Discussion
In contrast to the N limitation hypothesis, we showed that females preferred (Fig. 2), survived longer (Fig. 3), and laid heavier eggs (Fig. 4) on plants that had lower protein:carbohydrate ratios. Males showed no difference in preference or survival to the fertilization treatments. We ran the plant preference experiment five days after fertilizing the millet. Despite there only being a marginal effect on carbohydrate content at that point (Fig. 1A), female locusts selected control plants over plants treated with high fertilizer. At that time, there was no effect of fertilization on specific leaf area (SLA). After 12 days, SLA was lower in the control treatment than the high fertilization treatment, indicating that control plants were tougher. It is thus unlikely that leaf toughness played a significant role in plant choice or locust performance in this study. After 18 days, the addition of nitrogen to soil resulted in both higher protein and lower carbohydrate levels in the millet leaves (Fig. 1), most likely because carbon was mobilized for protein building (42).
Male body size may be more important than their diet in contributing to egg size. Typically, egg size is positively correlated with the female’s size and nutritional investment (43, 44); however, in this case total egg mass was correlated only with male body size (Table 3). Presumably this relationship is due to male contributing energetically to egg production via the spermatophore that is transferred to the females during mating. Nutrient transfer from the spermatophore to the eggs has previously been demonstrated for grasshoppers although the exact mechanism is poorly understood (45, 46). Females, relative to males, were more attuned to dietary macronutrients, potentially due to the significant impact on female survival (Figure 3) and/or the effect of female nutrition on egg size (Figure 4). Larger eggs yield offspring with higher performance traits across many species, suggesting egg size is a good correlate of fitness (44). To our knowledge this is the first time that increased plant protein:carbohydrates ratios have been shown to have a direct negative impact on reproductive output in field populations.
We hypothesize that migratory species are more likely to be carbohydrate limited because migration is energetically costly. Flying metabolic rates can be 20-100 times that of resting and insect thoracic muscles have the highest known mass-specific oxygen consumption rates (47–49). To accumulate fat reserves before and during migration animals usually rely on nutrient and diet selection (36, 49, 50). Accordingly, in the laboratory, several locust species have been reported to self-select carbohydrates-biased intakes both as nymphs and adults (18, 51) and locusts will increase carbohydrate consumption after long-distance flights (52). By contrast, a study on millet in the United States found that fertilization did not affect levels of herbivory for two non-migratory grasshopper species (53). Female locusts may have been more sensitive to carbohydrate limitation than males in this study because females likely have higher energetic demands for flight (54). Female grasshoppers, including locusts, are significantly larger than males (55). For many species migration occurs pre-reproductively (56, 57) but this is not always the case for locusts and few other insect species. For instance, female green darners (Odonata) migrate along the eastern seaboard of the United States while gravid (58). Female O. senegalensis (the current study species) can reproduce two to three times during their lifetime, amongst migration events, and an ootheca’s mass can exceed 150 mg, 20% or more of their body mass (59). These factors may contribute to females having higher carbohydrate demands, despite the established role of protein in optimization of reproductive output (14, 60, 61).
Free-living locust populations may have difficulty securing these preferred high carbohydrate, low protein diets. Despite the study taking place in an arid environment, which are usually described as being N poor (62, 63), most plants likely do not allow locusts to meet their carbohydrate demands. We previously showed that the majority of potential host plants across all major land use types in the region (millet, groundnut, fallow, and grazing) were protein biased relative to their preferred macronutrient balance of p1:c1.6 (64). Fields with the lowest soil organic matter hosted plants with the lowest N content, which attracted the most locusts (65). O. senegalensis is a major millet pest; however, the millet leaves in this experiment, including unfertilized millet, were protein biased relative to locusts’ carbohydrate biased demands (Figure 1; 62). For grasshoppers, including locusts, carbohydrates are more difficult to extract from plants than protein (66), potentially exacerbating the carbohydrate limitation further.
There is increasing evidence that field populations of locusts and other animals can be carbohydrate, not protein, limited (51, 67–69). Our study illustrates a field scenario where carbohydrate limitation can negatively affect fitness, suggesting far-reaching implications for population and evolutionary dynamics. While plant carbon and nitrogen are widely recorded for ecological studies, plant carbohydrate content is rarely measured in relation to herbivore performance (70), despite the demonstrated importance of macronutrient ratio on growth, survival, lifespan, and reproduction across many taxa (14, 14, 60, 61, 71–77). This study reinforces the realization that animals have different nutrient demands based on life history and the importance of non-protein energy for herbivore communities. This research provides new insights for a complementary hypothesis to the N-limitation paradigm: the carbohydrate limitation hypothesis.