Lepidoptera are important pollinators of both agricultural crops (Requier et al., 2023) and native plants (Walton et al., 2020), and their feeding behavior and fitness are therefore linked to yields of agricultural crops and conservation efforts, respectively. Crop-emptying rate and nectar allocation choices of insects affect foraging behavior (Piñero et al., 2021, Boggs, 1992). Crop-emptying refers to the release of consumed nectar from a storage organ, the crop. Nectar allocation is the distribution of consumed nectar nutrients to various functions in the body. Therefore, greater understanding of digestive physiology is the first step in predicting how nectar consumption can affect fitness of pollinators and their efficiency as pollinators. Studies on the internal crop organs of adult Lepidoptera and its effects on nutrient allocation are limited. Such studies have focused on crop-emptying rate effects on diuresis (excretion of excess water) after eclosion and feeding (Bushman, 1996, Hainsworth et al., 1991, Bushman et al., 2002). Rate of nectar release from the crop and nectar nutrient absorption have been calculated as “energy processing” in the butterfly Vanessa cardui L. (Hainsworth et al., 1991) and in blowflies (Hainsworth et al., 1990), but as of yet, no study has looked at the timing of when a nectar meal is completely emptied from the crop and allocated into specific body tissues. It is unknown how the crop-emptying rate affects the timing of allocation. This information is necessary for determining how long after the ingestion of nutrients they are used in fitness-increasing traits and behaviors. This study addresses this gap by investigating how a single nectar meal is stored, allocated, and metabolized over time.

Fitness of nectivorous insects is determined in part by when and how they allocate consumed nectar nutrients. Nutrients are allocated to various functions, such as flight, storage, somatic maintenance, growth, and reproduction (Boggs, 2009). Short-term behaviors, such as foraging, can have long-term effects on fitness (Mangel & Clark, 1988). Short-term foraging behaviors may be especially impactful on fitness in systems where food sources are sparsely distributed and feeding events may be rare. We use the model organism Manduca sexta hawkmoth (Sphingidae), which is ideal for this study since adults likely encounter bouts of starvation due to their preferred nectar plant’s variability in availability and sparse distribution (Alarcón et al., 2008, Levin et al., 2016, Raguso et al., 2003, Riffell et al., 2008, Raguso and Willis, 2003).

There is a lag period between the time of nutrient acquisition and the time of nutrient assimilation, affecting the timing of nectar allocation to fitness-increasing behaviors and traits. The rate limiting step in this process is the emptying of the crop (Treherne, 1967, Bernays and Simpson, 1982), an impermeable organ located in the foregut of insects where food is stored before being digested in the midgut (Maddrell & Gardiner, 1980). Once released from the crop, digested nutrients from consumed nectar can be allocated to fitness-related traits such as producing eggs, developing flight muscles, or fat body storage, which then contribute to reproductive output and lifespan (Murphy et al., 1983, Hill & Pierce 1989, Hill, 1989, Boggs and Ross, 1993, Leahy and Andow, 1994, O’Brien, 1999, Mevi-Schütz and Erhardt, 2005, Niitepõld and Boggs, 2015). Therefore, the timing of nectar release from the crop dictates how quickly it can be allocated to fitness-increasing traits or behaviors.

Both the timing of crop-emptying and allocation decisions could be altered by flight behavior. Locomotor activity in bees increases crop-emptying rate, likely due to its higher metabolic cost (Blatt and Roces, 2002a, Blatt and Roces, 2002b). Flight is energetically costly (Bartholomew and Casey, 1978, Casey et al., 1985), but necessary for locating food and mates while avoiding predators. Hawkmoths engage in hovering flight when feeding, one of the most metabolically costly behaviors (Biewener & Patek, 2018). The high metabolic cost of flight could increase the crop-emptying rate, leading to faster assimilation of consumed nutrients. Flight can affect when and how nutrients are allocated throughout the body for functions other than fueling flight itself. Nectar sugars are allocated to flight muscle to repair oxidative damage from flight in moths (Levin et al., 2017a), and flight increases the amount of consumed nectar sugars allocated to eggs in butterflies (Niitepõld and Boggs, 2015, Boggs and Niitepõld, 2014).

The fitness benefits of nutrient allocation decisions can vary by sex. Allocation strategies may be driven by sex-specific differences in behavior, including the types of flowers male and female Lepidoptera visit and their feeding frequency (Smith et al., 2019). In Manduca sexta, sex affects body size (Stillwell and Davidowitz, 2010), floral preference (Alarcón et al., 2010), feeding frequency (Ziegler, 1991), and lifespan (Wone et al., 2018). Nectar amino acid utilization is sex specific in M. sexta; for example, males allocate more consumed amino acids to fight muscle than do females (Levin et al., 2017b).

In this study we first determined how long it takes for consumed nectar to be released from the crop and allocated to body tissues in sedentary male and female moths over a period of four and a half days, about the estimated lifetime of a wild M. sexta moth (Levin et al., 2016). In this sedentary lifetime experiment, we measured the volume of nectar stored in the crop over time and sugar allocation after a single nectar meal in unmated sedentary male and female M. sexta hawkmoths. Second, we examined the effect of flight activity on crop-emptying and immediate pattern of sugar allocation to body tissues. In both the sedentary lifetime experiment and activity experiment, consumed sugars were tracked in two tissues, flight muscle and fat body. We used stable carbon isotopes to determine where moths allocate consumed nectar nutrients. Studies on sugar allocation have used natural differences in the 13C fractionation of sugars produced by plants that use the C3 and C4 photosynthetic pathways (O'Brien et al., 2004, Boggs and Niitepõld, 2014). Lepidoptera raised on a larval diet of C3 plant sugar (beet) have a different isotopic signature than that of C4 plant sugar (cane), making it possible to track where fed moths allocate cane sugar from an artificial nectar.

We predicted that males and females differ in both the rate at which they empty their crop as well as in the timing and location in the allocation of consumed nutrients to flight muscle and fat body. As fed M. sexta males have larger flight muscles than unfed males (Levin et al., 2016), we expected males to allocate more consumed sugar to flight muscles than females. We expected resting moths to allocate more consumed sugar to fat body, as it has been documented that sedentary flies store excess energy from the crop as lipids (Hainsworth et al., 1990). Additionally, we predicted that flight would increase the rate at which the crop is emptied as well as affect the allocation pattern, with nutrients preferentially sent to flight muscles to fuel flight or repair oxidative damage and to the fat body as lipids are used to fuel flight preferentially after 30 min of flight (Ziegler and Schulz, 1986a).