Ontogenetic changes in citrate synthase and lactate dehydrogenase activity in the jumping muscle of the American locust (Schistocerca americana)

Abstract

Intraspecific studies have repeatedly shown that muscle-specific oxidative enzyme activities scale negatively with body mass while muscle-specific glycolytic enzyme activities scale positively. However, most of these studies have not included juveniles. In this study, we examined how citrate synthase (CS, EC 2.3.3.1) and lactate dehydrogenase (LDH; EC 1.1.1.27) activity in the jumping muscle of Schistocerca americana grasshoppers varied with ontogeny across a 40-fold increase in body size. In contrast to the pattern observed when adult conspecifics are compared, we show that jumping muscle CS activity increased more than 2-fold from 2nd instars to adults, while jumping muscle LDH activity increased more than 5-fold. The increased LDH activity in older grasshoppers supports previous data that older grasshoppers have a reduced jumping endurance. The increased CS activity with age may help older grasshoppers efficiently produce aerobic ATP to bend cuticular springs for energy storage before a jump or alternatively recover from anaerobic metabolism after jumping. Metabolic changes in S. americana jumping muscle are similar to other developing taxa and highlight the importance of including juveniles within intraspecific studies. When compared to adults, juvenile locomotion may have increased selection pressure because of both greater energetic demands during growth and higher predation rates.

Introduction

When compared to adults, developing animals have increased energetic demands (reviewed in Glazier, 2005), undergo changes in body composition and physiology (mammals: Chappell et al., 2003; lizards: Garland and Else, 1987; insects: Greenlee et al., 2009; birds: Jackson and Diamond, 1995; fish: Young and Egginton, 2009), and have higher mortality rates (insects: Agnew et al., 2002; birds: Frederiksen and Bregnballe, 2000; fish: Gust et al., 2002; crabs: McDonald et al., 2001; mammals: Sendor and Simon, 2003). Therefore, natural selection may significantly influence species evolution during the juvenile stage when energetic or locomotory demands are greatest. While numerous studies have examined the scaling of whole animal resting metabolic rate across adult conspecifics (reviewed in Glazier, 2005), few have examined how development affects muscle biochemistry and physiology. Thus, it is not clear whether the intraspecific scaling patterns in the literature can be applied to the size changes occurring during ontogeny. Most intraspecific studies probably do not include juveniles likely due to the difficulty in collecting them (Blanckenhorn, 2000), determining their age (Walsh, 2010), and/or comparing them with adults that have significant differences in ecological life-history (Fonseca and Cabral, 2007, Juanes, 2007) or locomotory behavior (Blanckenhorn, 2000, Doligez and Pärt, 2008). While these complications are significant, insects provide an excellent model for performing ontogenetic studies because they have discrete developmental stages that can be clearly identified by morphological differences (Blossman-Myer and Burggren, 2010) and often show similar shape, ecological habitats, and locomotory strategies across all developmental stages. Although numerous studies have measured metabolic rate in developing insects (Greenlee and Harrison, 2004, Greenlee and Harrison, 2005, Hetz, 2007), we are not aware of an ontogenetic study that has measured muscle enzymatic activity in a hemimetabolous insect. In this study, we measure aerobic and anaerobic jumping muscle enzymes at different developmental stages of the American locust (Schistocerca americana) to determine whether the muscle biochemistry changes during ontogeny reflect locomotory performance. We also compare the ontogenetic scaling of muscle enzyme activity in S. americana to the enzyme scaling associated with both adult conspecifics and ontogeny in other taxa.

S. americana is an ideal insect to study how a muscle's metabolic profile changes during ontogeny. By molting through six distinct juvenile instars before becoming adults, developing S. americana increase in body mass 50-fold (Greenlee and Harrison, 2004). Throughout their life-history, S. americana have similar body shapes, exist in similar habitats, have similar diets, and can all jump. During repeated jumping, adults and older instars show greater fatigue rates than smaller/younger juveniles (Kirkton and Harrison, 2006). The increased fatigue rates during repeated jumping are positively correlated with over 4-fold greater lactate production rates in the 1st two minutes of repeated jumping (Kirkton et al., 2005). Although older grasshoppers fatigue more rapidly compared to juveniles, their jumping muscle is thought to have a greater mass-specific oxygen consumption (Kirkton et al., 2005). However, this increase in muscle oxygen consumption with age may not be accurate because it is calculated as the difference between the whole body metabolic rate during jumping with and without the autotomized hind legs.

Another approach to assess the metabolic potential of S. americana jumping muscle would be to examine enzymatic activities. Most studies use citrate synthase (CS) as an aerobic marker and lactate dehydrogenase (LDH) as an anaerobic marker of a muscle's metabolic potential (Vetter and Lynn, 1997, Seibel et al., 1998, Norton et al., 2000, Eme et al., 2009, Rosa et al., 2009). For example, in the migratory grasshopper (Locusta migratoria), the adult jumping muscle has over a two-fold greater activity of LDH than CS indicating a high reliance on anaerobic metabolism (Crabtree and Newsholme, 1975). It is unknown how CS or LDH activities vary with age in the jumping muscles of L. migratoria, S. americana, or other grasshopper species. Since older S. americana grasshoppers show reduced endurance with repeated jumping (Kirkton and Harrison, 2006), we predict that muscle-specific LDH activity will increase and muscle-specific CS activity will decrease with age. While not supporting the oxygen consumption data cited above, these enzymatic predictions would be similar to general trends seen in other well-studied conspecific adult taxa (e.g., fish white muscle, reviewed in Moyes and Genge, 2010; and cephalopod mantle muscle Seibel et al., 2000). Most studies have examined only adults of different sizes; when juveniles are included, the enzymatic scaling of both aerobic and anaerobic enzymes can vary significantly from conspecific adult scaling relationships (Vetter and Lynn, 1997, Seibel et al., 1998, Burness et al., 1999, Garenc et al., 1999, Norton et al., 2000, Overnell and Batty, 2000, Rosa et al., 2009).