PHYSIOLOGICAL ECOLOGY Age- and Density-Dependent Prophylaxis in the Migratory, Cannibalistic Mormon Anabrus simplex (: )

ROBERT B. SRYGLEY1

USDA-Agricultural Research Service, Northern Plains Agricultural Research Laboratory, 1500 N. Central Avenue, Sidney, MT 59270

Environ. Entomol. 41(1): 166Ð171 (2012); DOI: http://dx.doi.org/10.1603/EN11020 ABSTRACT As a result of the increased potential for disease transmission, are predicted to show an increased constitutive immunity when crowded. Cannibalistic aggressive interactions further increase the risk of wounding and pathogen transmission in crowds. Nymphal Mormon crickets Anabrus simplex Haldeman were collected in Montana and reared in the laboratory either solitarily or at densities similar to that experienced by Mormon crickets in migratory bands. As teneral adults, solitarily-reared Mormon crickets tended to have greater phenoloxidase activity than those reared in groups. Sampling enzyme activity a second time when the adults were nearing reproductive maturity, group-reared Mormon crickets had elevated levels of prophenoloxidase and encapsulated foreign objects faster than solitarily-reared insects. Rearing density did not have a signiÞcant effect on either the darkness of the cuticle or antibacterial activity. This is the Þrst report of age-related responses of adult immunity to crowding.

KEY WORDS cannibalism, disease, katydid, lysozyme, melanism

The density-dependent prophylaxis hypothesis is a insects do not (Cotter et al. 2004, Pie et al. 2005, predicted elevation of the baseline immunity of an Adamo 2006, Miller et al. 2009, Elliot and Hart 2010, organism as a response to crowding (Reeson et al. Miller and Simpson 2010). 1998, Wilson and Reeson 1998, Wilson et al. 2002, There are several reasons why organisms may not 2003). It is based on theoretical and empirical evi- respond to crowding with elevated levels of immunity. dence that pathogens are more likely to be transmitted For one, hosts may be more susceptible to infection at among conspeciÞcs in crowded environments. As a higher densities because of nutritional or physiologi- result of organisms evolving to minimize the chance of cal stress (Steinhaus 1958, Lindsey et al. 2009). For infection and maximize survivorship, they should re- example, Srygley et al. (2009) found that Mormon spond to their perception of crowding by increasing crickets, Anabrus simplex Haldeman (Orthoptera: Tet- their constitutive immunity (Møller and Erritzøe tigoniidae), had reduced phenoloxidase activity when 1996). Implicit is that there is a cost to constitutive their diets were deÞcient in protein. For another, immunity that prevents maintenance of this elevated allocation of resources to locomotion could limit al- response when the organism Þnds itself in uncrowded location to immunity. In some insects, antibacterial conditions (Sadd and Siva-Jothy 2006). activity can decrease with locomotor activity because Crowding also brings opportunities for cannibalistic the lipid transport protein apolipoprotein III, which attacks (Bazazi et al. 2008). For the attacker, canni- also has antibacterial activity, is shuttling lipid fuel to balism increases the risk of pathogen transmission muscles (Adamo et al. 2008, Srygley and Lorch 2011). above and beyond that because of crowding alone Many insects respond to crowding by increasing (Pfennig et al. 1998). For potential victims, defense of locomotor and migratory activities (Sword 2005). Lo- breaches in the exoskeleton from invasion by patho- custs, for example, exhibit a suite of life history traits gens and the rapid closure of wounds may be impor- that change between crowded and solitary rearing tant to minimize the risk of additional attacks. environments. Proximity to conspeciÞcs results in a Density-dependent prophylaxis has been investi- developmental regulatory switch that results in dark- gated in a number of insects. Many taxa appear to ened, warningly-colored cuticle, swarming behavior, respond to crowding as predicted with density-de- and migration. In Mormon crickets, proximity to con- pendent prophylaxis (Barnes and Siva-Jothy 2000, Bai- speciÞcs also results in aggregation behavior and mi- ley et al. 2008, Lindsey et al. 2009), whereas other gration, although unlike locusts, these responses to crowding are not the result of a phase-polyphenism 1 Corresponding author, e-mail: [email protected]. (Sword 2005).

0046-225X/12/0166Ð0171$04.00/0 ᭧ 2012 Entomological Society of America February 2012 SRYGLEY:CROWDING AND IMMUNITY OF MORMON CRICKETS 167

Some populations are composed of particularly prone to being cannibalized (Simpson et consistently solitary individuals that move a few tens al. 2006, R.B.S., unpublished data). A last-instar nymph of meters or less in a lifetime (Lorch and Gwynne for which the Þnal molt was delayed relative to the 2000, Lorch et al. 2005), whereas other populations others was cannibalized (R.B.S., unpublished data), have members that tend to aggregate into dense bands and three adults lost all or part of a hind leg on the day moving up to 2,000 m daily. The migratory Mormon that they molted. crickets also are characterized by darkened exoskel- Hemolymph was drawn on the same adults when etons relative to the solitary ones. Bailey et al. (2008) both young and older. On 16 June, eight group-reared showed that the migratory populations have greater Mormon crickets and eight solitary ones of each sex antibacterial lysozyme activity and encapsulate for- were selected to assay immunity of young adults be- eign objects faster relative to members of the solitary tween 0 and 4 d posteclosion. We measured body mass populations. These differences among populations of each cricket to the nearest mg with an Ohaus mi- might be physiological responses to crowding or more crobalance (model AV53, Ohaus Corp., Parsippany, deeply rooted in historical differences between the NJ), and then punctured the arthrodial membrane at solitary and migratory populations (Bailey et al. 2005, the base of the hindleg of each insect with a 26-gauge 2007). The purpose of this paper is to investigate hypodermic needle so that it exuded hemolymph. whether, within a single population, the immune sys- Puncturing again if necessary, 15 ␮l of hemolymph tems of migratory Mormon crickets respond to local were collected into a capillary tube. The hemolymph crowding consistently with density-dependent pro- was diluted 1:50 with phosphate buffered saline (PBS) phylaxis. solution to be used in assays of spontaneous phenoloxi- dase (PO) and total PO activity and total hemolymph Materials and Methods protein. Total PO activity is equal to spontaneous PO plus prophenoloxidase (proPO) activity. Because it is Fifth-instar Mormon crickets were collected at primarily the latter, we call total PO activity proPO in Lodgegrass Montana on 28 May 2009, placed in bug this paper. We immediately froze the hemolymph dorms and brought to the installation in Sidney, MT. samples at Ϫ20ЊC. The Mormon crickets were re- The next morning in the laboratory, we set up four turned to their respective group cages or solitary cups. groups of 12 nymphs (six of each sex) in four metal On 25 June, the same Mormon crickets that had screen cages (33 cm square by 46 cm high). Feces been bled as young adults were selected except two passed through the screened bottom to a tray that was group-reared ones that had died. They were now be- cleaned daily. In these cages, the density was Ϸ14.6 tween 9 and 13 d posteclosion and nearing reproduc- Mormon crickets per square meter of cage surface, a tive maturity as indicated by two males and two fe- density very similar to that of a band of Mormon males in the group cages mating the previous day. We crickets for which the density was measured in 2009 ␮ (R.B.S., unpublished data). measured body mass and extracted 15- l hemolymph, which was handled in the same way as for the younger On the same day, 24 Mormon crickets (12 of each ␮ sex) were placed in clear plastic, 0.95-liter cups. Eight adults. We collected an additional 10- l hemolymph cups were turned upside down on a 0.13-m2 tray and into a separate capillary tube and diluted it 1:4 with each cup had a paper sleeve placed around it to pre- PBS. This second sample was kept on ice for assaying vent neighbors from viewing one another. Cups were lysozyme activity later the same day. Mormon crickets screened at the top, and trays were cleaned daily. were returned to their respective group cages or sol- Mormon crickets in both treatments were reared in itary cups. designated insect rearing quarters at 30ЊC during the For immunity assays, we followed the protocols in day and ambient nights with ßuorescent lights on local Srygley et al. (2009). Samples of thawed hemolymph photoperiod. They were fed fresh Romaine lettuce diluted in PBS were centrifuged and activated with daily and given ample sunßower and mixed bird seeds, 10-mM dopamine solution to measure spontaneous wheat bran, and tropical Þsh ßakes. Molting and sur- PO activity. The plate was loaded into a temperature- vivorship were followed daily. The Mormon crickets controlled Biotek microplate reader (25ЊC), and ab- developed through two additional nymphal instars sorbance at 492 nm was read between 5 and 15 min. If sample absorbance was linearly related with time, we before reaching their adult molts, which occurred Ϫ between 12 and 17 June. Adult Mormon crickets were calculated mean V (change in absorbance min 1). uniquely marked with Bic quick-dry correction ßuid. One unit PO activity per ml hemolymph is deÞned as By 16 June, only 1Ð2 Mormon crickets had died in each the amount of enzyme resulting in a 0.001 increase in group cage, probably as a result of cannibalization, absorbance. whereas only one solitary female had died from a poor To measure proPO activity, we adapted the proto- molt. Survival was not signiÞcantly different between col of Goldsworthy et al. (2002). We dissolved 1-mg rearing treatments (␹2 test statistic ϭ 1.51, P ϭ 0.219). alpha-chymotrypsin from bovine pancreas (Sigma) in By 25 June, 2Ð5 Mormon crickets had died in each 1-ml PBS, combined an equal volume of this solution group cage, and signiÞcantly more of the group-reared with centrifuged hemolymph in PBS (1:50), and in- Mormon crickets had died than the solitarily-reared cubated for 30 min. In the plate wells, we added 5 ␮l ones (␹2 test statistic ϭ 6.27, P ϭ 0.012). Mormon of the incubated solution to 195-␮l 10-mM dopamine. crickets encountered by others during molting are As for PO, mean V was calculated from plate readings 168 ENVIRONMENTAL ENTOMOLOGY Vol. 41, no. 1

Table 1. Two-way analysis of variance for immunity assays of younger and older adults modelled with probabilities for sex, rearing density (Trt), and their interaction (Int.) where P < 0.05

Adult age class Assay F d.f. P Sex Trt Int. Younger Log PO 0.92 2,15 0.92 0.23 0.60 Younger ProPO 3.05 2,29 0.063 0.40 0.028 Younger Protein 2.87 3,28 0.054 0.64 0.087 0.030 Older Log PO 1.75 2,26 0.19 0.10 0.44 Older Log proPO 7.45 2,27 0.003a 0.17 0.001 Older Lysozyme 1.27 2,20 0.447 0.50 0.16 Older Log protein 7.60 2,27 0.002a 0.002 0.065 Older Log encapsulation 4.24 2,22 0.028a 0.18 0.021

a Indicates signiÞcant models (P Ͻ 0.05). between 5 and 15 min to measure proPO activity in We analyzed data from the two age classes (younger units mlϪ1 hemolymph. and older adults) separately. When we analyzed im- To measure lysozyme-like antibacterial activity, a munity measures to see how they were affected by sex, turbidimetric method was used. Thawed and PBS- rearing density, and body mass from the day of the diluted hemolymph (1:50) was added to a well with hemolymph draw, not all immunity assays were con- suspended gram-positive bacteria cells Micrococcus ly- ducted on all insects, and so we ran analysis of cova- sodeikticus (Worthington). Clearing of the well was riance (ANCOVA) on each measure of immunity sep- compared with a serial dilution of egg-white lysozyme arately. Spontaneous PO activity of younger and older (Sigma) added to the bacteria suspension. The plate adults, total protein of older adults, and proPO of older was loaded into a temperature-controlled Biotek mi- adults were log10 transformed to normalize these de- croplate reader (25ЊC), and absorbance at 450 nm read pendent variables. In building our models, body mass between 10 and 30 min. If the sample absorbance was was the covariate and sex and rearing density were linearly related with time, we calculated mean V. independent factors with all interactions evaluated. When sample activity fell below 6.5 ␮gmlϪ1, the To build the models, variance attributed to interac- sample was excluded because the standards showed tions that were not signiÞcant (alpha ϭ 0.05) was that the data were unreliable when sample activity was pooled with error. this weak. Changes in spontaneous PO and proPO activity as We measured total hemolymph protein in mg pro- adults aged were analyzed with Wilcoxon rank sums tein mlϪ1 hemolymph with a Total Protein Kit, Micro test. (Sigma) compared with a serial dilution of the human albumin standard. Results To measure encapsulation, we inserted, on 29 June, two quartz glass rods (National ScientiÞc Co., 1 mm Did body mass differ between rearing treatments? diameter ϫ 2 mm) dorsally between the Þrst and In the younger adults, body mass did not differ sig- second abdominal segments into the same Mormon niÞcantly between the sexes or rearing environments crickets that had been bled previously, except for two (F ϭ 0.96 P ϭ 0.40). In the older adults, body mass did males and two females that had died or disappeared not differ signiÞcantly between group and solitary from the group cages after the second blood draw. The reared adults (P ϭ 0.67), although females were sig- crickets were given water and tropical Þsh ßakes and niÞcantly heavier than males (3.75 relative to 3.10 g, kept isolated in 0.95-liter cups in the insect rearing on average, P ϭ 0.004). This result indicates that quarters. Twenty-four hours later (Ϯ8 min), we froze Mormon crickets in different treatments had similar the crickets to halt their encapsulation of the rods. To access to food. Furthermore in ANCOVAs for assays measure encapsulation response, rods were dissected of immunity and circulating protein, body mass was from the Mormon crickets, dried, and weighed. No not a signiÞcant factor in any model. Thus the co- rods were found in one group female. Weight of the variance was pooled with error, and I present the cleaned rod was subtracted. results from simpler two-way analysis of variance To measure cuticular darkness, we scanned a dorso- (ANOVA)s with sex and rearing treatment as inde- lateral view of the abdominal segments with an Epson pendent factors. Perfection 4990 Pro scanner (Seiko Epson Corp., Long In young adults, solitary individuals had signiÞ- Beach, CA). The abdomen was selected because it is cantly greater concentrations of proPO than group- more mottled and hence likely to vary more than the reared ones, but the general model was not signiÞcant pronotum. Using Adobe Photoshop CS2 (version 9.0, (P ϭ 0.063, Table 1). A signiÞcant interaction was Adobe Systems Inc., San Jose, CA), we excluded all observed between sex and treatment for total circu- but one side of the abdomen from the second to eighth lating protein, but again the general model was not abdominal segments between the dorsal midline and signiÞcant (P ϭ 0.054, Table 1). landmarks on the lateral side where the harder dorsal In contrast to when they were younger, older adults cuticle met the softer ventral one. This image was had signiÞcantly greater proPO titers when reared at converted to 256 shades of gray and the median shade higher densities (P ϭ 0.001, Fig. 1a). Mean PO did not of each insect was measured with Photoshop. differ signiÞcantly between treatments (Table 1), but February 2012 SRYGLEY:CROWDING AND IMMUNITY OF MORMON CRICKETS 169

Fig. 2. Changes in a) spontaneous phenoloxidase (PO) titers and b) proPO titers within Mormon crickets as they Fig. 1. Differences in mean a) proPO) activity and b) aged. Those reared in groups increased both spontaneous PO encapsulation mass between rearing environments of older and proPO blood titers more than those reared solitarily. adult Mormon crickets. Bars show standard errors, and num- bers in columns indicate sample sizes. Younger adults between 0 and 4 d posteclosion did not individuals reared in groups increased their PO levels respond by increasing their immune activity as ex- more as they aged than those reared solitarily (Wil- pected. If anything, the solitarily-reared adults tended coxon test, z ϭϪ2.57, n ϭ 16, P ϭ 0.010, Fig. 2a). The to have greater proPO activity than those reared in change in proPO levels with age also was much greater groups at this early age. Over the9dofadult life that in group-reared relative to solitarily-reared Mormon followed the Þrst sampling, group-reared Mormon crickets (Wilcoxon z ϭ 3.43, n ϭ 30, P ϭ 0.0006, Fig. crickets increased their PO and proPO concentrations 2b). Controlling PO and proPO levels for changes in more than the solitarily-reared adults. Older group- total protein as the insects aged did not qualitatively reared adults also encapsulated foreign bodies more affect the differences observed between rearing treat- quickly but did not have greater antibacterial activity. ments (Wilcoxon tests for protein-adjusted PO and Crowded Mormon crickets have more opportunities proPO: P ϭ 0.010 and P ϭ 0.012, respectively). for cannibalism. Thus, observed density-dependent Group-reared Mormon crickets encapsulated a for- prophylaxis in older adults might result from recog- eign object with more mass than solitary adults (P ϭ nition of conspeciÞcs through external contact or in- 0.021, Fig. 1b). Indeed, group-reared individuals had gestion, and potential or realized pathogen transmis- adhered on average 30% more mass on the introduced sion via these two routes (Pfennig et al. 1998, Barnes rods than those in solitarily-reared Mormon crickets. and Siva-Jothy 2000). Studies rarely sample insects However, encapsulation mass was not signiÞcantly more than once in a lifetime, and so to the best of my related to circulating spontaneous PO or proPO titers knowledge, this is the Þrst report of age-related re- (P ϭ 0.67 and P ϭ 0.30, respectively). sponses of adult insect immunity to crowding. Group-rearing did not result in signiÞcantly darker PO also occurs in an inactive zymogen called pro- individuals. When comparing abdominal coloration phenoloxidase (proPO), which is assayed along with between the two blood draws, Mormon crickets be- spontaneously-active PO in our measure of total PO came signiÞcantly darker as they aged (StudentÕs t-test activity. On average, circulating proPO was an order statistic ϭ 3.36, d.f. ϭ 28, P ϭ 0.0023), but there were of magnitude more concentrated than PO. However no signiÞcant differences between the sexes or treat- there was no relationship between the amount of ments in the amount of darkening (2-way ANOVA: active PO and that of proPO in the hemolymph (r ϭ F ϭ 0.34, d.f. ϭ 2,26, P ϭ 0.58). 0.05). Srygley and Lorch (2011) found a similar lack of association between circulating PO and proPO in Mormon crickets migrating in Nevada. Circulating PO Discussion titers increased with infection of Mormon crickets by There was a strong effect of adult age on the im- the entomopathogenic fungus Beauveria bassiana and mune response of Mormon crickets to crowding. were associated with attempted clearing of B. bassiana 170 ENVIRONMENTAL ENTOMOLOGY Vol. 41, no. 1 blastospores and hyphae from the hemolymph (Sry- response with rearing density indicates that at least gley and Jaronski 2011). some of the response that Bailey et al. (2008) observed Surprisingly, neither PO nor proPO titers were as- between populations can be explained by the physi- sociated with encapsulation. The Þrst stage of encap- ological elevation of immune defenses during growth sulation of a glass rod is recognition of the foreign and maturation of the teneral adults. object by hemocytes and hemocyte adhesion to the Cuticular darkness of the Mormon crickets was not object (Gillespie et al. 1997). The PO cascade is an inßuenced by their rearing environment. This result important part of encapsulation, particularly in the suggests that differences in darkness between solitary latter stages, because melanization causes the cell and migratory populations of Mormon crickets (Lorch mass to harden around the foreign body by cross- and Gwynne 2000) is attributed to historical differ- linkages. Thus, encapsulation involves both cellular ences rather than environmental effects. Chromatic and humoral responses to invasion. In Þeld studies of changes are a common feature of phase polyphenism Mormon crickets, the mass adhering to the rod over (Applebaum and Heifetz 1999), and the lack of cu- 24 h increased with the concentration of spontane- ticular darkening in group reared Mormon crickets is ously-active PO (Srygley et al. 2009, Srygley and Lorch further evidence that Mormon crickets differ from 2011). Whereas both the PO and proPO titers are their migratory locust cousins in that they do not show measures of constitutive immunity, the encapsulation phase polyphenism (Sword 2005). response is an induced measure. This difference be- tween background and induced levels of immunity may be one reason why neither enzyme assay was Acknowledgments correlated with the rate of encapsulation. The captive subjects were exposed to a more stable I thank L. Senior and C. Lindberg, USDA-Agricultural Research Service, for assistance with Mormon cricket rearing environment than Mormon crickets are likely to have and immunity assays. I also thank two anonymous reviewers available in the Þeld. 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