length of sarcomeres and distance of flight (F1, 29 ¼ 127.5, p < 0.0001, et al., 2013a, 2013b). In other species, such as mice and humans, R2 ¼ 0.82, f2 ¼ 4.6; Fig. 2B), as well as the percent area of mito- muscle function and strength decline with age (Carmeli et al., 2002; 2 chondria and distance of flight (F1, 29 ¼ 43.1, p < 0.0001, R ¼ 0.61, Haddad and Adams, 2006) and these changes are thought to be f2 ¼ 1.6; Fig. 2C). Significantly positive relationships were also found related to changes in signaling pathways (Haddad and Adams, 2006), between most of the other measures of ultrastructure, although increased abundance of cytoskeletal proteins (Piec et al., 2005; their R2 values indicate that they explain less of the variation in O'Connell et al., 2007), and mitochondrial dysfunction (Tribe and distance flown than do number of mitochondria and length of Ashhurst, 1972; Yarian and Sohal, 20 05; Hepple, 2014). Our results sarcomeres, as shown by the weaker positive coefficients of deter- show that flight distance was significantly shorter in advanced-aged mination between the number of sarcomeres and distance of flight moths (Day 6) compared to middle-aged moths (Day 3), which can be 2 2 (F1, 29 ¼ 14.5, p ¼ 0.0007; R ¼ 0.34, f ¼ 0.5: Fig. 2E), or percent area partially explained by the significantly lower number of mitochon- of sarcomeres and distance of flight (F1, 29 ¼ 16.7, p ¼ 0.0003; dria in the flight muscle cells of advanced-aged moths compared to R2 ¼ 0.37, f2 ¼ 0.6; Fig. 2D). No relationship was detected between middle-aged moths. This would suggest that the ability of unfed the size of mitochondria and distance flown (F1, 29 ¼ 0.27, p ¼ 0.61). M. sexta to find mates and suitable habitat to complete their life cycle is greatest within 3e4 d post-eclosion. Indeed, hawk moths are large, 3.2. Muscle ultrastructure powerful fliers that can reach speeds of 5e10 km/h (Willmott and Ellington, 19 97 ) and can fly distances greater than 20 km (Davido- The number of mitochondria in flight muscle changed with age witz, unpubl. data). Unfed moths flew up to 3200 m and averaged (F2, 27 ¼ 91.2, p < 0.0001). M. sexta collected on Day 3 post-eclosion 2903.5 m (±271.23 STD) on day 3 post-eclosion, but could still fly had significantly greater numbers of mitochondria compared to 600 m on day 6 (average 569.6 m ± 151.78 STD). moths collected on Day 1 or Day 6 (Figs. 1B and 3). The size of The results of the present study have implications for our mitochondria in the flight muscle changed with age as well understanding of the ecophysiological aspects of the life history of (F2, 27 ¼ 224.2, p < 0.0001). The average size of mitochondria was hawk moths. The adults used in the current study were raised in significantly larger in Day 6 post-eclosion moths than in Day 1 or colonies that have been maintained under laboratory conditions at Day 3 moths (Fig. 1C). In addition, percent area of mitochondria the University of Arizona since 1985 (Duch et al., 2000) in cages of increased as moths aged (F2, 27 ¼ 44.3, p < 0.0001). Specifically, about 1 m  1 m  1 m, and thus do not need to fly farther than 1 m 460 B.W.M. Wone et al. / Arthropod Structure & Development 47 (2018) 457e464

Fig. 1. Changes in flight muscle ultrastructures during starvation in Day 1, Day 3, and Day 6 post-eclosion Manduca sexta. (A) Mean distance flown. (B) Number of mitochondria. (C) Average mitochondrial size. (D) Percent area of mitochondria. (E) Number of sarcomeres. (F) Percent area of sarcomere. (G) Average length of sarcomere. Horizontal line ¼ median, diamond ¼ mean, and whiskers ¼ upper and lower quartiles. Lower case letters indicate significant pair-wise comparisons: a) compared to Days 1 and 6, adjusted p < 0.0001; b) compared to Days 3 and 6, adjusted p < 0.0001; c) compared to Days 1 and 3, adjusted p < 0.0001; and d) compared to Day 6, adjusted p < 0.05.

to find a mate, to feed, or to lay eggs. Moreover, adult M. sexta in this adult moths can experience periods of starvation (Levin et al., colony, especially males, fed irregularly whether nectar was avail- 2016), at least the males do not need to feed in order to find able freely or individually hand fed (Ziegler, 1991; Contreras et al., mates and complete their life cycle. However, more importantly, 2013; Wone et al., 2018). Ziegler (1991) reported that out of 40 because moth eggs contain ~70% water, females must drink (feed) males, 10 moths fed only once and only one moth fed twice during more to maintain viable eggs (Kawooya and Law, 1988). their lifetime. In contrast, 33 of 40 female moths fed from one to six times during their lifetime. This difference in feeding behavior 4.2. Muscle ultrastructure suggests that males normally do not feed. Wone et al. (2018) recently reported that the absence of diel shifts in the metabolic The changes in flight muscle ultrastructure, including the fusion profiles of male moths indicates that males generally do not feed, of mitochondria, reported in the present study are consistent with whereas females generally do feed. In addition, Ziegler (1991) those observed in other flying insects. The fusion of mitochondria observed that male moths are much more active than females, during aging has also been reported in flight muscles of other flying suggesting that in males, the drive to reproduce is much greater insects such as the housefly (Sohal et al., 1972; Sohal, 1976), black than the drive to feed. Furthermore, males mate up to six times carpet beetle (Butler and Nath, 1972), Drosophila (Sohal, 1975; and females only once (Levin et al., 2016). These observations, Miller et al., 2008), and the blowfly (Gregory et al., 1968). combined with results from the current study, indicate that as long We observed a significant decrease in the number of mitochondria as males are able to find females within 3e4 d post-eclosion, they in unfed advanced-aged moths (Day 6) compared to unfed middle- could mate even without having fed. During this relatively brief aged moths (Day 3). As expected, by Day 6 the flight muscle had active period, wild adult moths in the Southwest US feed on the deteriorated, as indicated by the enlarged mitochondria (Fig. 3C). nectar of flowering Datura wrightii and Agave palmeri, which may This increase in mitochondrial size is likely due to fusion of mito-  be of limited availability in their environment (Alarcon et al., 2008, chondria (which greatly reduces oxidative phosphorylation) and is 2010; Raguso et al., 2003; Riffell et al., 2008). Hence, although wild one of the age-related changes also observed in other animals (Seo B.W.M. Wone et al. / Arthropod Structure & Development 47 (2018) 457e464 461

Fig. 2. Relationship between distance flown and a subset of flight muscle ultrastructure in unfed Manduca sexta of different age classes. (A) Number of mitochondria and distance of flight. (B) Length of sarcomere and distance of flight. (C) Percent area of mitochondria and distance of flight. (D) Percent area of sarcomere and distance of flight. (E) Number of sarcomere and distance of flight. Further statistics are given in the text. Note: All the individuals that flew more than 2500 m were Day 3 moths, whereas Day 1 and Day 6 moths all flew less than 800 m. Unfilled diamonds e Day 1, Triangles e Day 3, and Circles e Day 6. Number of mitochondria, percent area of mitochondria, and percent area of sarcomere are measured in a 174 mm2 area. 462 B.W.M. Wone et al. / Arthropod Structure & Development 47 (2018) 457e464

Fig. 3. Representative Transmission Electron Microscopy (TEM) images (longitudinal view) of flight muscle in unfed Manduca sexta at different age classes (8,800 magnification and 174 mm2 area). (A) Day 1 (young). Young moths show fewer mitochondria and these organelles are small and distinct. In addition, myofibrils and sarcomeres (Z discs distinct, M lines visible) are organized (i.e., Microfilaments are distinct and parallel with each other). (B) Day 3 (middle-aged). Mitochondria are numerous and distinct. Myofibrils and sar- comeres (Z discs dark and distinct, M lines visible) are organized. (C) Day 6 (aged). Aged moths show fewer and larger mitochondria due to fusion. In addition, myofibrils and sarcomeres (Z discs faint, M lines absent) are disorganized (myofilaments are not parallel with each other). L, sarcomere length; Mf, myofibril; Mt, mitochondrion.

et al., 2010; Peterson et al., 2012). Mitochondrial fusion affecting identify any distinctions in flight muscle ultrastructure and function flight capacity might have accelerated under unfed conditions during aging between long- and short-lived lepidopteran species. in the current study. Fusion of aging mitochondria is thought to affect mtDNA integrity and respiratory function (Chen et al., 2005) Acknowledgements and is controlled by the optic atrophy 1 (Opa1) gene and the two GTPases mitofusin 1 and 2 (isoforms Mfn1 and Mfn2) in humans This work was supported by grants DARPA HI-MEMS Contract (Chan, 2006; Benard and Karbowski, 2009; Peterson et al., 2012). FA8650-07-C-7704 and NSF grant IOS-1053318 to G. Davidowitz. Other changes in flight muscle ultrastructure detected in unfed We thank David Byrne for discussions at the earlier stages of M. sexta include sarcomere and myofibril alterations. We found this project and the University of Arizona and the Southwest significant decreases in sarcomere size and myofibrillar changes Environmental Health Sciences Center Cellular Imaging Facility and disorganization in the flight muscle of unfed aged M. sexta Core for assistance in preparing the TEM slides. We thank Mary Ann (Fig. 3C). Such changes are thought to be associated with a decrease Cushman for technical editing assistance. in both muscle fiber types (Tomonaga, 1977; Scelsi et al., 1980; Kaminska et al., 1998), as well as their reduced function (Pastoret and Sebille,1995; Kirkendall and Garrett, 1998; Deschenes, 2004). References fi Decreases in muscle ber types likely explain the age-related  ~ muscle mass reduction seen in animals (Deschenes, 2004; Abramoff, M.D., Magalhaes, P.J., Ram, S.J., 2004. Image processing with image. Biophotonics Int. 11, 36e42. Demontis et al., 2013a; Nilwik et al., 2013). Interestingly, sarco- Åhman, M., Karlsson, B., 2009. Flight endurance in relation to adult age in the green-veined white butterfly Pieris napi. Ecol. Entomol. 34, 783e787. mere numbers and area increase after Day 1 are likely attributed to  additional biosynthesis post-eclosion (Sohal, 1976). Alarcon, R., Davidowitz, G., Bronstein, J.L., 2008. Nectar usage in a southern Arizona hawkmoth community. Ecol. Entomol. 33, 503e509. fl  Changes in muscle ultrastructure show how the ight muscle Alarcon, R., Riffell, J.A., Davidowitz, G., Hildebrand, J.H., Bronstein, J.L., 2010. Sex- breaks down with age, which results in decreased muscle size and dependent variation in the floral preferences of the hawkmoth Manduca sexta. flight duration over the lifespan of aging lepidopterans. Decreasing Anim. Behav. 80, 289e296. fl Andersen, N.M., 1973. Seasonal polymorphism and developmental changes in number, size, and length of sarcomeres in M. sexta ight muscle at organs of flight and reproduction in bivoltine pondskaters (Hem. gerridae). Day 6 post-eclosion all contribute to decreased flight capacity and Insect Systemat. Evol. 4, 1e20. muscle mass. Indeed, the total weights of fed or unfed adult M. sexta Arrese, E.L., Soulages, J.L., 2010. Insect fat body: energy, metabolism, and regulation. Annu. Rev. Entomol. 55, 207e225. continuously decrease during their adult lifespan (Ziegler,1991). One Bartholomew, G.A., Casey, T.M., 1978. Oxygen-consumption of moths during rest, likely reason for the decrease in sarcomere length is age-related pre-flight warm-up, and flight in relation to body size and wing morphology. decreases in muscle fiber types (Deschenes, 2004; Demontis et al., J. Exp. Biol. 76, 11e25. 2013a; Nilwik et al., 2013). Moreover, as mentioned above, body Benard, G., Karbowski, M., 2009. Mitochondrial fusion and division: regulation and role in cell viability. Semin. Cell Dev. Biol. 20, 365e374. mass also decreases with age in S. mormonia and C. eurytheme ~ Brinkhurst, R.O., 1959. Alary polymorphism in the gerroidea (Hemiptera-Hetero- (Niitepold et al., 2014), as well as in P. napi (Stjernholm and Karlsson, ptera). J. Anim. Ecol. 28, 211e230. 2008). However, some lepidopterans show no adverse effect of age Boggs, C.L., 2009. Understanding insect life histories and senescence through a resource allocation lens. Funct. Ecol. 23, 27e37. on flight capacity (Stjernholm et al., 2005). Such discrepancies likely Butler, L., Nath, J., 1972. Postemergence changes in ultrastructure of flight and leg reflect differing lifespans or life histories among species. That is, the muscle of the black carpet beetle. Ann. Ent. Soc. Amer. 65, 247e254. Carmeli, E., Coleman, R., Reznick, A.Z., 2002. The biochemistry of aging muscle. Exp. longer-lived species might not show the muscle breakdown e fl Gerontol. 37, 477 489. and concomitant decrease in ight capacity that is observed in the Chapman, J.A., 1956. Flight-muscle changes during adult life in a scolytid beetle. shorter-lived species such as M. sexta. Further studies are needed to Nature 177, 1183. B.W.M. Wone et al. / Arthropod Structure & Development 47 (2018) 457e464 463

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