Abstract

Migration often is associated with movement away from areas with depleted nutrients or other resources, and yet migration itself is energetically demanding. Migrating Mormon crickets Anabrus simplex (Orthoptera: Tettigoniidae) lack nutrients, and supplementation of deficient nutrients slows migratory movements and enhances specific aspects of their immune systems. Migrants deficient in proteins have less spontaneous phenoloxidase (PO) activity, whereas those deficient in carbohydrates have lower lysozyme-like anti-bacterial titers with a proposed compromise between migratory and anti-bacterial activities. To investigate the relationship between diet, movement, and immunity further, we removed Mormon crickets from a migratory band and offered each cricket one of five diets: high protein, high carbohydrate, equal weight of proteins and carbohydrates (P + C), vitamins only, or water only for 1 h. We then attached a radio, returned each to the migratory band, and recaptured them 18–24 h later. Mormon crickets fed protein moved the furthest, those with only water or only vitamins moved less, and those fed carbohydrates or P + C moved the least. Standard intake trials also indicated that the Mormon crickets were deficient in carbohydrates. Consistent with a previous study, lysozyme-like anti-bacterial activity was greatest in those fed carbohydrates, and there was no difference between those fed water, protein, or P + C. Crickets were removed from the same migratory band and fed one of four diets: high P, high C, P + C, or vitamins only, for 1 h. Then the crickets were held in captivity with water only for 4 or 24 h before blood was drawn. Immunity measures did not differ between times of draw. Diet treatments had no effect on anti-bacterial activity of captive Mormon crickets, whereas total PO was greater in those fed protein. These results support the hypothesis of a direct compromise between migratory and anti-bacterial activities, whereas PO is compromised by low protein independent of migratory activities. We discuss the potential effects of climate on nutritional deficits and susceptibility to different pathogens.

Introduction

Balancing dietary intake to optimize allocation to growth, maintenance, and reproduction is a challenge for organisms in a changing environment (Rapport 1980; Simpson et al. 2004). Scarcity of nutrients or other resources may result from changes in temperature, humidity, or other abiotic factors affecting primary productivity, or from interspecific or intraspecific competition, such as local crowding (Farrow 1990; Despeland et al. 2000). Scarcity in local nutrients or other resources can motivate animals to migrate. As a result of local scarcities, some or all members of the population move to sites where resources may be more abundant. However, moving long distances is also energetically demanding. If changes in primary productivity or consumer density can be predicted, then animals may prepare for their migratory behavior by depositing fat before food becomes scarce. However, changes in primary productivity and consumer density often occur without warning, and particularly when critical nutrients are scarce initially, animals find themselves poorly nourished (Mattson and Haack 1987). Animals in nutrient-poor habitats may redirect resources from other life-history traits, such as reproduction (Johnson 1969; Zera and Harshman 2001) or immunity (Srygley et al. 2009; Srygley and Lorch 2011), to fuel migration.

Migrating bands of Mormon “crickets” (actually katydids), common during the current outbreak in the U.S. Great Basin, are an excellent model organism for understanding mass migrations of grasshoppers, locusts, and other insects. They are also useful for understanding interactions between diet, migration, and immunity. Mormon crickets in some bands seek proteins over carbohydrates for their diet (Simpson et al. 2006; Srygley et al. 2009), whereas in other bands they prefer carbohydrates over proteins (Srygley and Lorch 2011). In locusts, preferences for particular nutrients depend on deficiencies due to past diets (Raubenheimer and Simpson 2003). Although studies of dietary effects on immunity are rare (Schmid-Hempel 2005), limitation of nutrients appears to be detrimental to immune function (Moret and Schmid-Hempel 2000; González-Tokman et al. 2011). Mormon crickets with different dietary needs also contrast strongly in their immune systems; protein seekers have less phenoloxidase (PO) enzyme circulating in their blood, whereas carbohydrate seekers have less anti-bacterial enzyme activity (Srygley et al. 2009; Srygley and Lorch 2011). Only two other studies have addressed the role of nutrition and immunity in Orthoptera (Jacot et al. 2005; Adamo et al. 2010), and only a few have investigated it in other insect orders (Coleoptera: Rantala et al. 2003; Lepidoptera: Ojala et al. 2005; Lee et al. 2006; Klemola et al. 2007; Lee et al. 2008; Povey et al. 2009). Parental diet has transgenerational effects in Plodia moths, with poor diets for either or both parents causing decreased PO titers and fewer circulating hemocytes in offspring (Triggs and Knell 2012).

In Mormon crickets, we hypothesize that a direct conflict exists between migratory and anti-bacterial activities and is most evident when dietary carbohydrates are limited and fats are shuttled to the muscles as fuel for migration (Srygley and Lorch 2011). We focus on lysozyme-like activity against Gram-positive bacteria for our measure of antibacterial activity in Mormon crickets because, unlike other insects, antimicrobial peptides (AMPs) are little known in Orthoptera. As far as we are aware, locustin is the only AMP identified to date (Swiss-Prot: P83428.1; characterized from migratory locust hemocytes with activity against Gram positive Micrococcus luteus). No AMPs have been found in other orthopterans. Adamo (2004) suggested that activity of the hemolymph of the cricket Gryllus against Gram negative bacteria indicates it has more than just lysozyme-like enzymes. However, this conclusion comes from the assumption that lysozyme-like enzymes are not thought to be active against Gram negative bacteria. In Lepidoptera, some lysozymes are active both against Gram-positive and Gram-negative bacteria (Gandhe et al. 2007; Wang et al. 2009). Hoffmann et al. (1996) suggested that the orthopterans might also rely on lysozyme-like enzymes for defense against both types of bacteria, but more work needs to be done on AMPs in Orthoptera.

In contrast, we hypothesize that PO is compromised by poor protein nutrition independent of migratory activities (Srygley et al. 2009). PO is a key enzyme in the generalized immune response of insects to wounding and invasion. PO catabolizes tyrosine to produce toxic quinones that polymerize to form melanin (for a recent review, see González-Santoyo and Córdoba-Aguilar 2012). The PO cascade is an important part of cellular encapsulation of foreign bodies because melanization hardens the cell mass and suffocates intruders (Kanost and Gorman 2008).

Seeking to compare the effects of protein and carbohydrate diets with that of diets lacking macronutrients, we offered a range of diets (with and without protein, carbohydrates, and essential vitamins, minerals, and oils) to Mormon crickets before releasing them back into a migrating band. For Mormon crickets with a carbohydrate deficiency, we predicted results consistent with those from the previous year: migratory velocity would decrease and lysozyme-like, anti-bacterial enzymatic activity would increase following consumption of a diet rich in carbohydrates compared with a diet rich in proteins. We also predicted that the two sets of controls (vitamins only versus wildtypes) would be similar in migratory velocity and enzymatic immunity. We predicted that (1) those fed a diet rich in carbohydrates would have a migratory velocity less than that of the two sets of controls, (2) anti-bacterial activity would be greater than for the two sets of controls, and (3) these activities of Mormon crickets given a diet rich in protein would not differ from the controls. Finally, we predicted that (4) a P + C diet would be intermediate between those fed either P or C. In other words, those fed a P + C diet would probably not differ substantially from the controls.

In addition, we investigated whether dietary effects on enzymatic immunity of captive insects reduced the effects of migration. Because we hypothesized that locomotion compromises anti-bacterial activity, we predicted that anti-bacterial activity would be independent of diet in captivity. Our prior studies differed in the time between dietary supplementation and sampling of hemolymph, and so we also explored whether this lag affects the PO and anti-bacterial enzymatic activities. Finally, we discuss possible consequences of nutritional deficits driven by abiotic factors for infections by fungi and bacteria.

Materials and methods

Mormon cricket bands and the study site

Mormon crickets, Anabrus simplex (Orthoptera: Tettigoniidae), are univoltine katydids that hatch in the spring from eggs laid singly in the soil. From the fourth instar onward they can begin to migrate, while completing their seven nymphal instars and molting to adults. As teneral adults, they gain additional weight and become reproductively mature in approximately 6–8 days in males and 10–12 days in females. Very little is known about the ecological mechanisms underlying outbreaks, aggregation, or migration. Outbreaks typically originate on rangeland and are characterized by spectacular migratory bands that can be over 10 km long, several kilometers wide, contain dozens of insects per square meter, and travel up to 2 km per day (Cowan 1929; Wakeland 1959; MacVean 1987; Lorch and Gwynne 2000; Gwynne 2001; Lorch et al. 2005). The current outbreak began in 1998 and is one of the most severe on record, with 5 million hectares in Nevada alone estimated to have been infested in 2004 (Knight 2007), which is six times the area recorded in 1939 for Nevada during another historic outbreak (Cowan 1990).

Between June 30 and July 7, 2009, we studied a band of pre-reproductive adult Mormon crickets in Independence Valley (latitude 41° 13′ 12″ N, longitude 116° 2′ 35″ W, elevation: 1940 m) of the Independence Range in Elko County approximately 50 km northwest of Elko, Nevada. This site was only 3 km from the band studied at Eagle Mountain in 2008 and described by Srygley and Lorch (2011). The density of adult Mormon crickets in the band at Independence Valley ranged from 10.3 to 14.0 m−2, and they were heading 70° from geographic North (R. B. Srygley, unpublished data). The floor of the valley is dominated by sagebrush (Artemisia tridentata), typical vegetation for high Great Basin desert scrubland. Mormon crickets are omnivorous; they eat broad-leafed plants, invertebrates, and fungi, scavenge on dead vertebrates and road kills, and cannibalize other Mormon crickets (Ueckert and Hansen 1970).

Intake trials

We characterized the dietary intake of field-collected Mormon crickets following the methods of Simpson et al. (2006). We prepared a 42% protein diet consisting of a 3:1:1 mix of casein, peptone, and albumen and a 42% carbohydrate diet consisting of equal parts of sucrose and dextrin. Both diets contained 54% cellulose and 1.8% Wesson’s salt mixture and 2.2% vitamins, linoleic acid, and cholesterol. In a free-choice experiment, four male and four female Mormon crickets were collected from the migratory band on June 30, housed individually with free access to water and 0.5 g of each diet for 24 h. After 24 h, the diets were removed and replaced with fresh diet, which remained with the insects for an additional 24 h. As a replicate, five males and three females were collected on June 30 from a second band that we did not radiotrack and given the same choice of diet. This band was 7 km east of the band used for release-recapture and for the laboratory experiments described below. The dry masses consumed from each diet is a measure of the relative intake of carbohydrates:protein over the first and second 24-h period. The diet provided during the second 24-h period is closer to the ideal intake for the band. A vector drawn from the mean intake during the first 24-h period and that for the second 24-h period indicates the dietary needs of members drawn from the migrating band relative to the same insects with free choice of diet. A migrating band is deficient in protein (or carbohydrate) when its members prefer to feed on proteins (or carbohydrates) when first captured and then switch to a more even ratio of proteins and carbohydrates (Simpson et al. 2006).

Manipulation of diet, radio-tracking, and collection of hemolymph

We captured 25 males and 25 females on July 3 and 20 males and 20 females crickets on July 6, 2009. Each was held in a 1-l translucent container and given one of the five diets: a 42% protein diet, a 42% carbohydrate diet (see above), a diet composed of equal parts protein and carbohydrate (21% for each constituent), a control diet lacking macronutrients (this we call the O diet: 96% cellulose, 1.8% Wesson’s salt mixture, and 2.2% vitamins, linoleic acid, and cholesterol), and another control group that did not receive any food (these we refer to as wildtypes). The insects were shaded beneath a tarpaulin and allowed to feed from their diet for 1 h between 15:30 and 16:30 h. In order to measure consumption, the food was weighed to the nearest milligram on an Ohaus field-portable microbalance (model AV53) before and after presentation to the crickets.

In order to follow each Mormon cricket’s migratory path, a 0.4 g radio-transmitter (Biotrack, Ltd., UK) was glued to the pronotum. The added mass of the radio did not affect locomotion in the laboratory (Lorch et al. 2005). Sex, treatment, and the unique radiofrequency that identified each insect were recorded. Using a Trimble GPS datalogger to record location and time for each cricket, we released the insects back into the band with each cricket initially separated from other marked individuals by ca. 8 m on a linear transect perpendicular to the general direction of movement of the band at that time. On July 3, crickets were released into the band between 18:30 and 18:55 h and retrieved the following day between 11:00 and 17:00 h (PDT) for the first group (six were not recaptured until July 5 between 11:00 and 17:00). Two from this first group were excluded because they were either predated or lost their radio within 7 m of release. On July 6, crickets were returned to the band between 18:15 and 18:35 and recovered the following day between 10:30 and 15:15 (PDT). One from this second group was predated within 12 m of release. The location (±1 m) and time of recapture were recorded, and each cricket was placed in a 50-ml Corning plastic centrifuge tube. Velocity (mm s−1) was calculated as the straight-line distance between release and recapture over time.

Returning to our field laboratory in Taylor Canyon, Nevada, in the early afternoon, we measured body mass of each cricket to the nearest milligram with the Ohaus microbalance. We drew hemolymph by puncturing the arthrodial membrane at the base of each insect’s hind leg with a 26-gauge hypodermic needle. In Orthoptera, prophenoloxidase (proPO) is primarily held in circulating hemocytes until wounding or infection (Kanost and Gorman 2008). Hence lysis of hemocytes at the wound may cause local elevation of proPO and initiation of the proPO cascade to yield spontaneously-active PO. Puncturing again, if necessary, 15 μl of hemolymph was collected into a capillary tube. The hemolymph was diluted 1:50 with phosphate buffered saline (PBS) solution to be used in assays of spontaneous PO and proPO enzymatic activity and total hemolymph protein. An additional capillary tube with 10 μl of hemolymph diluted 1:4 with PBS was collected for assay of anti-bacterial activity. We immediately froze the hemolymph samples at −18°C in a field-portable Engel freezer (model MT45F-U1).

We captured a second set of 48 Mormon crickets from the same band and held each in a 1-l translucent container. Each was given one of the four diets: P, C, P + C, or O. The insects were allowed to feed from their diet for 1 h between 13:20 and 14:20. In order to measure consumption, diets were weighed to the nearest milligram on the microbalance before and after presentation to the crickets. These were held in captivity in the field laboratory on natural photoperiod for an additional 4 or 24 h when hemolymph was drawn and stored, as described for the released-recaptured crickets above.

Immunocompetence assays

We followed the protocols detailed by Srygley and Lorch (2011). Briefly, samples of thawed hemolymph diluted in PBS were centrifuged and activated with 10 mM dopamine solution to measure spontaneous PO activity. The plate was loaded into a temperature-controlled Biotek microplate reader (25°C), and absorbance at 492 nm was read between 5 and 15 min. If absorbance of the sample was linearly related with time, we calculated mean V (change in absorbance min1). One unit PO activity per milliliter of hemolymph is defined as the amount of enzyme resulting in a 0.001 increase in absorbance.

To measure total combined activity of PO and proPO, we adapted the protocol of Goldsworthy et al. (2002). We dissolved 1 mg alpha-chymotrypsin from bovine pancreas (Sigma) in 1 ml PBS, combined an equal volume of this solution with centrifuged hemolymph in PBS (1:50), and incubated for 30 min. In the plate wells, we added 5 µl of the incubated solution to 195 µl 10 mM dopamine. As above, mean V was calculated from plate readings between 5 and 15 min to measure total PO activity in units ml−1 of hemolymph.

To measure lysozyme-like antibacterial activity, a turbidimetric method was used. Ten microliters of thawed and PBS-diluted hemolymph (1:4) was added to a well with 140 µl of Gram-positive cells of the bacterium Micrococcus lysodeikticus (Worthington) suspended in PBS (0.5 µg/µl). Clearing of the well was compared with a serial dilution of egg-white lysozyme (Sigma) added to the bacterial suspension. The plate was loaded into a temperature-controlled Biotek microplate reader (25°C) and absorbance at 450 nm read between 10 and 30 min. If the sample absorbance was linearly related with time, we calculated mean V. When activity of the sample fell below 6.5 μg ml−1, the sample was excluded because the standards showed that such data were unreliable.

Statistical analyses

To analyze the effects on marching velocity, which did not differ from a normal distribution (P = 0.66), we conducted a forward stepwise regression analysis using JMP 6.0.2 (SAS Inc.) with velocity as the dependent variable, body mass as the covariate, sex, day of release, and diet as independent factors. The analysis first divided the five diet treatments to make two group levels with the greatest separation between means of the responses, and then those two groups were further divided to maximize the separation within each group, continuing hierarchically until all levels fell into four terms. All two-way and three-way interactions were evaluated. We selected the model that minimized the Akaike Information Criterion.

When we analyzed immunity measures of released-recaptured Mormon crickets to see how they were affected by sex, diet, and body mass, not all immunity assays were conducted on all insects, and so we ran analysis of covariance (ANCOVA) on each measure of immunity separately. Anti-bacterial activity was log10 transformed to normalize this dependent variable (P = 0.41), whereas total PO did not differ from a normal distribution (P = 0.50). In building our models, body mass was entered as a covariate and sex and diet were independent factors with all interactions evaluated. Interactions that were not significant were removed from the model. For significant factors, multiple comparisons of the least squares means of the dependent variable were conducted post hoc using Student’s t-tests.

Because the distribution of spontaneous PO activity in released-recaptured Mormon crickets was bimodal, we used the non-parametric Kruskall–Wallis test to evaluate differences among treatments within each sex.

We used ANCOVA to evaluate effects of body mass, lag-time, sex, and diet on anti-bacterial activity, total PO, and spontaneous PO activities of captive insects. Anti-bacterial activity and total PO were log10 transformed to normalize their distributions. We evaluated three-way interactions, which were removed from the model if not significant. Then we evaluated two-way interactions, which also were removed from the model if not significant before evaluating the main effects of the factors. Multiple comparisons of the least squares means were conducted as stated above.

Results

Intake trials

Mormon crickets from the band in Independence Valley ate more carbohydrate than protein with about 1.5 times the consumption of C to P on the first day, followed by a more even consumption of C and P on the second (approximately 1.2:1 C:P, Fig. 1). The disparity in consumption of C and P on the first day was not as great as for the band studied the previous year, which consumed a C:P ratio as great as 5:1 on the first day of the experiment. Convergence of intake vectors from both years (Fig. 1) suggests an intake target of approximately 1.3:1. Hence, the Mormon crickets from Independence Valley showed compensatory feeding behaviors consistent with a deficiency of carbohydrates in their natural diets (Simpson and Abisgold 1985).

Fig. 1

Results of intake trials of Mormon cricket bands near Tuscarora Nevada showing vectors drawn from carbohydrate:protein dietary intake on day 1 (diamonds) to that on day 2 (squares). The bold arrow indicates the band whose members were released and recaptured, and the vector for a second band from Independence Valley measured simultaneously is also shown. For comparison, intake vectors of two replicates from a band at Eagle Mountain in the previous year (open symbols) is adapted from Srygley and Lorch (2011). The dashed line indicates consumption of an even ratio of carbohydrates to proteins.

Fig. 1

Results of intake trials of Mormon cricket bands near Tuscarora Nevada showing vectors drawn from carbohydrate:protein dietary intake on day 1 (diamonds) to that on day 2 (squares). The bold arrow indicates the band whose members were released and recaptured, and the vector for a second band from Independence Valley measured simultaneously is also shown. For comparison, intake vectors of two replicates from a band at Eagle Mountain in the previous year (open symbols) is adapted from Srygley and Lorch (2011). The dashed line indicates consumption of an even ratio of carbohydrates to proteins.

Diet and migratory movement

Only the first hierarchical split in diet treatment was selected to best explain migratory velocity. The two diet levels that significantly separated the mean migratory velocities were C and P + C combined, contrasted with P, O, and W combined (Fig. 2, Table 1). The estimated mean velocity for C and P + C combined (5.58 mm s−1) was slower than that for the other diet treatments combined (6.84 mm s−1).

Fig. 2

Least-squares means of migratory velocity (±s.e.) of Mormon crickets covaried with body mass were dependent on diet (P: protein, C: carbohydrate, PC: protein + carbohydrate, O: vitamins + salt control, W: unfed wildtype).

Fig. 2

Least-squares means of migratory velocity (±s.e.) of Mormon crickets covaried with body mass were dependent on diet (P: protein, C: carbohydrate, PC: protein + carbohydrate, O: vitamins + salt control, W: unfed wildtype).

Table 1

Model that best explained migratory velocity of Mormon crickets following stepwise regression on date of release, dietary treatments, gender, size, and all two-way and three-way interactions

Factor SS MS d.f. F P 
Diet (P&W&O-C&PC) 32.9 32.9 4.25 0.042 
Error 650.1 7.7 84   
Factor SS MS d.f. F P 
Diet (P&W&O-C&PC) 32.9 32.9 4.25 0.042 
Error 650.1 7.7 84   

Diet and immunity in released and recaptured Mormon crickets

Log lysozyme activity increased significantly with body mass, and diet was also a significant factor (Fig. 3, Table 2). As predicted, Mormon crickets fed a C diet had greater anti-bacterial activity than did those fed a P diet. Also as predicted, the two controls were not significantly different in anti-bacterial activity and their means were intermediate between those fed C and those fed P. As predicted, anti-bacterial activity of those fed P did not differ significantly from either set of controls. However, those fed a C diet were not significantly different from those fed vitamins only, but did differ significantly from the wildtypes. Finally, Mormon crickets fed a diet of both P and C were not significantly different from those fed P alone, or from the wildtype controls.

Fig. 3

Least-squares means of antibacterial activity (±s.e.) covaried with body mass were dependent on dietary treatment in the release-recapture experiment (open bars), whereas the effect of dietary treatment was not significant in the laboratory experiment (gray bars; see legend of Fig. 2 for abbreviations). Different letters above the columns indicate significant differences in multiple comparisons of the means (P < 0.05) within each experiment. For comparison with the field results, the dashed line indicates the mean for the Mormon crickets in the laboratory experiment.

Fig. 3

Least-squares means of antibacterial activity (±s.e.) covaried with body mass were dependent on dietary treatment in the release-recapture experiment (open bars), whereas the effect of dietary treatment was not significant in the laboratory experiment (gray bars; see legend of Fig. 2 for abbreviations). Different letters above the columns indicate significant differences in multiple comparisons of the means (P < 0.05) within each experiment. For comparison with the field results, the dashed line indicates the mean for the Mormon crickets in the laboratory experiment.

Table 2

ANCOVA of log antibacterial activity following different dietary treatments of captured-released Mormon crickets of differing gender and size

Factor SS d.f. F P 
Mass 0.044 5.01 0.029 
Sex 0.000 0.00 0.99 
Diet 0.124 3.49 0.013 
Model 0.166 3.13 0.0105 
Error 0.479 54   
Factor SS d.f. F P 
Mass 0.044 5.01 0.029 
Sex 0.000 0.00 0.99 
Diet 0.124 3.49 0.013 
Model 0.166 3.13 0.0105 
Error 0.479 54   

Total PO (proPO + spontaneous PO) increased significantly with body mass but did not vary significantly with sex or diet (Fig. 4, Table 3). Within each sex, spontaneous PO activity was not significantly related to body size (n = 36: F = 0.11, P = 0.74; n = 33 males: F = 3.29, P = 0.08) nor did it differ significantly between dietary treatments (Fig. 5; Kruskal–Wallis tests: females: χ2 = 5.27, d.f. = 4, P = 0.26; males: χ2 = 1.03, d.f. = 4, P = 0.90).

Fig. 4

Least-squares means of total PO activity (±s.e.) covaried with body mass were not significantly dependent on diet in the release-recapture experiment (open bars), whereas the effect of diet was significant in the laboratory experiment (gray bars; see legend of Fig. 2 for abbreviations). Different letters above the columns indicate significant differences in multiple comparisons of the means (P < 0.05). For comparison with the laboratory results, the dashed line indicates the mean for the Mormon crickets in the release-recapture experiment.

Fig. 4

Least-squares means of total PO activity (±s.e.) covaried with body mass were not significantly dependent on diet in the release-recapture experiment (open bars), whereas the effect of diet was significant in the laboratory experiment (gray bars; see legend of Fig. 2 for abbreviations). Different letters above the columns indicate significant differences in multiple comparisons of the means (P < 0.05). For comparison with the laboratory results, the dashed line indicates the mean for the Mormon crickets in the release-recapture experiment.

Fig. 5

Spontaneous PO activity (mean ± s.e.) for females (open bars) and males (gray bars) in the release-recapture experiment were independent of diet (see legend of Fig. 2 for abbreviations).

Fig. 5

Spontaneous PO activity (mean ± s.e.) for females (open bars) and males (gray bars) in the release-recapture experiment were independent of diet (see legend of Fig. 2 for abbreviations).

Table 3

ANCOVA of total PO activity following dietary treatments of captured-released Mormon crickets of differing gender and size

Factor SS d.f. F P 
Mass 25,316,730 6.60 0.0122 
Sex 2,504,258 0.65 0.42 
Diet 31,030,382 2.02 0.0997 
Model 54,601,894 2.37 0.0373 
Error 291,543,658 76   
Factor SS d.f. F P 
Mass 25,316,730 6.60 0.0122 
Sex 2,504,258 0.65 0.42 
Diet 31,030,382 2.02 0.0997 
Model 54,601,894 2.37 0.0373 
Error 291,543,658 76   

Diet and immunity in captive Mormon crickets

Mass, sex, diet, and draw time did not have significant effects on log anti-bacterial activity of captive insects (P = 0.81, 0.60, 0.12, and 0.49, respectively, Fig. 3; two-way and three-way interactions were not significant). On average, log-transformed anti-bacterial activity was 3.215 ± 0.018 (mean ± s.e.) at 4 h and 3.205 ± 0.017 at 24 h. Log total PO activity increased significantly with mass and was significantly affected by diet, but not by sex nor draw time (Table 4, two-way and three-way interactions were not significant). On average, log-transformed total PO activity was 4.092 ± 0.025 (mean ± s.e.) at 4 h and 4.045 ± 0.022 at 24 h. Multiple comparison of the mass-adjusted means indicated that animals on P and P + C diets had significantly higher total PO activity than did those fed vitamins only (Fig. 4). P, C, and P + C were not significantly different, and C and vitamins-only were not significantly different. Spontaneous PO activity was significantly greater in females than in males, but it was not significantly affected by mass, draw time, or by diet (Table 5; two-way and three-way interactions were not significant). Circulating titers of PO averaged 663 ± 39 units/ml hemolymph in blood drawn at 4 h and 601 ± 45 units/ml hemolymph at 24 h.

Table 4

ANCOVA of log total PO activity of captive Mormon crickets with blood drawn 4 or 24 h following dietary treatment

Factor SS d.f. F P 
Mass 0.0974 7.76 0.0076 
Sex 0.0140 1.11 0.30 
Diet 0.1178 3.13 0.0340 
Drawtime 0.0111 0.89 0.35 
Model 0.2527 3.36 0.0075 
Error 0.6151 49   
Factor SS d.f. F P 
Mass 0.0974 7.76 0.0076 
Sex 0.0140 1.11 0.30 
Diet 0.1178 3.13 0.0340 
Drawtime 0.0111 0.89 0.35 
Model 0.2527 3.36 0.0075 
Error 0.6151 49   
Table 5

ANCOVA of PO activity of captive Mormon crickets with blood drawn 4 or 24 h following dietary treatment

Factor SS d.f. F P 
Mass 969 0.03 0.86 
Sex 493473 15.3 0.0003 
Diet 43307 0.45 0.72 
Drawtime 58911 1.82 0.18 
Model 683840 3.52 0.0065 
Error 1358134 42   
Factor SS d.f. F P 
Mass 969 0.03 0.86 
Sex 493473 15.3 0.0003 
Diet 43307 0.45 0.72 
Drawtime 58911 1.82 0.18 
Model 683840 3.52 0.0065 
Error 1358134 42   

Discussion

Lacking carbohydrates in their diet, the Mormon crickets we studied probably were migrating in search of scarce macronutrients. Mormon crickets that were removed from the band ate more carbohydrates than protein; and those that ate protein moved more quickly after returning to the band than did those that fed on carbohydrates. Those that were fed vitamins only as well as the unfed wildtypes moved like those fed only carbohydrates, which suggests that those fed a protein diet moved more. Protein-fed Mormon crickets behaved as if they had found an unsuitable patch in which to forage and consequently migrated faster than did those fed carbohydrates. Those fed equal parts of carbohydrates and proteins behaved like those fed carbohydrates alone, suggesting that it was an insufficiency of protein in their diet that spurred them on. We conclude that the Mormon crickets were migrating through an environment relatively abundant in protein and scarce in carbohydrates.

In contrast to dietary effects on migratory activity, lysozyme-like anti-bacterial activity of Mormon crickets that were fed carbohydrates was greater than in those that were fed proteins. Anti-bacterial activity of those that were fed vitamins only and the unfed wildtypes were not significantly greater than in those fed protein alone, which suggests that those fed a carbohydrate diet showed an increase in anti-bacterial activity. Those fed equal parts carbohydrates and proteins had enzyme concentrations like those fed protein alone, suggesting that an insufficiency of carbohydrate in their diet reduced their anti-bacterial activity. These results are consistent with the hypothesized trade-off between locomotion and immune activity mediated by apolipophorin III, which has roles in both corporal functions (Weisner et al. 1997; Halwani and Dunphy 1999; Weers and Ryan 2006; Adamo et al. 2008, 2010; Srygley and Lorch 2011). When sugar concentrations in the hemolymph are low, apolipophorin III associates reversibly with high-density proteins that transport lipids from the fat body. When sugar concentrations are high, lipid-free apolipophorin III increases anti-bacterial lytic activity (Weisner et al. 1997; Halwani and Dunphy 1999).

In further support of a trade-off between locomotion and anti-bacterial activity, diet did not have a significant effect on anti-bacterial activity of captive Mormon crickets. However, we predicted that in the absence of migratory activity, anti-bacterial activity would increase for all diet treatments and approach that of the Mormon crickets fed carbohydrates and released back into the band. However, we found that the average for captive animals was well below that for animals fed carbohydrates and was nearer to values for those fed P or P + C and released back into the band (Fig. 3). This suggests that apolipophorin III in Mormon crickets in the laboratory is not in the free state. The insects were able to move freely within the container, but it is not likely that they moved as much as those in the field. They were not observed struggling to escape, except when handled.

We also found that total PO activity did not differ among the dietary treatments for those Mormon crickets released back into the band. However, total PO activity was significantly affected by diet in the captive animals. Those fed macronutrients, whether P, C, or P + C diets, in captivity had greater total PO activity than those fed vitamins only. Spontaneous PO activity was not affected by diet in Mormon crickets released back into the band or in those held captive. This result supports the conclusion that carbohydrate-seeking bands are not limited in PO activity (Srygley and Lorch 2011). It is interesting that total PO activity, but not spontaneous PO activity, increased with body mass. In a previous study of migrating Mormon crickets, spontaneous PO increased with body mass, and we hypothesized that smaller individuals might be younger or malnourished (Srygley et al. 2009). From the day of eclosion to adulthood, Mormon crickets under laboratory conditions gain mass until at least 13 days old. Spontaneous and total PO activities also increase with age in laboratory-reared Mormon crickets (Srygley 2012). Although gain in mass in the field is likely to be more variable than in the laboratory, the association between body mass and total PO is probably due to an underlying association between body mass and age.

Protein-limited Mormon crickets, such as those found in migratory bands in Idaho (Simpson et al. 2006) and Utah (Srygley et al. 2009), are deficient in spontaneous PO. When offered a protein-rich diet, protein-limited crickets increase circulating PO titers but did not elevate anti-bacterial activity. Spontaneous PO titers are directly proportional to the rate of encapsulation of foreign bodies introduced into the hemolymph (Srygley et al. 2009; Srygley and Lorch 2011), and they are also associated with attempted clearing of blastospores and hyphae of the entomopathogenic fungus Beauveria bassiana from the hemolymph (Srygley and Jaronski 2011).

In direct contrast, Mormon crickets that are carbohydrate-limited, such as those we found in Nevada, increase anti-bacterial activity but not spontaneous PO activity following ingestion of carbohydrate-rich foods. Thus, we hypothesize that nutritional deficiencies result in the development of two types of migratory Mormon cricket, one that shows a general reduction in immunocompetence, making it more susceptible to pathogenic fungi, and another that is susceptible to bacteria. The challenge for the Mormon cricket is to balance intake of carbohydrates and proteins in ways that maximize resistance to both types of pathogen.

Effects of climate on macronutrient availability

Temperature and precipitation can have profound effects on the availability of protein and carbohydrates (Jones and Coleman 1992). Studies of dietary intake integrate behavioral decisions and physiological processes at the organismal level, demographic processes at the population level, and patch dynamics of the metapopulation at the landscape level (Simpson et al. 2010). However, in a simpler system in which the Australian plague locust Chortoicetes terminifera eats only two species of grass, measures of protein and carbohydrate in the host plants failed to predict assimilation or growth (Clissold et al. 2006). The precise effects of abiotic changes on the protein and carbohydrate sources sought by the Mormon crickets have not been investigated, but we know that seeds, flower heads, and invertebrates are protein-rich sources sought by Mormon crickets and that fungi are a carbohydrate-rich source. In the water-limited rangeland of the western United States, nitrogen limits net primary productivity and soil moisture increases the ability of plants to absorb nitrogen from the soil (Hooper and Johnson 1999; Yahdjian et al. 2011). As a result, foliage becomes richer in protein in wetter years, and the plants produce more protein-rich flowers and seeds. Rainfall not only influences the amount of protein accessible to Mormon crickets directly from plants but may also influence the abundance of high-protein, rangeland invertebrates upon which Mormon crickets also forage. High density bands of Mormon crickets will generally deplete macronutrients locally and nutrients may become limiting both in wet and in dry years. In drier habitats or years, we would expect Mormon crickets to be protein-limited and less resistant to pathogenic fungi, whereas in wetter habitats or years they should be carbohydrate-limited and more susceptible to bacterial pathogens. We are currently investigating this hypothesized relationship between environment, locomotion, and immunity in Mormon crickets.

This relationship between the environment, diet, and immunity might apply to herbivorous insects in general because they all share these innate defenses: the PO cascade, including the proPO cascade, and anti-bacterial enzymatic activity. Environmental stress, such as moderate drought, can cause mobilization of nitrogen from plant tissues, thereby providing a better source of food for some herbivorous insects (White 1984), but chewing and galling insects generally perform less well on stressed plants (Koricheva et al. 1998). Because we hypothesize that anti-bacterial activity is compromised directly by movement, carbohydrate-limited insects might need to be dispersing or migrating to new habitats to show increased susceptibility to bacteria. Few studies have incorporated movement into investigations of resistance to disease (e.g., Adamo et al. 2008; González-Tokman et al. 2011). Thus, differences in diet and movement between insects in the laboratory and those in nature call into question the validity of applying the results of pathogen tests conducted in a laboratory to field conditions.

Acknowledgments

We gratefully acknowledge Laura Senior (USDA-ARS) for assistance with fieldwork and with assays conducted in the laboratory, and Neil Drinkard for assistance in the field. We thank J. Gaskin and anonymous reviewers for critiquing earlier versions of this manuscript.

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Author notes

From the symposium “Coping with Uncertainty: Integrating Physiology, Behavior, and Evolutionary Ecology in a Changing World” presented at the annual meeting of the Society for Integrative and Comparative Biology, January 3–7, 2013 at San Francisco, California.