Maturation of the immune system of the male house cricket, Acheta domesticus

Journal of Insect Physiology 59 (2013) 752–760

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Maturation of the immune system of the male house cricket, Acheta domesticus Angelica V. Piñera, Heather M. Charles, Tracy A. Dinh, Kathleen A. Killian ⇑ Department of Zoology and Center for Neuroscience, 212 Pearson Hall, Miami University, Oxford, OH 45056, United States

a r t i c l e

i n f o

Article history: Received 30 December 2012 Received in revised form 21 May 2013 Accepted 22 May 2013 Available online 30 May 2013 Keywords: Immune Cricket Phenoloxidase Lysozyme Hemocyte Encapsulation

a b s t r a c t The immune system functions to counteract the wide range of pathogens an insect may encounter during its lifespan, ultimately maintaining fitness and increasing the likelihood of survival to reproductive maturity. In this study, we describe the maturation of the innate immune system of the male house cricket Acheta domesticus during the last two nymphal stages, and during early and late adulthood. Total hemolymph phenoloxidase enzyme activity, lysozyme-like enzyme activity, the number of circulating hemocytes, and encapsulation ability were all determined for each developmental stage or age examined. The number of circulating hemocytes and lysozyme-like enzyme activity were similar for all developmental stages examined. Nymphs and newly molted adult males, however, had significantly lower total phenoloxidase activity than later adult stages, yet nymphs were able to encapsulate a nylon thread just as well as adults. Encapsulation ability would thus appear to be independent of total phenoloxidase activity. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Animals encounter a myriad of stressors in their environment, many of which can be detrimental to an animal’s fitness. The immune system functions to deter disease-causing pathogens and parasites and to help repair injuries, thereby maintaining health and increasing an animal’s ability to survive to reproductive age (see Rolff and Siva-Jothy, 2003). Insects have an immune system similar in function to the innate immune system of vertebrates (reviewed in Loker et al., 2004; Strand, 2008), There is also some evidence for an inducible immunological memory (Moret and SivaJothy, 2003; Sadd and Schmid-Hempel, 2006) and this enhanced ability to respond to pathogens after exposure to a non-lethal immune challenge has been called ‘immunological priming’ (Little and Kraaijeveld, 2004). The insect innate immune response is composed of both cellular and humoral defense pathways that interact to mount an effective immune response. The cellular defense pathway is mediated by circulating hemocytes, which are involved in processes such as phagocytosis, encapsulation, and nodulation (reviewed in Gillespie et al., 1997; Jiravanichpaisal et al., 2006; Lavine and Strand, 2002; Strand, 2008). When a foreign object enters the body, if it is small enough, specific hemocytes can phagocytize it and eliminate the

⇑ Corresponding author. Address: Department of Zoology, 260 Pearson Hall, Miami University, Oxford, OH 45056, United States. Tel.:+1 513 529 3310; fax: +1 513 529 6900. E-mail address: [email protected] (K.A. Killian). 0022-1910/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinsphys.2013.05.008

threat. However, if the object is too large to phagocytize, hemocytes adhere and aggregate to form multiple layers coating the surface of the foreign object, effectively isolating it from the rest of the body. This encapsulation response also requires the activity of the humoral defense pathway, but this pathway is involved in a later step—the melanization and hardening of the hemocyte layers in a process known as nodulation (for examples, see: Brookman et al., 1989; Gunnarsson and Lackie, 1985; Miller and Stanley, 2004). This is accomplished in large part by an enzyme called phenoloxidase (PO). In the absence of an immune challenge, PO can be found in the plasma and hemocytes as an inactive zymogen, prophenoloxidase (proPO). In response to an immune challenge, a cascade of serine proteases is activated until the final protease, phenoloxidase activating enzyme (PAE), cleaves proPO into its active form, PO. PO then catalyzes the hydroxylation of tyrosine to DOPA, and production of quinones from DOPA and the major dopamine metabolite, N-acetyldopamine (NADA), leading to melanization or sclerotinization (reviewed in Cerenius et al., 2008; Cerenius and Söderhall, 2004; Christensen et al., 2005; GonzálezSantoyo and Córdoba-Aguilar, 2012; Kanost and Gorman, 2008). Another component of the humoral defense pathway response is antimicrobial peptides such as lysozyme, which break down bacterial cell walls (reviewed in Hultmark, 1996). These antimicrobial peptides are produced and secreted by the insect fat body, an organ that has functions similar to vertebrate liver and adipose tissue (Arrese and Soulages, 2010). An insect’s habitat (Adamo and Lovett, 2011; Sorvari et al., 2008) and available resources (Alaux et al., 2010; Myers et al.,

A.V. Piñera et al. / Journal of Insect Physiology 59 (2013) 752–760

2011; Yang et al., 2007) can influence the strength and effectiveness of an insect’s immune response to a pathogenic challenge. The efficacy of the insect immune system can also be influenced by an animal’s developmental stage (Shi and Sun, 2010; Wilson-Rich et al., 2008), age (Anand and Lorenz, 2008; Rolff, 2001), and gender (Rantala and Roff, 2006; Rolff, 2001). These factors may affect how well the immune system can respond and counteract a threat (immunocompetence), as well as regulate which immune parameters are involved in carrying out a given response. In this report, we describe the maturation of the immune system of male Acheta domesticus crickets from the last two nymphal stages (8th and 9th instar) through late adulthood (21 days after the imaginal molt). The house cricket is a hemimetabolous insect and under our laboratory rearing conditions, crickets undergo nine developmental molts, the ninth being the imaginal molt into adulthood (10th instar). The number of molts may vary depending on the environmental conditions in which crickets are reared (Masaki and Walker, 1987). During molting, or ecdysis, the old cuticle is shed to allow for growth. This process is regulated by the hormones 20-hydroxyecdysone and juvenile hormone; the relative concentrations of these hormones dictate the developmental stage in which an insect remains or into which it molts (reviewed in Riddiford, 2012; Jindra et al., 2013). Since different immune parameters may be differentially regulated, it is important to measure multiple immune parameters in order to develop a complete picture of an animal’s physiological immunocompetence (see Adamo, 2004a; b). We thus collected hemolymph samples from male crickets in different developmental stages and assayed the samples for total phenoloxidase enzyme activity, lysozyme-like enzyme activity, and the number of circulating hemocytes. In addition, as a functional test of the male cricket’s immune defense response at each of these developmental stages, encapsulation ability assays were performed. 2. Methods 2.1. Insects Animals used were male A. domesticus crickets purchased from Fluker’s Cricket Farm (Port Allen, LA) or acquired from our laboratory colony. Crickets arrived weekly as 6th–8th instar nymphs and groups of approximately 100–150 nymphs were kept in large rectangular plastic containers with food (autoclavable rat chow) and water (moist cotton balls) provided ad libitum. Containers were maintained in incubators at 29°C under a 12hr light:12hr dark cycle. Animals were checked every Monday, Wednesday, and Friday for newly molted adults. Adult crickets were isolated into small (500 ml) circular containers with food and water ad libitum until the day of experiments. Upon arrival, the weekly cricket shipment was also staged for 6th, 7th, and 8th instar nymphs. Two containers were created to house approximately 50 nymphs, with one (box 1) containing 6th–7th instar males and females (since gender could not be determined externally at this stage without anesthetization) and the other (box 2) containing 8th instar males only (since gender was easily discernible). Cricket 8th and 9th instars could be determined by the structure of the immature wings, and both containers of nymphs were staged daily for either 8th (box 1) or 9th (box 2) instar male nymphs that had molted within the past 24hr. These newly molted nymphs were removed from the large box and placed as groups of 4–6 into small (500ml) circular deli containers until the day of the experiment. Under our housing conditions, the 8th instar stage lasted approximately 7–9 days while the 9th instar stage lasted approximately 11–14 days, after which the male molted into an adult.

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2.2. Experimental groups For immune assays, male nymphs had blood removed exactly 2 days after molting into an 8th instar (D2–8th), 5 days after molting into an 8th instar (D5–8th), 2 days after molting into a 9th instar (D2–9th), or 9 days after molting into a 9th instar (D9–9th). For characterization of adult immune parameters, blood was removed from adult males that were 1–3 days in age (designated D1–3), 5–7 days in age (D5–7), 9–12 days in age (D9–12), 14– 16 days in age (D14–16), or 21–23 days in age (D21–23) past the adult (10th) molt. Since we had previously found that multiple blood sampling from the same individual had a negative impact on survival, hemolymph of each nymph and adult animal was only sampled once. This resulted in animals in ten differently aged groups, four from the last two nymphal stages, and six from the adult stage. In addition, in order to determine the critical time point for the developmental change in total phenoloxidase enzyme activity (see results), blood was removed from adults exactly 4 days (D4), 5 days (D5), or 6 days (D6) in age after the adult molt. These samples were assayed for total hemolymph PO activity and protein content only. Lastly, crickets from different developmental stages were used for an encapsulation assay (see below) to determine if there were developmental differences in the ability of male A. domesticus to respond to an immune challenge.

2.3. Hemolymph removal for immune assays On the day of each experiment, crickets were anesthetized on ice for 7–10 min and weighed. A 25-gauge needle sterilized with 70% ethanol and dried was used to puncture the soft tissue between each cricket’s pro- and mesothoracic leg, on the right side of the thorax. In our initial experiments, blood from this sample was only used to determine total PO enzyme activity and protein content. For these crickets, 3 ll of hemolymph was quickly removed by touching the end of a 3 ll Drummond capillary tube to the wound site. Upon visual confirmation that the tube was filled, the hemolymph was immediately dispensed into a 1.5 ml eppendorf tube containing 147 ll phosphate buffered saline (PBS, pH 7.4; Sigma, St. Louis, MO), and vortexed for 15 s. Immediately after vortexing, two duplicates of 50 ll of the PBS-hemolymph mixture were placed into two separate 1.5 ml plastic cuvettes for the PO assay, while the remaining 50 ll was stored at 20 °C until a Bradford protein assay was performed. In our later experiments, cricket blood samples were also used to determine lysozyme enzyme activity and to estimate the number of circulating hemocytes. For these males, a 1 ll hemolymph sample was first removed to determine hemocyte number. This sample was removed by applying a 1 ll Drummond capillary tube to the open wound and the blood from the filled tube was immediately dispensed into a resin embedding capsule (8 mm ID  20 mm high, EMS, PA) containing 12.5 ll of anticoagulant buffer (98 mM NaOH, 146 mM NaCl, 16 mM EGTA, 10 mM citric acid, pH 6.5; modified from da Silva et al. (2000). Another 2 ll of hemolymph was then removed by touching the end of a 2 ll Drummond capillary tube to the same wound site. Hemolymph from the filled tube was then dispensed into a 1.5 ml eppendorf tube containing 98 ll PBS (pH 7.4; Sigma) and vortexed for 15 s. Immediately after vortexing, 50 ll of the PBS-hemolymph mixture was placed in a 1.5 ml plastic cuvette, while the remaining 50 ll was stored at 20 °C until a Bradford protein assay was performed. Finally, 1 ll of hemolymph was quickly removed by applying a 1 ll Drummond capillary tube to the same wound site. This sample was immediately dispensed into a 1.5 ml eppendorf tube containing 9 ll of PBS (pH 6.2; Sigma), vortexed for 15 s, and stored at 20 °C until lysozyme-like enzyme activity could be determined.

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Removal of hemolymph for the various immune assays took no longer than 3 min per cricket. All hemocyte counts and phenoloxidase assays were performed within 1 h of hemolymph collection. In initial experiments, hemolymph samples for hemocyte counts were dispensed into 12.5 ll of insect physiological saline. We found that our hemocyte counts were 50% lower in saline than in anticoagulant buffer. Accordingly, hemocyte counts performed in saline were discarded and are not reported here. All Bradford protein assays and lysozyme-like enzyme activity assays were performed within two months of hemolymph collection. A detailed description of each assay is provided below. 2.4. Hemolymph protein content A standard Bradford assay was used to measure hemolymph protein content. Hemolymph used for this assay was stored at 20 °C, and thawed at the time of the experiment. All experimental samples were run in duplicate. Two 15 ll samples were removed from the 50 ll hemolymph-PBS mixture, and placed into individual cuvettes followed by 750 ll of Bradford reagent (Sigma), and left to incubate for 30 min. Standards of 1, 1.5, 2, 2.5, 5, 10, 15 lg/ll bovine serum albumin (BSA, Fisher Scientific, Pittsburgh, PA) were read simultaneously with blood samples at 595 nm, using a Beckmann DU530 Spectrophotometer. Absorbances of the duplicates of each sample were averaged and this value was used to determine the protein content using the standard curve.

grids was counted. The number of hemocytes per ll hemolymph was estimated using the formula: cells per ll = (total number cells counted/5)  dilution factor of 13.5 ll  volume factor of 10. Hemocytes were classified as plasmatocytes, granulocytes, fibroblasts, or ‘undetermined’ based on observed morphology and in comparison with a previous report describing A. domesticus hemocytes by da Silva et al. (2000). To better visualize hemocyte morphology, hemocytes collected from a separate group of adult crickets, D9–12 in age, were plated onto poly-L-lysine-coated coverslips. Coverslips were flame sterilized, covered with poly-L-lysine (Sigma) for 15 min, then washed twice with anticoagulant buffer. Crickets were anesthetized and hemolymph removed as described above and 4–6 ll of hemolymph from each cricket was added to 1000 ll of anticoagulant buffer in a 1.5 ml eppendorf tube. The hemolymph-buffer mixture was agitated by gentle tapping and then 300 ll was pipetted onto a coverslip. Hemocytes were allowed to fall and adhere to the coverslips for 1 h at room temperature. Coverslips were then submerged in Carnoys fixative (6:3:1 ratio chloroform:100% ethanol:acetic acid) overnight at 4 °C. Cells were then rehydrated in a descending ethanol series, washed in distilled water, and then each coverslip was flipped over onto a clean slide covered with Vectashield containing the nuclear dye 40 ,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame CA). Hemocytes were visualized using a Zeiss 710 confocal microscope with Zen software and digital interference contrast images were taken of the cells.

2.5. Total phenoloxidase enzyme activity Total phenoloxidase enzyme activity in a 1 ll hemolymph sample was determined using a spectrophotometric assay modified from Adamo (2004a) and Shoemaker et al. (2006). Each 50 ll PBS-hemolymph sample was first vortexed to disrupt hemocytes and then placed into a 1.5 ml plastic cuvette. Then, 70 ll of 1.3 mg/ml a-chymotrypsin from bovine pancreas (Sigma) made up fresh in pH 7.4 PBS was added to the hemolymph-PBS mixture, vortexed for 10 s, and left to incubate at room temperature for 20 min. Phenoloxidase activity in blood samples from both nymphs and adults could not be detected without the addition of chymotrypsin, which enzymatically activates PO from its inactive zymogen, pro-PO (Ohnishi et al., 1970); we thus measured total PO activity (TPA) in all samples. A similar inability to measure basal PO without the addition of chymotrypsin had been reported previously for A. domesticus crickets (da Silva et al., 2000), as well as the crickets Gryllus texensis (Adamo, 2004a) and Teleogryllus oceanicus (Bailey and Zuk, 2008). At the end of the incubation period, 600 ll of 0.15M 3,4-Dihydroxy-L-phenylalanine (L-DOPA; Sigma) made up fresh in pH 7.4 PBS was added and the change in absorbance was read at 490 nm using a Beckmann DU530 Spectrophotometer. Absorbances were determined at 0, 8, 15, 23, 30, 45, and 60 min following the addition of L-DOPA, with the 0 time point being immediately after L-DOPA addition. The assay was performed in a darkened room due to the light-sensitive nature of the reaction. Total PO activity was reported as the slope of the absorbance from the linear phase of the reaction (8–30 min for adults and 15–60 min for nymphs) multiplied by 103. 2.6. Circulating hemocytes A Neubauer hemocytometer composed of nine 1 mm2 grids was used to estimate the number of circulating hemocytes in the hemolymph. The 13.5 ll hemolymph-anticoagulant buffer mixture was gently mixed with a pipette to prevent cell lysis and 10 ll of the mixture was loaded into the hemocytometer chamber. Using an Olympus U-CA compound microscope at 20, the total number of hemocytes within a predetermined area consisting of five 1 mm2

2.7. Encapsulation ability We used an encapsulation assay modified from Rantala and Kortet (2004) and Niemela et al. (2012). A 3-mm nylon thread (0.3 mm diameter) was knotted at one end, roughened with sandpaper to increase hemocyte affinity, soaked in 70% ethanol for sterilization, and dried. Crickets were immobilized on ice for 7–10 min and weighed. The area between the second and third abdominal sternite on the left side of each cricket was punctured with a sterile 27½ gauge needle, after which the prepared thread was inserted parallel to the anteroposterior axis until the knot was touching the cricket’s external cuticle. After 24 h, the thread was dissected from a small incision in the abdomen, and placed in a 1.5 ml eppendorf tube at room temperature to air dry overnight. Of 181 threads inserted into crickets in select developmental time points (D2–8ths, D5–8ths, D2–9ths, D9–9ths, and adult ages D1–3, D9–12, D21–23), 131 could be successfully retrieved. The knot of an inserted thread had to remain completely flush with the external body wall in order for the thread to be included in the analysis. Dried threads were stored at 20 °C until ready to be photographed. Three different faces of each thread were photographed at 315 zoom under a dissecting microscope equipped with a Canon SZX12 camera. ImagePro software was used to analyze the level of encapsulation by taking the average pixel values for a predetermined rectangular box (1500  201 pixels) overlaying each thread face. This box was placed 300 pixels from the cut end of each thread to prevent inclusion of scar tissue which could sometimes accumulate near the knotted end of the thread. Using bitmap analysis, the pixel value within each box was determined and the three views averaged. The same analysis was performed for the un-inserted control threads. The experimental value was then subtracted from the averaged values of the control threads to produce an encapsulation score. Darker values corresponded to smaller numerical values, therefore, larger differences between control and experimental threads indicated a greater level of encapsulation (modified from Rantala and Kortet, 2004).

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2.8. Lysozyme-like enzyme activity Lysozyme is an enzyme that breaks down linkages in the peptidoglycan layer of gram-positive and gram-negative bacterial membranes (reviewed in Herreweghe and Michiels, 2012). A turbidometric assay (modified from Drayton et al., 2011) was used to measure lysozyme-like activity in the hemolymph. 10 ll of hemolymph-PBS mixture (1 ll hemolymph in 9 ll PBS, pH 6.2) was loaded into the well of a 96-well microplate followed by 90 ll of the substrate Micrococcus lysodeikticus (0.5 lg/ml, Sigma) made up fresh at the time of the experiment. The absorbance at 450 nm was read at 0, 5, 10, 12, 15, 17, 20, 25, and 30 min using a Spectramax Plus 384 plate reader with temperature at 30 °C. Lysozyme-like activity was reported as the change in absorbance from the linear phase of the reaction (10–25 min) multiplied by 103. Multiplying by a negative number was done to enable easier, more intuitive visualization of the results—i.e. that a greater decrease in absorbance indicated greater lytic activity. Standards of increasing units of enzyme activity were made using lysozyme from chicken egg whites (Sigma). Standards were thawed from 6.59 unit and 5.27 unit stocks stored at 20 °C. The 5.27 unit stock was diluted to make 3.51, 2.64, and 1.76 unit stocks fresh on the day of the assay. A unit was defined as the amount of enzyme needed to decrease absorbance by 0.001 per min at 450 nm. The standards were read simultaneously with the hemolymph samples to confirm that the assay was progressing as expected (i.e. absorbance values decreasing). 2.9. Statistical methods All measured parameters (body weight, hemolymph protein content, PO enzyme activity, lysozyme-like enzyme activity, and

A

encapsulation ability) were statistically analyzed using a oneway ANOVA. If there were significant differences found, a TukeyKramer post-hoc test was performed with a = 0.05. All values are reported as mean ± sem. All statistical analyses were performed with JMP Pro 10.0.0 software (2012, SAS Institute, Inc.). 3. Results 3.1. Developmental changes in body condition Under our laboratory rearing conditions, 8th instar nymphs took approximately 8–9 days to develop and molt into the 9th instar following their isolation as an 8th instar, while 9th instar nymphs took 14–15 days to reach the imaginal (adult) molt. There was a consistent and significant increase in body mass across these developmental stages (F8,338 = 181.22, P < 0.0001). D2–8th nymphs had the lowest body mass and each subsequent nymphal age and stage examined had a significantly greater body mass than the next (Fig. 1A). However, no significant differences in body mass were found between D9–9th instar nymphs and adults. There was also no significant change in body mass of adults during the first three weeks of adulthood. The hemolymph protein content of males in each of these nymphal and adult stages was also determined (Fig. 1B), with significant differences observed (F8,328 = 35.42, P < 0.0001). D2–8th instar male nymphs had the lowest hemolymph protein content while 8th instar nymphs that were examined just 3 days later (D5–8th instar) had the highest protein content. There was a significant increase in hemolymph protein content within each specific 8th and 9th instar nymphal stage; D5–8th instar males had higher hemolymph protein content than D2–8th instar males (P < 0.001). Similarly, D9–9th instar nymphs had higher protein con-

0.40

d

0.35

Body mass (g)

0.30 0.25

c 0.20

b 0.15

a 0.10 0.05 51

40

36

45

46

18

46

16

41

15

40

0.00

Hemolymph Protein Content (µg/µl)

B

60

b

50

40

d c

30

c 20

a

10 52

40

33

45

40

18

46

D2 8th

D5 8th

D2 9th

D9 9th

D1-3 ad

D5-7 ad

D9-12 ad

0 D14-16 D21-23 ad ad

Fig. 1. Developmental changes in body mass (A) and hemolymph protein content (B) for male cricket nymphs (open bars) and adults (solid bars). Numbers in bars indicate sample sizes. All values are mean ± sem and different letters indicate significant differences.

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A.V. Piñera et al. / Journal of Insect Physiology 59 (2013) 752–760

A

B

10

d

9 8

10 9 8

c

7

6

5

5

4

3

a,b 1

c

4

b

3 2

c

7

6

a 52

40

a,b

a

36

45

a

2 45

18

46

16

1

41

0

11

15

13

D4 ad

D5 ad

D6 ad

0 D2 8th

D5 8th

D2 9th

D9 9th

D1-3 ad

D5-7 ad

D9-12 ad

D14-16 D21-23 ad ad

Fig. 2. (A) Total phenoloxidase (PO) enzyme activity (mean ± sem) for nymphs (open bars) and adults (solid bars) at different developmental ages. (B) Total phenoloxidase enzyme activity for adults that were exactly 4 days (D4), 5 days (D5), or 6 days (D6) in age past the imaginal molt. Numbers in bars indicate sample sizes and different letters indicate significant differences.

A

Ud Pl Fi

B

Hemocytes in 1µl hemolymph

Gr

14000 a 12000

a a,b

10000 8000

a,b

a,b a,b

6000

b

4000 2000

15

14

17

14

14

N/A

D2 8th

D5 8th

D2 9th

D9 9th

D1-3 ad

D5-7 ad

15

N/A

14

0 D9-12 D14-16 D21-23 ad ad ad

Fig. 3. Hemocyte types and estimated number of circulating hemocytes in the hemolymph of male crickets. (A) Confocal micrographs of hemocytes classified as granulocytes (Gr, left), fibroblasts (Fi, middle), plasmatocytes (Pl, right) and undetermined (Ud, right) in a hemolymph sample from an adult male cricket. Arrowheads indicate the characteristic cytoplasmic extensions of the fibroblasts and plasmatocytes. (B) Estimated number of hemocytes per ll hemolymph (mean ± sem) for male nymphs (open bars) and adults (filled bars). Numbers in bars indicate sample sizes and different letters indicate significant differences. N/A: data not available for this stage.

tent than D2–9th instar males (P < 0.001). After the imaginal molt, there was no significant change in hemolymph protein content during the first three weeks of adulthood (Fig. 1B).

3.2. Total phenoloxidase activity To characterize developmental changes in the humoral immune response of male A. domesticus crickets, total phenoloxidase activity (TPA) of 8th and 9th instar nymphs and of differently aged adults was determined (Fig. 2A). Significant differences in TPA were detected (F8,338 = 106.89, P < 0.0001), with nymphs and D1– 3 adults exhibiting significantly lower hemolymph TPA compared to adult males between 5 and 23 days in age. D5–8th also had significantly higher TPA than D2–8th instar (P = 0.002) and D9–9th instar (P = 0.004) nymphs. However, these nymphs also had significantly higher hemolymph protein content (Fig. 1B) and if we expressed TPA at each stage on a per mg protein basis, this difference between D5–8th instar nymphs and the other nymphal stages was no longer apparent.

Because of the large increase in TPA found to occur between D1–3 and D5–7 adults, we closely examined the critical timing of this change by determining TPA for exact D4, exact D5 and exact D6 adult males (Fig. 2B). There was a significant difference in TPA among these three adult groups (F2,38 = 14.14, P < 0.0001) with the critical time point of significant TPA change occurring between adult day 4 and 5 (P = 0.0003).

3.3. Number of circulating hemocytes Hemocytes are mediators of the insect cellular immune defense response (Gillespie et al., 1997), and so in order to characterize developmental changes in cricket cellular immunity, hemocytes were visualized, classified, and quantified. Four morphologically distinct types of hemocytes were observed (Fig. 3A). Granulocytes (Gr) were large, 10–15 lm diameter, spherical cells with a large, irregularly-shaped nucleus (Fig. 3A, left panel). Granulocytes also had a distinct cell membrane that was easily distinguishable in the hemocytometer from the cell membrane of the other large

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A.V. Piñera et al. / Journal of Insect Physiology 59 (2013) 752–760 Table 1 Classification of hemocytes in 1 ll of hemolymph from male Acheta domesticus nymphs and adults. Stage

Age

n

Nymphs

D2–8th D5–8th D2–9th D9–9th D1–3 D9–12 D21–23

15 14 17 14 14 15 14

Adult

% Total hemocytes Granulocytes

Fibroblasts

Plasmatocytes

Undetermined

93.0 88.7 96.2 96.3 93.0 95.3 96.5

1.0 2.9 0.5 0.6 2.1 0.7 0.3

4.3 3.3 2.3 2.1 4.3 2.8 3.0

1.7 5.1 1.0 1.0 0.6 1.2 0.2

80 b

Encapsulation Score

70 a,b 60 50

a,c a,c c

40

c c

30 20 10 14

11

23

11

18

N/A

20

N/A

28

D2 8th

D5 8th

D2 9th

D9 9th

D1-3 ad

D5-7 ad

D9-12 ad

D14-16 ad

D21-23 ad

0

Fig. 4. Encapsulation ability (mean ± sem) of male nymphs (open bars) and adults (solid bars). Numbers in bars indicate sample sizes and different letters indicate significant differences. N/A: data not available.

Lyc acvity (∆ absorbance/min x -103)

hemocyte, the plasmatocytes (Pl), which tended to flatten as they attached to the hemocytometer surface. Plasmatocytes, which were similar in size to the granulocytes, contained a large spherical nucleus and exhibited cytoplasmic extensions, leading these cells to have a crescent-shaped appearance (Fig. 3A, right panel). Fibroblasts, at 5–7 lm diameter, were the smallest hemocytes. Fibroblasts had an elongated shape and contained a thin, oval nucleus with a striated appearance (Fig. 3A, middle panel). Finally, we observed a fourth type of small hemocyte (6–7 lm diameter) with a round nucleus and very little cytoplasm (Fig. 3A, right panel). These cells, few in number, were classified as ‘undetermined’. Granulocytes were the most common type of hemocyte observed, comprising approximately 90% of all hemocytes counted in 1 ll hemolymph samples obtained from late nymphal and adult male crickets (Table 1). The number of hemocytes counted in the

different hemolymph samples from individual crickets was highly variable (Fig. 3B). However, a significant difference in the number of circulating hemocytes was observed (F6,101 = 2.72, P = 0.02) with both D5–8th and D9–9th instar nymphs exhibiting a significantly greater number of hemocytes than D9–12 adults (P = 0.03 for both; Fig. 3B). There were no significant differences in the number of circulating hemocytes between males of the two nymphal stages or among the differently aged adult males (Fig. 3B). 3.4. Encapsulation ability Though A. domesticus nymphs had much lower total phenoloxidase enzyme activity than adult males (Fig. 2A), their ability to encapsulate a nylon thread was similar to that of the adult (Fig. 4). Significant differences in the ability to encapsulate a nylon

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 33

14

26

34

16

D2 8th

D5 8th

D2 9th

D9 9th

D1-3 ad

0.0

N/A D5-7 ad

27 D9-12 ad

N/A

14

D14-16 D21-23 ad ad

Fig. 5. Hemolymph lysozyme-like enzyme activity (mean ± sem) of male nymphs (open bars) and adults (solid bars). Numbers in bars indicate sample sizes. No significant differences were found among the groups. N/A: data not available.

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thread implant were found among the developmental stages (F6,124 = 6.54, P < 0.0001), including the specific nymphal stages, with D2–8th instar nymphs exhibiting a lower encapsulation ability than D5–8th nymphs (P = 0.02) and D2–9th instar nymphs exhibiting a significantly greater encapsulation ability than D9–9th nymphs (P = 0.007). D5–8th instar nymphs encapsulated a nylon thread to a significantly greater degree than all other developmental stages/ages except D2–9ths. Adult males of all ages examined exhibited similar encapsulation ability (Fig. 4).

3.5. Lysozyme-like enzyme activity Lysozyme-like enzyme activity in 1 ll hemolymph samples was also determined and compared for all four nymphal groups and for D1–3, D9–12, and D21–23 adults (Fig. 5). No significant differences in lysozyme-like activity were found (F6,163 = 1.42, P = 0.21).

4. Discussion We describe the maturation of the innate immune system of the male house cricket A. domesticus during the last two nymphal stages, and early and late adult stages. To date, relatively few studies have compared the immune function of both the larval/nymphal and the adult stages of insects (Gryllus integer: Adamo et al., 2001; Apis mellifera: Gätschenberger et al., 2012; Laughton et al., 2011; Wilson-Rich et al., 2008; Locusta migratoria: Mullen and Goldsworthy, 2003; Periplaneta americana: Rheins and Karp, 1985; Dendroctonus valens: Shi and Sun, 2010; Anabrus simplex, Syrgley, 2012). Here, we report a significant increase in hemolymph PO activity during the male cricket’s transition from a late instar nymph to a mature adult. Though total hemolymph PO activity was lowest during the two nymphal stages we examined, male A. domesticus nymphs tended to exhibit the strongest encapsulation response. In contrast, both the number of circulating hemocytes and hemolymph lysozyme-like activity varied little among the developmental stages examined, and did not change significantly during the first three weeks of adulthood. In addition to our examination of total PO activity, hemocyte number, lysozyme-like activity and encapsulation ability, we monitored developmental changes in body mass and hemolymph protein content. As expected, body mass increased during development, reaching a maximum of about 300 mg in the late last nymphal stage, just a few days before the imaginal molt. Woodring (1983) previously reported that the attainment of a body weight of at least 280–300 mg by last instar A. domesticus nymphs is necessary to initiate the transition into the adult molt and he proposed that this critical weight gain triggered the release of prothoraciotropic hormone (PTTH) from the brain; PTTH signals the insect prothoracic glands to release ecdysteroids required for the imaginal molt (reviewed in Gilbert et al., 2002). Both body mass and hemolymph protein content changed little during adulthood. Total hemolymph protein content has been positively correlated with overall body condition in the cricket Gryllus campestris (Jacot et al., 2005) and with the ability to counteract a pathogenic challenge in the cricket G. texensis (Adamo 2004a). During nymphal stages, however, we found hemolymph protein content to be at its lowest level when measured a few days after ecdysis. A similar change in hemolymph protein concentration was reported for late instar female A. domesticus nymphs (Woodring et al., 1977a). Female nymphs were found to decrease food intake a few days before and a few days after ecdysis, while exhibiting the greatest consumption of food near the midpoint of the nymphal stage (Roe et al., 1985; Woodring et al., 1977b). Though we did not measure food intake in the current study, our

results for hemolymph protein content suggest male nymphs may follow a similar pattern of feeding behavior. The enzyme PO is a key component of the insect innate immune response that is synthesized as the inactive zymogen ProPO by hemocytes (reviewed in Cerenius and Söderhall, 2004; GonzálezSantoyo and Córdoba-Aguilar, 2012). During nodulation and encapsulation responses, hemocytes aggregate around and surround foreign bodies, such as bacterial cells, within the hemocoel; the ensuing activation of PO results in melaninzation of the surrounding hemocyte layers and death of the targeted invader by starvation or asphyxiation (reviewed in Christensen et al., 2005; Gillespie et al., 1997; Strand, 2008). We found the total hemolymph PO activity of late instar male A. domesticus nymphs and recently molted (D1–4) adults to be less than half that of mature (i.e., day 5 or older) adults. What could account for such a large increase in hemolymph PO activity following the imaginal molt? One possible explanation could be the changing hormonal milieu associated with the transition from the nymphal to the adult stage. During nymphal stages, juvenile hormone (JHIII) secretion from the corpora allata ensures that each molt will produce another immature nymph; when the nymph reaches a critical body mass, however, a large decrease in JHIII levels and concurrent increase in ecdysteroid levels induces the imaginal molt (reviewed in Gilbert et al., 2002; Jindra et al., 2013). Juvenile hormone has been shown to negatively impact insect immune function. Injection of JHIII into the adult mealworm Tenebrio molitor decreased hemolymph PO activity and suppressed encapsulation ability, but did not affect lysozyme-like activity (Rantala et al., 2003). Similarly, juvenile hormone administration inhibited the increase in PO activity usually observed in adult Rhodnius prolixus following a bacterial challenge (Nakamura et al., 2007). Such findings suggest that a decrease in JHIII levels during the adult transition could permit the elevation of PO activity that we observed in mature adult male crickets. In A. domesticus crickets, the main route of JHIII degradation is through the action of the enzyme juvenile hormone esterase (JHE; Woodring and Sparks, 1987). In females, hemolymph JHE levels were found to be relatively constant during the first 3 days of adulthood, but showed a sharp increase on day 4 (Renucci et al., 1984); we propose that a similar change in male JHE levels could produce a reduction in JHIII levels sufficient to permit the sudden large increase in PO activity that we first observed 5 days after the imaginal molt. Though the hormonal changes which take place during female cricket development have been the focus of intense study, hormonal changes occurring during the development of male crickets, and the ability of those hormones to influence both male and female immune function, need further study. A significant increase in hemolymph PO activity following the molt from a larva or nymph to an adult has also been reported for the honeybee A. mellifera (Wilson-Rich et al., 2008) and the Mormon cricket A. simplex (Syrgley, 2012). However, a decrease in hemolymph PO activity occurred after the transition into adulthood for the red turpentine beetle D. valens (Shi and Sun, 2010) and for male G. texensis crickets (Adamo et al., 2001). Species-specific variation could be one explanation for such differences in the development of PO activity; the different housing conditions under which insects are reared, however, may also play a role. For example, the male crickets of our study remained isolated following the imaginal molt and were thus socially naïve, while the male G. texensis crickets of Adamo et al. (2001), which showed a significant decrease in PO activity following the imaginal molt, were maintained in social groups. Mating was shown to significantly decrease the hemolymph PO activity of male and female mealworm beetles (T. molitor) through a mating-induced increase in juvenile hormone secretion (Rolff and Siva-Jothy, 2002). Interestingly, virgin adult male Gryllus bimaculatus crickets exhibited lower hemolymph JHIII

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levels than same-aged males housed with a conspecific female (Klein et al., 1993); we suggest that an isolation-induced reduction in JHIII hemolymph levels may have contributed to the greater hemolymph PO activity observed in the socially naïve adult male crickets of our study. Hemolymph samples from the adult male crickets of our study that were three weeks in age past the imaginal molt had significantly higher PO activity than younger adults, 1–2 weeks in age. Many studies on adult insects have reported a decline in immune function with age, a process referred to as immunosenescence (reviewed in Leips, 2009). For example, in adult male G. texensis crickets, hemolymph PO activity decreased with age (Adamo et al., 2001) while in adult male Gryllus assimilis crickets, both the number of circulating hemocytes and the nodulation response to injection of bacterial lipopolysaccharides decreased with age (Park et al., 2011). As discussed above, social isolation may have also contributed to the increase in PO activity we observed in the three week old adult male crickets of our study. It would be of interest to examine hemolymph PO activity of even older males and to determine if social interaction could, in fact, produce the decrease in PO activity with advancing age as has been observed for other species of crickets. Hemocytes are required for encapsulation and nodulation (reviewed in Gillespie et al., 1997; Strand, 2008). To classify the circulating hemocytes we observed in male cricket nymphs and adults, we referred to the previous morphological description of A. domesticus hemocytes by da Silva et al. (2000). We identified three main types of hemocytes: granulocytes, plasmatocytes, and fibroblasts. The most prevalent hemocytes found in blood sampled from male crickets in each age group were granulocytes. Plasmatocytes were classified into both spherical and fibroblastic forms by da Silva et al. (2000). We only observed spherical plasmatocytes. Cells that we classified as fibroblasts were elongated in shape, and contained an oval nucleus with a distinctly striated appearance, leading us to place these cells into a class of their own, separate from the much larger plasmatocytes. These cells may be similar to the vermiform cells of lepidoptera (Ribeiro and Brehélin, 2006). We also observed a fourth type of hemocyte that we classified as ‘undetermined’; these cells were small in size and contained little cytoplasm. It is possible that these are either the remnants of damaged cells or may be similar to the prohemocytes reported for other insect species (reviewed in Lavine and Strand, 2002; Ribeiro and Brehélin, 2006). We did not observe the hemocytes classified as coagulocytes by da Silva et al. (2000). We observed little change in the number of circulating hemocytes during the transition from the nymphal to adult stage of the male cricket, though the number of hemocytes counted could vary greatly, even among crickets of the same age. Hemocytes have been reported to be the primary site of ProPO synthesis (reviewed in Cerenius and Söderhall, 2004; González-Santoyo and CórdobaAguilar, 2012), and so the low hemolymph PO activity that we observed for nymphal male crickets could not be attributed to the presence of fewer circulating hemocytes. However, among insects, the actual cell type responsible for PO synthesis can vary from species to species (see González-Santoyo and Córdoba-Aguilar, 2012), and the hemocyte type (or types) responsible for PO synthesis in our insect model is currently unknown. Nymphs and newly emerged adult males had significantly lower total hemolymph PO activity than older adults, yet were able to encapsulate a nylon thread just as well as, if not better, than older adult male crickets. Encapsulation ability was thus independent of total PO activity. A similar lack of correlation between hemolymph PO activity and encapsulation ability had been reported for other insects including the Mormon cricket (Syrgley, 2012) and honeybee (Wilson-Rich et al., 2008). However, the encapsulation response itself can lead to an increase in hemolymph PO activity

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(and corresponding decrease in lysozyme-like activity) in a variety of insects, including A. domesticus crickets (Ardia et al., 2012). It would be interesting to compare the relative changes in hemolymph PO activity of both nymphs and adults following the initiation of an encapsulation response. Lysozyme, like PO, is an enzyme involved in the insect innate immune response. Lysozyme is synthesized and secreted by the insect fat body and aids in breaking down bacterial cell walls (reviewed in Callewaert and Michiels, 2010; Hultmark, 1996). Hemolymph lysozyme-like activity was highly variable but did not differ significantly among the developmental groups examined in our study. In summary, though late instar nymphs and newly emerged adult male crickets exhibited much lower total hemolymph PO activity than mature adults, their response in encapsulating a foreign body was just as effective. In addition, the number of circulating hemocytes and lysozyme-like activity in the hemolymph of nymphs and adults varied little, indicating that A. domesticus nymphs may respond as well as adults to immune challenges they may encounter in their environment. However, to confirm this, a thorough examination of the ability of both nymphs and adults to respond to a variety of immune challenges must be performed. Acknowledgements The authors thank Kerry Orton and Michael Schmees for their contributions to this research study and Harmin Chima and Joshua Schiets for maintaining our cricket colony. We also thank Mollie Sorrel and an anonymous reviewer for their editorial comments on the manuscript. Funds for this research were provided by the Zoology Department of Miami University. References Adamo, S.A., 2004a. Estimating disease resistance in insects: phenoloxidase and lysozyme-like activity and disease resistance in the cricket, Gryllus texensis. Journal of Insect Physiology 50, 209–216. Adamo, S.A., 2004b. How should behavioural ecologists interpret measurements of immunity? Animal Behaviour 68, 1443–1449. Adamo, S.A., Jensen, M., Younger, M., 2001. Changes in lifetime immunocompetence in male and female Gryllus texensis (formerly G. integer)⁄: trade-offs between immunity and reproduction. Animal Behaviour 62, 417–425. Adamo, S.A., Lovett, M.M.E., 2011. Some like it hot: the effects of climate change on reproduction, immune function and disease resistance in the cricket Gryllus texensis. Journal of Experimental Biology 214, 1997–2004. Alaux, C., Ducioz, F., Crauser, D., Le Conte, Y., 2010. Diet effects on honeybee immunocompetence. Biology Letters 6, 562–565. Anand, A.N., Lorenz, M.W., 2008. Age-dependent changes of fat body stores and the regulation of fat body lipid synthesis and mobilization by adipokinetic hormone in the last larval instar of the cricket, Gryllus bimaculatus. Journal of Insect Physiology 54, 1404–1412. Ardia, D.R., Gantz, J.E., Schneider, B.C., Strebel, S., 2012. Costs of immunity in insects: an induced immune response increases metabolic rate and decreases antimicrobial activity. Functional Ecology 26, 732–739. Arrese, E.L., Soulages, J.L., 2010. Insect fat body: energy, metabolism, and regulation. Annual Review of Entomology 55, 207–225. Bailey, N.W., Zuk, M., 2008. Changes in immune function of male field crickets with mobile parasitoid larvae. Journal of Insect Physiology 54, 96–104. Brookman, J.L., Rowley, A.F., Ratcliffe, N.A., 1989. Studies on nodule formation in locusts following injection of microbial products. Journal of Invertebrate Pathology 53, 315–323. Callewaert, L., Michiels, C.W., 2010. Lysozymes in the animal kingdom. Journal of Bioscience 35, 127–160. Cerenius, L., Söderhall, K., 2004. The prophenoloxidase-activating system in invertebrates. Immunological Reviews 198, 116–126. Cerenius, L., Lee, B.L., Soderhall, K., 2008. The proPO-system: pros and cons for its role in invertebrate immunity. Trends in Immunology 29 (6), 263–271. Christensen, B.M., Li, J., Chen, C., Nappi, A.J., 2005. Melanization immune responses in mosquito vectors. Trends in Parasitology 21, 192–199. da Silva, C., Dunphy, G.B., Rau, M.E., 2000. Interaction of hemocytes and prophenoloxidase system of fifth instar nymphs of Acheta domesticus with bacteria. Developmental and Comparative Immunology 24, 367–379. Drayton, J.M., Milner, R.N., Hall, M.D., Jennions, M.D., 2011. Inbreeding and courtship calling in the cricket Teleogryllus commodus. Journal of Evolutionary Biology 24, 47–58.

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