CELLULAR IMMUNOSENESCENCE in ADULT MALE CRICKETS, Gryllus Assimilis

Article

CELLULAR IMMUNOSENESCENCE IN ADULT MALE CRICKETS, Gryllus assimilis

Youngjin Park Department of Entomology, University of Georgia, Athens, Georgia Yonggyun Kim Department of Bioresource Sciences, Andong National University, Andong, Korea David Stanley Biological Control of Insects Research Laboratory, USDA/Agricultural Research Service, Columbia, Missouri

Ecological immunity studies in invertebrates, particularly insects, have generated new insights into trade-offs between immune functions and other physiological parameters. These studies document physiologically directed reallocations of immune costs to other high-cost areas of physiology. Immunosenescence, recognized as the age-related deteriora- tion of immune functions, is another mechanism of radically altering immune systems. We investigated the hypothesis that aging brings on immunosenescence in adult males of the cricket, Gryllus assimilis. Our data show that the intensity of melanotic nodule formation decreased with adult age from after 3-week post-adult emergence. Circulating hemocyte populations similarly decreased from about 5,000 hemocytes/ml hemolymph to about 1,000 hemocytes/ml hemolymph. The numbers of damaged hemocytes in circulation increased from less than 10% at 1-week post- adult emergence to approximately 60% by 3-week post-adult emergence. The composition of hemocyte types changed with age, with increasing proportions of granulocytes and decreasing proportions of plasmatocytes. The declines in nodule formation were not linked to the adult age of sexual behaviors, which begin shortly after entering adulthood in this species. We infer that age-related senescence, rather than cost reallocations, may account for observed declines in various parameters of

Grant sponsor: Korea Science & Engineering Foundation. Correspondence to: David Stanley, Biological Control of Insects Research Laboratory, USDA/Agricultural Research Service, Columbia, MO 65203. E-mail: [email protected]

ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 76, No. 4, 185–194 (2011) Published online in Wiley Online Library (wileyonlinelibrary.com). & 2011 Wiley Periodicals, Inc. DOI: 10.1002/arch.20394 186 Archives of Insect Biochemistry and Physiology, April 2011

immune functions in insects, as seen in other animals. C 2011 Wiley Periodicals, Inc.

Keywords: immune; Gryllus assimilis

INTRODUCTION

Similar to most invertebrates, insects manifest robust innate, but not adaptive, immune reactions to microbial and parasitic challenge. Insect immunity often is resolved into two broad areas, humoral and cellular immunity (Stanley et al., 2009). Humoral immunity involves induced expression of genes encoding a large number of anti- microbial proteins, which appear in the hemolymph of infected insects 6–12 h after infection (Kanost et al., 2004), although such proteins have not been reported for orthopterans (Adamo and Parsons, 2006). Cellular immunity is characterized by direct interactions between circulating hemocytes and invading microbes (Strand, 2008). Specific interactions include phagocytosis, nodulation and for invaders much larger than hemocytes, such as parasitoid eggs, encapsulation. Encapsulation is the broader term, which includes nodulation. The cellular defense reactions and prophenoloxidase (PPO) activation are launched immediately after an infection is detected and these reactions are responsible for clearing the bulk of infectious organisms from hemolymph circulation (Dunn and Drake, 1983; Cerenius and So¨derha¨ll, 2004). Circulating hemocytes and the PPO activating system represent the constitutive immune system of insects, thought to be present at any life stage without stimulation by previous infection (Brown et al., 2003). Recent research into ecological and evolutionary forces responsible for shaping invertebrate immune systems indicates that immune effector mechanisms are tradable features of insect biology. Although robust immune defense mechanisms are vital to insects, they are also costly physiological functions (Schmid-Hempel, 2003; Rolff and Siva-Jothy, 2003). For example, nodulation reactions to bacterial infection can cause loss of thousands or even millions of hemocytes, which entails very high replacement costs (Miller et al., 1994; Doums et al., 2002). Reallocation of costs associated with immune functions toward several other physiological parameters has been reported. Direct trade-offs between immunity and reproductive behavior (Adamo et al., 2001; Rolff and Siva-Jothy, 2003), foraging (Ko¨nig and Schmid-Hempel, 1995) and learning (Alghamdi et al., 2008) also are documented. In honeybees, adult foragers appear to reallocate costs of cellular immune mechanisms to the energetically expensive costs of foraging (Schmid et al., 2008), mediated by an endocrine-driven hemocyte apoptosis mechanism (Amdam et al., 2003). We infer from these studies that the various immune mechanisms are not fixed features of invertebrates, but rather are physiological functions subject to change according to evolutionary ecological optimizations. Another mechanism of immune function plasticity is the progressive decline in immune function with age, known as immunosenescence. Immunosenescence is well documented in mammals and regarded an impairment in elderly humans (Agarwal and Busse, 2010). Insects also undergo immunosenescence. For a single example, Zerofsky et al. (2005) reported that, compared with younger females, older Drosophila females exhibited reduced capacity to induce the gene encoding diptericin. This finding is confounded with reproductive capacity as immune-challenged females also produced fewer eggs than control females. Immunosenescence may be more

Archives of Insect Biochemistry and Physiology Immunosenescence 187 complicated among insect species; however, as Lesser et al. (2006) reported that virgin females of some Drosophila lines exhibited increased immune performance with age while others showed no change or declined performance as they aged. As it stands, the distinctions between physiological trade-offs and immunosenescence are not thoroughly established. With a view to differentiating between immunosenescence and physiological trade-offs, we selected an insect species with a fairly simple adult life history pattern that is not confounded by ontological task changes or ovarian physiology. In this article, we report quantitative declines in cellular immune functions and in circulating hemocytes associated with aging in adult male crickets, Gryllus assimilis.

MATERIALS AND METHODS

Organisms

The Jamaican ﬁeld cricket, Gryllus assimilis, was maintained on oatmeal and tap water at 25711C, 6075% RH and a photoperiod of 16:8 (L:D). The age of male crickets was recorded starting on the day of adult eclosion. Only crickets of known age were used in experiments. Each animal was sampled only one time.

Dose–Response Curve for Injected Lipopolysaccharide

The nodulation assay followed general protocols described elsewhere (Miller and Stanley, 1998). Crickets were anesthetized by chilling on ice and surface-sterilized with 95% ethanol. Lipopolysaccharide (LPS) prepared from the bacterium Serratia marcescens was used to stimulate nodulation (Bedick et al., 2000). LPS was dissolved in phosphate-buffered saline (PBS, pH 8.0) and injected into the hemocoels of crickets between the second and third abdominal sclerites. All injections were carried out in 4 ml volumes with a Hamilton 701 microsyringe (Hamilton, Reno, NV). Crickets were injected with 0, 50, 100, 200, or 400 mg LPS/cricket, n 5 5 crickets/ dose. At 24 h post-injection (PI), the crickets were anesthetized by chilling on ice, then the hemocoels were exposed. Melanized, dark nodules were counted under a stereomicroscope at 60 Â (Olympus, Tokyo, Japan). After the initial count in abdomens and thoraces, the alimentary canal was removed and nodulation was assessed in the newly exposed area. The internal tissues, including alimentary canal, Malpighian tubules, and accessory glands, were then checked for possible nodules. Nodules were distinct, and direct counting reliably reﬂected the extent of the nodulation reactions to challenges.

Time Course of Nodulation

Adult crickets, (1 day after adult emergence) were injected with 100 mg of LPS, n 5 5 crickets per time point. Nodulation was assessed at 0, 1, 2, 4, 8, 16, and 24 h PI. Controls were injected with 4 ml of PBS.

The Inﬂuence of Aging on Nodulation Reactions

Adult males of ages 1, 7, 14, 21, 28, and 35 days were challenged with 100 mg LPS/ cricket, n 5 5 crickets per age. At 24 h PI, the crickets were anesthetized and the numbers of nodules were determined as just described.

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The Inﬂuence of Aging on Total and Differential Hemocyte Counts Adult males of ages 1, 7, 14, 21, 28, and 35 days (n 5 5 crickets per age) were surface- sterilized, then decapitated with sterilized scissors. Hemolymph was collected into chilled 1.5-ml microcentrifuge tubes, mixed with 0.2% bromophenol blue dye (1:12, v/v), and incubated for 5 min. Cells were identiﬁed as plasmatocytes, granulocytes, and others, and then counted on a hemocytometer (Superior, Marienfeld, Germany).

Statistical Analysis Mean and variances of the treatments were analyzed by one-way ANOVA by PROC GLM within SAS (SAS Institute, 1989). All mean were compared by least squared difference with a signiﬁcance set at Pr0.05.

RESULTS

We began this study by determining the time course of nodule formation after LPS challenge and optimal experimental LPS challenge doses for the Jamaican field cricket, G. assimilis. It can be seen in Figure 1 that the formation of melanotic nodules began within the first hour PI and increased to a maximum of about 12 nodules/cricket by 8 h PI. Longer incubation periods did not yield significant increases in nodulation reaction. As a practical matter, we used 24 h incubation periods in subsequent experiments on nodulation. The data presented in Figure 2 show that numbers of melanized nodules formed by 24 h PI increased with increasing LPS doses. We recorded very few nodules in untreated control crickets (o2 nodules/cricket). Experimental crickets formed significantly more nodules in response to larger LPS challenge doses, up to approximately 15 nodules/cricket at 200 and 400 mg LPS/cricket. We used 200 mg challenge doses in subsequent experiments on nodulation. The intensity of nodulation reactions was assessed in terms of numbers of melanotic nodules formed at 24 h after treating crickets with the standard LPS

15 c c c 12

9 b b b 6

3 Number of nodules a 0

012481624 Time (hr) after LPS injection

Figure 1. Time-course for nodulation reactions to LPS challenge in adult male Gryllus assimilis. Crickets were injected with 100 mg of LPS and nodulation was assessed at the indicated times PI. Each point represents the mean number of nodules (7SEM, n 5 5) per individual. Points annotated with the same letter are not signiﬁcantly different (F 5 20.62; df 5 6, 14; P 5 0.0001).

Archives of Insect Biochemistry and Physiology Immunosenescence 189

18 c 15 c

12 b 9 ab 6

3 a Number of nodules Number

0 50 100 200 400 LPS injection dosages (µg)

Figure 2. The inﬂuence of LPS challenge dosage on nodulation reactions in adult male Gryllus assimilis. Crickets were injected with the indicated LPS dosage and nodulation was assessed at the 24 h PI. Each point represents the mean number of nodules (7SEM, n 5 5) per individual. Points annotated with the same letter are not signiﬁcantly different (F 5 21.22; df 5 4, 10; P 5 0.0001).

18 a a a a 15 b 12

9 c 6 Number of nodules 3

0 012345 Time (week) after adult molting

Figure 3. The influence of age on nodulation reactions to a standard LPS challenge in adult male Gryllus assimilis. Crickets at the indicated ages after adult emergence were injected with the LPS (100 mg) and nodulation was assessed at the 24 h PI. Each point represents the mean number of nodules (7SEM, n 5 5) per individual. Points annotated with the same letter are not significantly different (F 5 21.39; df 5 4, 10; P 5 0.0001). challenge. G. assimilis males produced about 15 nodules/cricket at the first, second, and third week after adult molt, which declined to about nine nodules/cricket at the fourth week and to about five nodules/cricket after the fifth week (Fig. 3). The data on nodulation capacity are similar to our observations on hemocyte population census in adult male crickets (Fig. 4). The data displayed in Figure 4 indicate G. assimilis males maintain high numbers of hemocytes for the first 2 weeks after the adult molt (ca. 5 Â 106 cells/ml). Hemocyte numbers declined to about 3 Â 106 cells/ml after the third week and to approximately 106 cells/ml hemolymph at weeks four and five. Gross examination of the hemocytes revealed many damaged cells in older males. Approximately 60% of hemocytes from G. assimilis males at 3, 4, and 5 weeks after adult molt featured surface blebbing and broken membranes, whereas less than 20% of the cells from younger males were similarly damaged (Fig. 5). Aside from increasing

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6000 a a 5000

b 4000

l hemolymph) 3000 µ

2000 c c 1000 THC (cells/

0 12345 Time (week) after adult molting

Figure 4. The inﬂuence of age on total hemocyte counts (THCs) in hemolymph circulation in adult male Gryllus assimilis. Hemocyte counts were assessed in crickets at the indicated ages after adult emergence. Each point represents the mean number of nodules (7SEM, n 5 5). Points annotated with the same letter are not signiﬁcantly different (F 5 37.65; df 5 4, 10; P 5 0.0001).

100

80 bb b

40 a 20

Hemocyte damage (%) Hemocyte a

0 12345 Time (week) after adult molting

Figure 5. The inﬂuence of age on proportions of damaged hemocytes circulated in hemolymph of adult male Gryllus assimilis. Each point represents the mean proportion of damaged hemocytes at the indicated age after adult emergence (7SEM, n 5 5). Points annotated with the same letter are not signiﬁcantly different (F 5 11.30; df 5 4, 10; P 5 0.0010). proportions of damaged hemocytes, the composition of hemocyte types changed with age, with increasing proportions of granulocytes and decreasing proportions of plasmatocytes (Table 1).

DISCUSSION

In this article, we document declines in immune function, registered as nodulation, and hemocyte numbers through 5 weeks of adult life in males of the cricket, G. assimilis, and provide strong support for the concept of age-related immunosenescence in insects. G. assimilis is a widely distributed species and there is no general description of its life cycle. In South Dakota, adults appear in late July and live about 7 weeks, although some live until a solid freeze kills them (Severin, 1926). Although the

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Table 1. The Inﬂuence of Aging on Hemocyte Composition in Adult Males of the Cricket, Gryllus assimilis

Time (week) after adult molting

Hemocyte types (%) 1 2 3 4 5 Ã Ã GR 20.571.2 18.572.0 28.179.3 30.1714.7 38.178.4 PL 24.672.2 20.773.1 23.575.6 16.977.5 18.277.2 Others 54.973.2 60.874.6 48.479.0 45.076.5 51.7719.2

Values are proportions of total counted hemocytes7SEM. Means annotated with an asterisk are significantly different (LSD; Po0.05) within each cell type. GR, granulocytes; PL, plasmatocytes. data in this article are not directly correlated to the sexual phase of adult male crickets, the time course of our study reasonably reflects their overall life span in the field. The idea of age-related declines in immune functions is well established. Immunosenescence has been documented with respect to the adaptive and innate immune systems in mammals (Hakim et al., 2004; Agarwal and Busse, 2010). One of the predominant mechanisms of immunosenescence in humans is the influence of age on hematopoietic stem cells. As humans age, there is a general reduction in capacity for renewing the cells responsible for replacing dead and damaged immune cells. Moreover, immune cells themselves lose capacity for specific cell functions. For example, rat T cells undergo reduced capacity for cell migration in the rat intestine (Ogino et al., 2004) and vaccine responses within the adaptive immune system decline with age (Fisman et al., 2002). Age-related immunosenescence probably occurs in all animals that express an immune system and live long enough for their immune functions to decline (Kurz and Tan, 2004). Immunosenescence has been recorded for a couple of social insect species. In their study of eicosanoid actions in honeybee cellular immune reactions to bacterial infection, Bedick et al. (2001) noted that newly emerged honeybees, those performing in-hive functions, mounted intense melanotic nodulation reactions to bacterial infection. Contrarily, foraging adults captured while returning to their nests did not form any nodules in reaction to similar challenge. Upon closer inspection, it was very difficult or impossible to collect hemolymph from foragers, from which the authors concluded that the older bees were virtually without hemolymph, hemocytes, and cellular immunity. Schmid et al. (2008) studied honeybee immunosenescence in more detail, reporting that age-related reductions in hemocyte numbers occurred in adult workers, drones, and queens. Moreover, the loss of hemocyte-based immunity may be compensated, in part, by increased phenoloxidase-based immunity in workers and queens. Based on the Bedick et al. (2001) article, it was unclear whether this loss of hemocyte-based immune functions followed from direct senescence of immune effector tissues or from endocrine-driven changes in task allocations. This was resolved using the single cohort colony procedures of Robinson et al. (1989), which showed that the reduction in hemocyte numbers is due to aging and not to ontological task allocation. The conclusions drawn by Schmid et al. (2008) differ from the findings of Amdam et al. (2005), who found increased hemocytes in foragers that reverted to nurse behaviors. They also noted that increased hemocyte numbers necessarily involves the production of new hemocytes. The process appears to be endocrine-driven because they demonstrated that juvenile hormone titers of reverted bees were similar to the titers in nurse bees. These variances may be due to differences in task reversion

Archives of Insect Biochemistry and Physiology 192 Archives of Insect Biochemistry and Physiology, April 2011 protocols; however, we infer that the mechanisms, and associated cost reallocations, of rapid depletion of hemocytes in honeybee workers, drones, and queens are considerably more subtle than appears so far. Doums et al. (2002) reported substantial decreases in immunocompetence in bumble bee workers of two species, Bombus terrestris and B. lucorum. They used the capability of cellular encapsulation of a foreign object as a general and quantitative measure of immunocompetence. They inserted a standardized piece of nylon monofilament as the foreign object and later determined the degree of melanization by changes in light transmission on a stereomicroscope. Bumble bee workers of both species underwent substantial and statistically significant reductions in melanization of the foreign objects as they aged from adult emergence to approximately 25 days old. Although the melanization reactions declined with age, the authors did not record changes in hemocyte population census during the same aging period. Again, these findings are confounded by a possible trade-off between foraging costs and the costs of sustaining an immune system. The authors argued, however, that all experiments were conducted in the laboratory and the bumble bees were not able to engage in foraging flights. They therefore favored the idea of immunosenescence during the adult lives of bumble bee workers. While their argument makes sense, it is not rock-solid because the absence of opportunity for flight behavior does not necessarily mean the workers did not experience age-related, endocrine-driven changes in organismal physiology, including trading off immune functions. Adamo et al. (2001) reported on a related study in crickets, G. texensis. Their main goal was to determine whether males reallocate the energetic costs of maintaining immune functions to the costs of reproduction. They measured immunocompetence in terms of phenoloxidase activity, hemocyte numbers, and ability to survive bacterial infection. Their data showed that the proportions of males and females able to survive bacterial challenges declined significantly as the crickets aged beyond 2 weeks, with survival of males declining more than females. They interpreted their data with respect to the onset of sexual behavior, i.e. males trade-off immunocompetence in favor of reproductive efforts. For this cricket species, the declining immunocompetence was not associated with reductions in hemocyte numbers. For males and females, hemocyte numbers did not decline over a 4-week aging period. Our data indicate substantial reductions in hemocyte numbers and changes in hemocyte composition in G. assimilis. The variance from the work with G. texensis just discussed may be appreciated as differences in hemocyte population sizes in young adult crickets. We recorded approximately 5,000 hemocytes/ml hemolymph in crickets at 1- and 2-weeks post-molt, which declined to about 1,000 hemocytes/ml hemolymph by weeks 4 and 5 post-molt. Adamo et al. (2001) recorded considerably lower hemocyte counts in G. texensis, in the range of 2,000 to 3,000 hemocytes/ml hemolymph, from which there was no decline. Ramsden et al. (2008) introduced another mechanism for age-related immunosenescence in Drosophila. They investigated the influence of age on the ability to clear bacterial infections, finding that age did not reduce bacterial clearance. Paradoxically, they also found that older flies are reduced in capacity to survive infections. They posed the hypothesis that aging does not impair the ability to clear infections, but does increase susceptibility to bacterially derived chemical factors. They cultured E. coli overnight, sterilized the culture medium, and challenged experimental flies with the medium. The sterile medium, amended with bacterial factors from overnight culture, was more lethal to older flies. In Drosophila, then, aging may exert more influence on the ability to survive bacterial products than the ability to survive the bacteria, per se. In addition to the ability to handle microbial products, aging may influence the ability of

Archives of Insect Biochemistry and Physiology Immunosenescence 193 older insects to downregulate the biosynthesis of anti-microbial proteins. Zerofsky et al. (2005) reported that young Drosophila females (1 week) terminate the expression of diptericin before 24 h after infection, whereas older females continued expressing the gene for up to 72 h. These ﬁndings are consistent with immunosenescence in fruit ﬂies. The main point is that insect immunity is complex and we can discern what appear to be a wide range of effects of the aging process on immunity. A couple of possible mechanisms may account for the absence of hemocytes in foraging honeybees and for steeply reduced numbers of hemocytes in older individuals in a cricket species. For one, hemocytes may undergo increasing damage with aging, which may be coupled to apoptosis, as reported for honeybees (Amdam et al., 2003, 2005). This is congruent with the data of Schmid et al. (2008), who reported that adult drones, workers, and queens undergo sharp declines in hemocyte numbers while maintaining phenoloxidase-based immunity. Our data from G. assimilis showing increasing numbers of damaged hemocytes with advancing age also support this model. For another, as seen in mammals, insects may undergo alterations in hematopoiesis, either through senescence of hematopoietic organs or loss of stem cells (Hakim et al., 2004). To the extent senescence of hematopoietic organs or of stem cells operates in invertebrate immune effector systems, age-related loss or reduction in the ability to replace hemocytes may be taken as an instance of immunosenescence rather than a trade-off favoring another physiological system.

ACKNOWLEDGMENTS

This research was supported, in part, by the Post-doctoral Fellowship Program of the Korea Science & Engineering Foundation to Y. Park. This article reports the results of research only and mention of a proprietary product does not constitute an endorsement or recommendation for its use by the USDA.

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