A r t i c l e
IMPACT OF IMMUNE RESPONSE OF A PARASITIC BEETLE Dastarcus helophoroides ON ITS HOST BEETLE Monochamus alternatus Xiao-juan Li, Guang-ping Dong, Jian-min Fang, Hong-jian Liu, Li Yang, and Wan-lin Guo Institute of Forest Protection, Anhui Provincial Academy of Forestry, Hefei, People’s Republic of China
Dastarcus helophoroides is an ectoparasitoid beetle of Monochamus alternatus, and the parasitism by D. helophoroides larvae remarkably influenced on the immune responses of M. alternatus larvae in many aspects. The hemolymph melanization reactions in the hosts were inhibited 1 h and 24 h postparasitization. The phenoloxidase activities of hemolymph were significantly stimulated 4 h postparasitization and inhibited 12 h postparasitization, and back to control level. The antibacterial activities of hemolymph in the parasitized hosts were significantly lower than that in the unparasitized ones 1 h postparasitization. By 72 h postparasitism, the total hemocyte numbers of the parasitized larvae declined to not more than one-seconds of the number collected from the unparasitized larvae. All sampled hemolymph held the capability of nodulation, and there were fluctuations in the number of nodules the hemocytes made. However, there were no significant differences between unparasitized and parasitized larvae at each time point in the hemagglutination activity and the ratios of spreading hemocytes. In conclusion, D. helophoroides larvae could regulate M. alternatus immune system and resulted in the changes in host immune responses. C 2015 Wiley Periodicals, Inc. Keywords: Dastarcus helophoroides; Monochamus alternatus; immune response
Grant sponsor: National Natural Science Foundation of China; Grant number: Project 31300547; Grant sponsor: Anhui Provincial Natural Science Foundation; Grant number: Project no. 1208085QC72. Correspondence to: Xiao-juan Li, Institute of Forest Protection, Anhui Provincial Academy of Forestry, Hefei, People’s Republic of China. E-mail: [email protected] ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 90, No. 1, 28–42 (2015) Published online in Wiley Online Library (wileyonlinelibrary.com). C 2015 Wiley Periodicals, Inc. DOI: 10.1002/arch.21242
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INTRODUCTION Pine sawyer beetle Monochamus alternatus Hope (Coleoptera: Cerambycidae) is the primary vector of the destructive forest pest pine wood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle (Aphelenchida: Parasitaphelenchidae) and it also causes serious damage to several pine species (Mamiya and Enda, 1972). The parasitoid Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) (a biotype of the species special on M. alternatus) was found and confirmed as an important natural enemy of M. alternatus (Taketsune, 1982; Inoue, 1993; Yang et al., 2014). D. helophoroides larvae are ecto-parasitoids of M. alternatus, and they parasitize larvae, pupae, and young adults of the longhorned beetle (Qin and Gao, 1988). First instar larvae of D. helophoroides have legs and can actively move to search for hosts. Their legs degenerate once parasitism has occurred (Wei et al., 2009). Due to the studies on biology (Qin and Gao, 1988) and mass-rearing techniques (Ogura et al., 1999; Wang and Ogura, 1999, Lei et al., 2005), the laboratory mass production of D. helophoroides have been realized. Here, we give a picture to illustrate this unusual antagonistic beetle system (Fig. 1). Insects defend themselves against pathogens and parasites with their effective innate immune system, which is divided into humoral and cellular responses (Beck and Strand, 2007; Strand, 2008). The humoral responses refer to the production of antimicrobial peptides, complement-like proteins, reactive intermediates of oxygen or nitrogen, and the complex enzymatic cascades that regulate coagulation and melanization of hemolymph (Alves et al., 2013). The cellular responses in contrast involve phagocytosis, nodulation, and encapsulation that are mediated by hemocytes (Er et al., 2011). However, there is an overlap between humoral and cellular responses, since many humoral factors regulate hemocyte activity and hemocytes are important sources of many humoral defense molecules (Strand, 2008; Er et al., 2011). Researches about immune response of insect against parasitism were mainly conducted on Lepidopteran larvae or pupae parasitized by Hymenoptera parasitic wasps. Here, we summarize some of the studies relative to our experiments, which will be introduced below. ¨ The hemolymph melanization reaction in larvae of Trichoplusia ni (Hubner) (Lepi¨ doptera: Noctuidae), Helicoverpa armigera Hubner (Lepidoptera: Noctuidae), Ostrinia furnacalis Guene (Lepidoptera: Pyralidae), and Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) were significantly inhibited after parasitization by Hyposoter exiguae (Viereck) (Hymenoptera: Ichneumonidae), Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae), Macrocentrus cingulum Brischke (Hymenoptera: Braconidae), and Diadegma semiclausum (Hellen) (Hymenoptera: Ichneumonidae), respectively (Stoltz and Cook, 1983; Yin et al., 2001; Feng et al., 2004; Huang et al., 2011a). The hemolymph hemagglutination activity in Pieris rapae (Linnaeus) (Lepidopetera: Pieridae) pupae parasitized by Pteromalus puparum (Linnaeus) (Hymenoptera: Pteromalidae) was always higher than that in unparasitized ones (Cai et al, 2001). The phenoloxidase activities in host hemolymph were more or less inhibited by parasitism in most of the reports (Stoltz and Cook, 1983; Beck et al., 2000; Yin et al., 2001; Feng et al., 2004; Li et al., 2006; Huang et al., 2011a), for example, Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) larvae parasitized by Venturia canescens (Gravenhorst) (Hymenoptera: Ichneumonidae) (Beck et al., 2000) and P. xylostella larvae parasitized by Diadromus collaris (Gravenhorst) (Hymenoptera: Ichneumonidae) (Li et al., 2006). Nevertheless, parasitism did not induce the inhibition of phenoloxidase activity in other insect species (Sroka and Vinson, 1978; Cai et al., 2001; Li et al., 2011). The antibacterial activity in P. rapae pupae
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Figure 1.
Archives of Insect Biochemistry and Physiology, September 2015
Antagonistic system between D. helophoroides and M. alternatus.
hemolymph postparasitization by P. puparum became higher (Cai et al., 2001), whereas it was decreased in P. xylostella larvae parasitized by D. semiclausum (Huang et al., 2011a). The total hemocyte numbers (THC) in P. xylostella pupae parasitized by D. collaris increased (Li et al., 2006), but those numbers declined in the same host larvae parasitized by D. semiclausum (Huang et al., 2009). The spreading behaviors of granulocytes and plasmatocytes were inhibited in P. xylostella pupae parasitized by D. collaris, and never recovered (Li et al., 2006). While in P. xylostella larvae parasitized by D. semiclausum, the spreading ability of hemocytes from the host larvae was obviously inhibited in the early stage of the parasitism, and recovered at 24 h postparasitization (Huang et al., 2009). Both the phagocytosis and encapsulation abilities were suppressed in P. xylostella larvae in the early stage of the parasitism by D. semiclausum, and those abilities were recovered at 24 h postparasitization (Huang et al., 2009; Huang et al., 2011b). In this article, we examined the immune response in the larval hemolymph of pine sawyer beetle M. alternatus, to parasitism by its parasitoids D. helophoroides. Both the Archives of Insect Biochemistry and Physiology
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humoral immune responses include melanization, hemagglutination, phenoloxidase activity, and antibacterial activity and the cellular immune responses include THC, cell spreading, and nodulation were investigated.
MATERIALS AND METHODS Insect Collection Pinus massoniana Lamb infested with M. alternatus were cut down in the Huanglishu Forest Farm of Quanjiao County, Anhui province, China, in March, 2014. M. alternatus larvae were collected from the xylem of the host trees. These activities were conducted with permission from the Huanglishu Forest Farm of Quanjiao County. Larvae were held singly in small sterile tubes, kept in a box out of the sun, and returned to the Anhui Provincial Academy of Forestry within 8 h after collection. Larvae were stored at 8 ± 0.5 °C and 50 ± 5% RH. Mature larvae in the fifth instars, based on head capsule measurements (Liu et al., 2008), were used in the experiments, to separate age effect from parasitism effect on host immune responses.
Parasitoid Rearing D. helophoroides colony was provided by the Institute of Forest Protection, Anhui Provincial Academy of Forestry, Hefei, China. They were the laboratory-reared offspring of the mother adults provided by the Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing, China. The ancestors parasitized larvae or pupae of M. alternatus in the trucks or branches of P. massoniana, which were collected in 2003 at Guangzhou (23°80 N, 113°17 E, and altitude 120 m), Guangdong Province, China. D. helophoroides adults were reared on artificial diet and the larvae were reared on live M. alternatus larvae. D. helophoroides larvae used in the experiments were the third generation of the beetles reared in laboratory.
Parasitization Six newly hatched D. helophoroides larvae were inoculated larva using a small brush. One parasitized or unparasitized was put into a sterile tube (with a small hole drilled in bated at 25 ± 0.5 °C and 50 ± 5% RH for 1, 2, 4, 72 h.
to a M. alternatus M. alternatus larva its lid) and incu8, 12, 24, 48, or
Bleeding Procedure M. alternatus larvae were surface sterilized with 75% ethanol for a few seconds and rinsed with sterile water, and then, dried. Hemolymph was obtained from the host larvae by cutting their posterior part with sterile ophthalmic scissors. Archives of Insect Biochemistry and Physiology
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Melanization Reaction Experiment of melanization reaction was conducted by the method of Yin et al., (2001). In order to access the capacity of melanization reaction in the whole hemolymph of the host, cohorts of ten M. alternatus larvae were selected at 1, 2, 4, 8, 12, 24, 48, or 72 h postparasitization. Hemolymph sample from each parasitized or unparasitized larva was collected and put on a Parafilm. The drop of undiluted hemolymph was left for 1 h at ambient room temperature. A change in coloration of the hemolymph from opaque to brown–black was recorded as normal melanization, whereas the maintenance of the previous color or change in an intermediate color was considered as reflecting an inhibition of melanization. Hemagglutination Assay Hemagglutination activity test method referenced to Drif and Breh lin (1989) cited by Cai et al., (2001). Hemolymph of each M. alternatus larva was collected and put into a sterile tube with a few phenlythioura (PTU) crystals, and then, centrifuged at 8,000 g for 10 min at 4 °C. The supernatant was diluted with 0.1 M PBS (pH 7.4) according to different dilution factor. To conduct the assay, 15 μl undiluted hemolymph or twofold serial dilutions of the hemolymph, mixed with 15 μl 2% chicken red blood cells suspension, and added to the wells of a 96-well V-bottom plate. The red blood cells mixed with the PBS were used as controls. The plate was incubated at room temperature for 1 h. The titers were expressed as geometric mean titers (GMT) of various treatments. Assay for Phenoloxidase (PO) Activity The PO activity was measured spectrophotometrically according to Xylander and Bogusch (1992) as cited by Beck et al., (2000). As substrate, a 0.02 M solution of L-3,4dihydroxyphenylalanine (L-DOPA) (Alfa Aesar, America) in 0.1 M PBS (pH 7.4) was used. Hemolymph of each M. alternatus larva was collected and put into a sterile tube. After centrifugation at 14, 000 g for 10 min at 4 °C, 50 μl supernatant was taken and mixed with 100 μl substrate solution in a 96-well plate. PO activity was monitored by measuring absorbance at 490 nm using a microplate reader (Thermo Scientific Multiskan GO, America) after 30 min incubation at 30 °C. One unit of phenoloxidase activity was defined as A490 = 1. Antibacterial Test Antibacterial test was conducted by the method of Huang et al. (2011a). The mixture of serum-free insect cell culture medium (Hyclone, America), Escherichia coli suspension (A600 = 0.5), and hemolymph from each larva, at a ratio of 18:1:1, were incubated at 37 °C for 30 min, and then, measured the absorbance at 600 nm using a microplate reader (Thermo Scientific Multiskan GO, America). One unit of antibacterial activity was defined as A600 = 1. Hemocyte Counting Method for counting the hemocytes was as described by Manachini et al. (2011). To count the hemocytes, 10 μl pure hemolymph from each M. alternatus larva was put, immediately Archives of Insect Biochemistry and Physiology
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after bleeding, into a sterile tube with 90 μl anticoagulant solution (0.098M NaOH, 0.186M NaCl, 0.017M EDTA, 0.041M Citric acid, pH 4.5) added. After blending, the diluted hemolymph was put in a 0.0025 mm2 hemocytometer under an inverted phase contrast microscope (Olympus CX41, Japan). The THC from parasitized and unparasitized larvae was recorded at designated times postparasitization.
Assay for Cell Spreading Larva hemolymph was sampled at 1, 2, 4, 8, 12, 24, 48, or 72 h postparasitization. 10 μl hemolymph of each larva was put on a clean slide immediately after bleeding and covered with a cover glass. The spreading of granulocytes and plasmatocytes was observed using an inverted phase contrast microscope (Olympus CX41, Japan). We counted all granulocytes and plasmatocytes in ten randomly chosen fields of view at 400×magnification, and gave the counts of spreading granulocytes and plasmatocytes, which were identified according to Jones (1962) and Ribeiro and Breh´elin (2006). The spreading percentage of granulocytes and plasmatocytes were calculated as follows: % spreading = (number of spreading granulocytes and plasmatocytes observed)/(total number of granulocytes and plasmatocytes observed)×100.
Assay for Nodulation Heat-killed E. coli were used as nodulation targets. Fluorescein isothiocyanate (FITC) labeling of bacteria was performed by the method of Hed & Stendahl (1982) as cited by Beck & Strand (2005) with slight modifications. Heat-killed E. coli (109 /ml) cells were incubated in 0.2 M carbonate buffer (pH 9.5) containing FITC (0.1 mg/ml) for 30 min at 37 °C. Thereafter, the FITC-conjugated bacteria were washed four times in 0.2 M PBS (pH 7.2), and then, kept at 4 °C until use. 10 μl hemolymph of each larva, 10 μl serum-free insect cell culture medium (Hyclone, America), and 10 μl FITC labeled E. coli suspension were added to a sterile tube. Cultures were maintained at 25 °C for 2 h. At this time, multiple hemocytes adhered to aggregations of E. coli and formed a nodule. 10 μl of the culture was put on a clean slide and covered with a cover glass. We scored the number of nodules from ten randomly selected fields of view by use of an inverted fluorescence microscope (Olympus CX41, Japan).
Statistics The number of samples used for each experimental condition is indicated in the figure legends. The significant of differences in melanization reaction was determined by the Chi-square test. The differences of hemagglutination activity, phenoloxidase activity, antibacterial activity, THC, the spreading percentage of granulocytes and plasmatocytes, and the number of capsules between parasitized larvae and unparasitized ones at each time point were analyzed using the Pairwise t-test. We also took a time zone test: early parasitism stage (1, 2, 3, 4, 8, 12, and 24 h) versus late parasitism stage (48 and 72 h) by using the Pairwise t-test as well. All data were performed on SPSS 19.0. Archives of Insect Biochemistry and Physiology
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Table 1. Hemagglutination Activity in M. alternatus Larvae Parasitized or Unparasitized by D. helophoroides (n = 3 Replicates for Each Time Points. GMT Means Geometric Mean Titers) Hours after parasitization
1 2 4 8 12 24 48 72
Unparasitized Parasitized Unparasitized Parasitized Unparasitized Parasitized Unparasitized Parasitized Unparasitized Parasitized Unparasitized Parasitized Unparasitized Parasitized Unparasitized Parasitized
Titer (log2 ) Range
Average
GMT
0–4 0–3 0–0 0–0 0–2 0–0 0–2 0–1 0–0 0–3 0–1 0–0 0–1 0–2 0–2 2–2
1.67 1 0 0 0.67 0 0.67 0.33 0 1 0.33 0 0.67 0.67 0.67 2
3.18 2 1 1 1.59 1 1.59 1.26 1 2 1.26 1 1.59 1.59 1.59 4
RESULTS Effect of Parasitization on Melanization reaction Not all sampled hemolymph were melanized normally in 1 h, and only 20–70% from unparasitized larvae and 0–40% from parasitized larvae were melanized in given time (Fig. 2). There were variations of melanization both in parasitized larvae and the controls, however, the differences were not significant according to the Chi-square test results (parasitized X2 = 8.686, DF = 7, P = 0.276; unparasitized X2 = 10, DF = 7, P = 0.189; Chisquare test). The differences between unparasitized and parasitized larvae in melanization reaction were significant only at 1 h and 24 h postparasitization, while they were not at other time points (1 h, X2 = 7.500, DF = 1, P = 0.006; 2 h, X2 = 3.810, DF = 1, P = 0.051; 4 h, X2 = 2.400, DF = 1, P = 0.121; 8 h, X2 = 0.952, DF = 1, P = 0.329; 12 h, X2 = 0.392, DF = 1, P = 0.531; 24 h, X2 = 5.495, DF = 1, P = 0.019; 48 h, X2 = 1.250, DF = 1, P = 0.264; 72 h, X2 = 3.529, DF = 1, P = 0.060; Chi-square test). Effect of Parasitization on Hemagglutination Activity Hemolymph hemagglutination activity of M. alternatus larvae was checked by using 2% chicken red blood cells suspension. Almost 100% of undiluted hemolymph from both unparasitized and parasitized larvae could cause the red blood cells hemagglutination. The hemagglutination titers were indicated in Table 1. There were no significant differences between unparasitized and parasitized larvae (1 h, T = 2.000, DF = 2, P = 0.184; 2 h, T not available, DF = 2, P not available; 4 h, T = 1.000, DF = 2, P = 0.423; 8 h, T = 0.378, DF = 2, P = 0.742; 12 h, T = −1.000, DF = 2, P = 0.423; 24 h, T = 1.000, DF = 2, P = 0.423; 48 h, T = 0, DF = 2, P = 1.000; 72 h, T = −2.000, DF = 2, P = 0.184; Pairwise t-test). No significant differences were found between early parasitism stage and late parasitism stage (T = −1.000, DF = 5, P = 0.363; Pairwise Archives of Insect Biochemistry and Physiology
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Figure 2. Melanization reaction in M. alternatus larvae parasitized or unparasitized by D. helophoroides. Times postparasitization were shown on the left. Bars in the graphs indicate the number melanized or nonmelanized.
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Figure 3. Phenoloxidase activity in M. alternatus larvae parasitized or unparasitized by D. helophoroides (The error bars correspond to SE. * indicates significantly different between parasitized and unparasitized samples at P
IMPACT OF IMMUNE RESPONSE OF A PARASITIC BEETLE Dastarcus helophoroides ON ITS HOST BEETLE Monochamus alternatus Xiao-juan Li, Guang-ping Dong, Jian-min Fang, Hong-jian Liu, Li Yang, and Wan-lin Guo Institute of Forest Protection, Anhui Provincial Academy of Forestry, Hefei, People’s Republic of China
Dastarcus helophoroides is an ectoparasitoid beetle of Monochamus alternatus, and the parasitism by D. helophoroides larvae remarkably influenced on the immune responses of M. alternatus larvae in many aspects. The hemolymph melanization reactions in the hosts were inhibited 1 h and 24 h postparasitization. The phenoloxidase activities of hemolymph were significantly stimulated 4 h postparasitization and inhibited 12 h postparasitization, and back to control level. The antibacterial activities of hemolymph in the parasitized hosts were significantly lower than that in the unparasitized ones 1 h postparasitization. By 72 h postparasitism, the total hemocyte numbers of the parasitized larvae declined to not more than one-seconds of the number collected from the unparasitized larvae. All sampled hemolymph held the capability of nodulation, and there were fluctuations in the number of nodules the hemocytes made. However, there were no significant differences between unparasitized and parasitized larvae at each time point in the hemagglutination activity and the ratios of spreading hemocytes. In conclusion, D. helophoroides larvae could regulate M. alternatus immune system and resulted in the changes in host immune responses. C 2015 Wiley Periodicals, Inc. Keywords: Dastarcus helophoroides; Monochamus alternatus; immune response
Grant sponsor: National Natural Science Foundation of China; Grant number: Project 31300547; Grant sponsor: Anhui Provincial Natural Science Foundation; Grant number: Project no. 1208085QC72. Correspondence to: Xiao-juan Li, Institute of Forest Protection, Anhui Provincial Academy of Forestry, Hefei, People’s Republic of China. E-mail: [email protected] ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 90, No. 1, 28–42 (2015) Published online in Wiley Online Library (wileyonlinelibrary.com). C 2015 Wiley Periodicals, Inc. DOI: 10.1002/arch.21242
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INTRODUCTION Pine sawyer beetle Monochamus alternatus Hope (Coleoptera: Cerambycidae) is the primary vector of the destructive forest pest pine wood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle (Aphelenchida: Parasitaphelenchidae) and it also causes serious damage to several pine species (Mamiya and Enda, 1972). The parasitoid Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) (a biotype of the species special on M. alternatus) was found and confirmed as an important natural enemy of M. alternatus (Taketsune, 1982; Inoue, 1993; Yang et al., 2014). D. helophoroides larvae are ecto-parasitoids of M. alternatus, and they parasitize larvae, pupae, and young adults of the longhorned beetle (Qin and Gao, 1988). First instar larvae of D. helophoroides have legs and can actively move to search for hosts. Their legs degenerate once parasitism has occurred (Wei et al., 2009). Due to the studies on biology (Qin and Gao, 1988) and mass-rearing techniques (Ogura et al., 1999; Wang and Ogura, 1999, Lei et al., 2005), the laboratory mass production of D. helophoroides have been realized. Here, we give a picture to illustrate this unusual antagonistic beetle system (Fig. 1). Insects defend themselves against pathogens and parasites with their effective innate immune system, which is divided into humoral and cellular responses (Beck and Strand, 2007; Strand, 2008). The humoral responses refer to the production of antimicrobial peptides, complement-like proteins, reactive intermediates of oxygen or nitrogen, and the complex enzymatic cascades that regulate coagulation and melanization of hemolymph (Alves et al., 2013). The cellular responses in contrast involve phagocytosis, nodulation, and encapsulation that are mediated by hemocytes (Er et al., 2011). However, there is an overlap between humoral and cellular responses, since many humoral factors regulate hemocyte activity and hemocytes are important sources of many humoral defense molecules (Strand, 2008; Er et al., 2011). Researches about immune response of insect against parasitism were mainly conducted on Lepidopteran larvae or pupae parasitized by Hymenoptera parasitic wasps. Here, we summarize some of the studies relative to our experiments, which will be introduced below. ¨ The hemolymph melanization reaction in larvae of Trichoplusia ni (Hubner) (Lepi¨ doptera: Noctuidae), Helicoverpa armigera Hubner (Lepidoptera: Noctuidae), Ostrinia furnacalis Guene (Lepidoptera: Pyralidae), and Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) were significantly inhibited after parasitization by Hyposoter exiguae (Viereck) (Hymenoptera: Ichneumonidae), Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae), Macrocentrus cingulum Brischke (Hymenoptera: Braconidae), and Diadegma semiclausum (Hellen) (Hymenoptera: Ichneumonidae), respectively (Stoltz and Cook, 1983; Yin et al., 2001; Feng et al., 2004; Huang et al., 2011a). The hemolymph hemagglutination activity in Pieris rapae (Linnaeus) (Lepidopetera: Pieridae) pupae parasitized by Pteromalus puparum (Linnaeus) (Hymenoptera: Pteromalidae) was always higher than that in unparasitized ones (Cai et al, 2001). The phenoloxidase activities in host hemolymph were more or less inhibited by parasitism in most of the reports (Stoltz and Cook, 1983; Beck et al., 2000; Yin et al., 2001; Feng et al., 2004; Li et al., 2006; Huang et al., 2011a), for example, Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) larvae parasitized by Venturia canescens (Gravenhorst) (Hymenoptera: Ichneumonidae) (Beck et al., 2000) and P. xylostella larvae parasitized by Diadromus collaris (Gravenhorst) (Hymenoptera: Ichneumonidae) (Li et al., 2006). Nevertheless, parasitism did not induce the inhibition of phenoloxidase activity in other insect species (Sroka and Vinson, 1978; Cai et al., 2001; Li et al., 2011). The antibacterial activity in P. rapae pupae
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Figure 1.
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Antagonistic system between D. helophoroides and M. alternatus.
hemolymph postparasitization by P. puparum became higher (Cai et al., 2001), whereas it was decreased in P. xylostella larvae parasitized by D. semiclausum (Huang et al., 2011a). The total hemocyte numbers (THC) in P. xylostella pupae parasitized by D. collaris increased (Li et al., 2006), but those numbers declined in the same host larvae parasitized by D. semiclausum (Huang et al., 2009). The spreading behaviors of granulocytes and plasmatocytes were inhibited in P. xylostella pupae parasitized by D. collaris, and never recovered (Li et al., 2006). While in P. xylostella larvae parasitized by D. semiclausum, the spreading ability of hemocytes from the host larvae was obviously inhibited in the early stage of the parasitism, and recovered at 24 h postparasitization (Huang et al., 2009). Both the phagocytosis and encapsulation abilities were suppressed in P. xylostella larvae in the early stage of the parasitism by D. semiclausum, and those abilities were recovered at 24 h postparasitization (Huang et al., 2009; Huang et al., 2011b). In this article, we examined the immune response in the larval hemolymph of pine sawyer beetle M. alternatus, to parasitism by its parasitoids D. helophoroides. Both the Archives of Insect Biochemistry and Physiology
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humoral immune responses include melanization, hemagglutination, phenoloxidase activity, and antibacterial activity and the cellular immune responses include THC, cell spreading, and nodulation were investigated.
MATERIALS AND METHODS Insect Collection Pinus massoniana Lamb infested with M. alternatus were cut down in the Huanglishu Forest Farm of Quanjiao County, Anhui province, China, in March, 2014. M. alternatus larvae were collected from the xylem of the host trees. These activities were conducted with permission from the Huanglishu Forest Farm of Quanjiao County. Larvae were held singly in small sterile tubes, kept in a box out of the sun, and returned to the Anhui Provincial Academy of Forestry within 8 h after collection. Larvae were stored at 8 ± 0.5 °C and 50 ± 5% RH. Mature larvae in the fifth instars, based on head capsule measurements (Liu et al., 2008), were used in the experiments, to separate age effect from parasitism effect on host immune responses.
Parasitoid Rearing D. helophoroides colony was provided by the Institute of Forest Protection, Anhui Provincial Academy of Forestry, Hefei, China. They were the laboratory-reared offspring of the mother adults provided by the Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing, China. The ancestors parasitized larvae or pupae of M. alternatus in the trucks or branches of P. massoniana, which were collected in 2003 at Guangzhou (23°80 N, 113°17 E, and altitude 120 m), Guangdong Province, China. D. helophoroides adults were reared on artificial diet and the larvae were reared on live M. alternatus larvae. D. helophoroides larvae used in the experiments were the third generation of the beetles reared in laboratory.
Parasitization Six newly hatched D. helophoroides larvae were inoculated larva using a small brush. One parasitized or unparasitized was put into a sterile tube (with a small hole drilled in bated at 25 ± 0.5 °C and 50 ± 5% RH for 1, 2, 4, 72 h.
to a M. alternatus M. alternatus larva its lid) and incu8, 12, 24, 48, or
Bleeding Procedure M. alternatus larvae were surface sterilized with 75% ethanol for a few seconds and rinsed with sterile water, and then, dried. Hemolymph was obtained from the host larvae by cutting their posterior part with sterile ophthalmic scissors. Archives of Insect Biochemistry and Physiology
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Melanization Reaction Experiment of melanization reaction was conducted by the method of Yin et al., (2001). In order to access the capacity of melanization reaction in the whole hemolymph of the host, cohorts of ten M. alternatus larvae were selected at 1, 2, 4, 8, 12, 24, 48, or 72 h postparasitization. Hemolymph sample from each parasitized or unparasitized larva was collected and put on a Parafilm. The drop of undiluted hemolymph was left for 1 h at ambient room temperature. A change in coloration of the hemolymph from opaque to brown–black was recorded as normal melanization, whereas the maintenance of the previous color or change in an intermediate color was considered as reflecting an inhibition of melanization. Hemagglutination Assay Hemagglutination activity test method referenced to Drif and Breh lin (1989) cited by Cai et al., (2001). Hemolymph of each M. alternatus larva was collected and put into a sterile tube with a few phenlythioura (PTU) crystals, and then, centrifuged at 8,000 g for 10 min at 4 °C. The supernatant was diluted with 0.1 M PBS (pH 7.4) according to different dilution factor. To conduct the assay, 15 μl undiluted hemolymph or twofold serial dilutions of the hemolymph, mixed with 15 μl 2% chicken red blood cells suspension, and added to the wells of a 96-well V-bottom plate. The red blood cells mixed with the PBS were used as controls. The plate was incubated at room temperature for 1 h. The titers were expressed as geometric mean titers (GMT) of various treatments. Assay for Phenoloxidase (PO) Activity The PO activity was measured spectrophotometrically according to Xylander and Bogusch (1992) as cited by Beck et al., (2000). As substrate, a 0.02 M solution of L-3,4dihydroxyphenylalanine (L-DOPA) (Alfa Aesar, America) in 0.1 M PBS (pH 7.4) was used. Hemolymph of each M. alternatus larva was collected and put into a sterile tube. After centrifugation at 14, 000 g for 10 min at 4 °C, 50 μl supernatant was taken and mixed with 100 μl substrate solution in a 96-well plate. PO activity was monitored by measuring absorbance at 490 nm using a microplate reader (Thermo Scientific Multiskan GO, America) after 30 min incubation at 30 °C. One unit of phenoloxidase activity was defined as A490 = 1. Antibacterial Test Antibacterial test was conducted by the method of Huang et al. (2011a). The mixture of serum-free insect cell culture medium (Hyclone, America), Escherichia coli suspension (A600 = 0.5), and hemolymph from each larva, at a ratio of 18:1:1, were incubated at 37 °C for 30 min, and then, measured the absorbance at 600 nm using a microplate reader (Thermo Scientific Multiskan GO, America). One unit of antibacterial activity was defined as A600 = 1. Hemocyte Counting Method for counting the hemocytes was as described by Manachini et al. (2011). To count the hemocytes, 10 μl pure hemolymph from each M. alternatus larva was put, immediately Archives of Insect Biochemistry and Physiology
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after bleeding, into a sterile tube with 90 μl anticoagulant solution (0.098M NaOH, 0.186M NaCl, 0.017M EDTA, 0.041M Citric acid, pH 4.5) added. After blending, the diluted hemolymph was put in a 0.0025 mm2 hemocytometer under an inverted phase contrast microscope (Olympus CX41, Japan). The THC from parasitized and unparasitized larvae was recorded at designated times postparasitization.
Assay for Cell Spreading Larva hemolymph was sampled at 1, 2, 4, 8, 12, 24, 48, or 72 h postparasitization. 10 μl hemolymph of each larva was put on a clean slide immediately after bleeding and covered with a cover glass. The spreading of granulocytes and plasmatocytes was observed using an inverted phase contrast microscope (Olympus CX41, Japan). We counted all granulocytes and plasmatocytes in ten randomly chosen fields of view at 400×magnification, and gave the counts of spreading granulocytes and plasmatocytes, which were identified according to Jones (1962) and Ribeiro and Breh´elin (2006). The spreading percentage of granulocytes and plasmatocytes were calculated as follows: % spreading = (number of spreading granulocytes and plasmatocytes observed)/(total number of granulocytes and plasmatocytes observed)×100.
Assay for Nodulation Heat-killed E. coli were used as nodulation targets. Fluorescein isothiocyanate (FITC) labeling of bacteria was performed by the method of Hed & Stendahl (1982) as cited by Beck & Strand (2005) with slight modifications. Heat-killed E. coli (109 /ml) cells were incubated in 0.2 M carbonate buffer (pH 9.5) containing FITC (0.1 mg/ml) for 30 min at 37 °C. Thereafter, the FITC-conjugated bacteria were washed four times in 0.2 M PBS (pH 7.2), and then, kept at 4 °C until use. 10 μl hemolymph of each larva, 10 μl serum-free insect cell culture medium (Hyclone, America), and 10 μl FITC labeled E. coli suspension were added to a sterile tube. Cultures were maintained at 25 °C for 2 h. At this time, multiple hemocytes adhered to aggregations of E. coli and formed a nodule. 10 μl of the culture was put on a clean slide and covered with a cover glass. We scored the number of nodules from ten randomly selected fields of view by use of an inverted fluorescence microscope (Olympus CX41, Japan).
Statistics The number of samples used for each experimental condition is indicated in the figure legends. The significant of differences in melanization reaction was determined by the Chi-square test. The differences of hemagglutination activity, phenoloxidase activity, antibacterial activity, THC, the spreading percentage of granulocytes and plasmatocytes, and the number of capsules between parasitized larvae and unparasitized ones at each time point were analyzed using the Pairwise t-test. We also took a time zone test: early parasitism stage (1, 2, 3, 4, 8, 12, and 24 h) versus late parasitism stage (48 and 72 h) by using the Pairwise t-test as well. All data were performed on SPSS 19.0. Archives of Insect Biochemistry and Physiology
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Table 1. Hemagglutination Activity in M. alternatus Larvae Parasitized or Unparasitized by D. helophoroides (n = 3 Replicates for Each Time Points. GMT Means Geometric Mean Titers) Hours after parasitization
1 2 4 8 12 24 48 72
Unparasitized Parasitized Unparasitized Parasitized Unparasitized Parasitized Unparasitized Parasitized Unparasitized Parasitized Unparasitized Parasitized Unparasitized Parasitized Unparasitized Parasitized
Titer (log2 ) Range
Average
GMT
0–4 0–3 0–0 0–0 0–2 0–0 0–2 0–1 0–0 0–3 0–1 0–0 0–1 0–2 0–2 2–2
1.67 1 0 0 0.67 0 0.67 0.33 0 1 0.33 0 0.67 0.67 0.67 2
3.18 2 1 1 1.59 1 1.59 1.26 1 2 1.26 1 1.59 1.59 1.59 4
RESULTS Effect of Parasitization on Melanization reaction Not all sampled hemolymph were melanized normally in 1 h, and only 20–70% from unparasitized larvae and 0–40% from parasitized larvae were melanized in given time (Fig. 2). There were variations of melanization both in parasitized larvae and the controls, however, the differences were not significant according to the Chi-square test results (parasitized X2 = 8.686, DF = 7, P = 0.276; unparasitized X2 = 10, DF = 7, P = 0.189; Chisquare test). The differences between unparasitized and parasitized larvae in melanization reaction were significant only at 1 h and 24 h postparasitization, while they were not at other time points (1 h, X2 = 7.500, DF = 1, P = 0.006; 2 h, X2 = 3.810, DF = 1, P = 0.051; 4 h, X2 = 2.400, DF = 1, P = 0.121; 8 h, X2 = 0.952, DF = 1, P = 0.329; 12 h, X2 = 0.392, DF = 1, P = 0.531; 24 h, X2 = 5.495, DF = 1, P = 0.019; 48 h, X2 = 1.250, DF = 1, P = 0.264; 72 h, X2 = 3.529, DF = 1, P = 0.060; Chi-square test). Effect of Parasitization on Hemagglutination Activity Hemolymph hemagglutination activity of M. alternatus larvae was checked by using 2% chicken red blood cells suspension. Almost 100% of undiluted hemolymph from both unparasitized and parasitized larvae could cause the red blood cells hemagglutination. The hemagglutination titers were indicated in Table 1. There were no significant differences between unparasitized and parasitized larvae (1 h, T = 2.000, DF = 2, P = 0.184; 2 h, T not available, DF = 2, P not available; 4 h, T = 1.000, DF = 2, P = 0.423; 8 h, T = 0.378, DF = 2, P = 0.742; 12 h, T = −1.000, DF = 2, P = 0.423; 24 h, T = 1.000, DF = 2, P = 0.423; 48 h, T = 0, DF = 2, P = 1.000; 72 h, T = −2.000, DF = 2, P = 0.184; Pairwise t-test). No significant differences were found between early parasitism stage and late parasitism stage (T = −1.000, DF = 5, P = 0.363; Pairwise Archives of Insect Biochemistry and Physiology
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Figure 2. Melanization reaction in M. alternatus larvae parasitized or unparasitized by D. helophoroides. Times postparasitization were shown on the left. Bars in the graphs indicate the number melanized or nonmelanized.
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Figure 3. Phenoloxidase activity in M. alternatus larvae parasitized or unparasitized by D. helophoroides (The error bars correspond to SE. * indicates significantly different between parasitized and unparasitized samples at P
