Introduction
Unlike vertebrates, insects only have an innate immune system to defend against the invading pathogens (Siva-Jothy et al., Reference Siva-Jothy, Moret and Rolff2005). Insect innate immunity is highly developed and divided into humoral and cellular immune defenses (Gillespie et al., Reference Gillespie, Kanost and Trenczek1997; Lavine & Strand, 2002). Cellular immune responses include phagocytosis in which small particles are phagocytized by hemocytes, nodulation and encapsulation (Little et al., Reference Little, Hultmark and Read2005). However, humoral responses are mediated by producing antimicrobial peptides following a challenge (Bulet et al., Reference Bulet, Hetru, Dimarcq and Hoffmann1999), the activation of proPhenoloxidase (proPO) cascade (Cerenius et al., Reference Cerenius, Lee and Söderhäll2008), and the synthesis of reactive oxygen species and nitric oxide (Siva-Jothy et al., Reference Siva-Jothy, Moret and Rolff2005). In the recent decades, extensive progresses have been gained on the molecular and biochemical mechanisms underlying the recognition of invading pathogens and the activation of immune responses in insect. However, because most studies only focused on one stage of holometabolous insects such as larvae or adults, the relevance of immune defenses across every stage of holometabolous insects has received little attention.
The oriental fruit fly, Bactrocera dorsalis (Hendel), is a highly polyphagous destructive pest for fruit and vegetable production in many Southeast Asian and Pacific countries, with female adults causing direct damage by laying eggs under the skin of fruits and vegetables and the larvae feeding in decaying flesh of the crop (Clarke et al., Reference Clarke, Armstrong, Carmichael, Milne, Raghu, Roderick and Yeates2005; Benelli et al., Reference Benelli, Daane, Canale, Niu, Messing and Vargas2014). For holometabolous insects, it is well known that many dramatic physiological changes often occur during the development from an actively feeding larva to an immobile pupa and sexually mature adult. B. dorsalis adult females oviposit into the fruits, within which the larvae develop as the fruits rapidly become rotten with the proliferation of different kinds of microbes (Behar et al., Reference Behar, Jurkevitch and Yuval2008). Subsequently, the older larvae jump into the soil and pupate. Therefore, the environments in which the different life stages of B. dorsalis reside are often distinct from each other. On the other hand, each stage of holometabolous insects including B. dorsalis tends to be attacked by different species of specific natural enemies, and so resistance mechanisms and strategies also tend to be stage-specific (Kraaijeveld et al., Reference Kraaijeveld, Barker and Godfray2008). Because insect immune defense has evolved in the context of costs and benefits (Ardia et al., Reference Ardia, Gantz, Schneider and Strebel2012), we predicted that the immunocompetence of B. dorsalis at the different life stages before emergence would increase to adapt to the deteriorating ecological environments.
Mating and immunity are both central for fitness, and the two processes influence each other in multiple ways (Lawniczak et al., Reference Lawniczak, Barnes, Linklater, Boone, Stuart and Chapman2007). Furthermore, mating has been observed to either induce or suppress the distinct components of female immune defense in many invertebrate species (Rolff & Siva-Jothy, Reference Rolff and Siva-Jothy2002; Lawniczak et al., Reference Lawniczak, Barnes, Linklater, Boone, Stuart and Chapman2007; Valtonen et al., 2010). For example, defense against systemic infection by many bacterial pathogens is reduced by mating in female Drosophila melanogaster Meigen (Fedorka et al., Reference Fedorka, Linder, Winterhalter and Promislow2007; Short & Lazzaro, Reference Short and Lazzaro2010). However, recent evidence showed that mating enhanced resistance against the infection of entomopathogenic fungus Beauveria bassiana (Bals.-Criv) Vuill and the effect was stronger on males than females of Tenebrio molitor Linnaeus (Valtonen et al., 2010). Mating has mixed effects on the immune system of the cricket Allonemobius socius Scudder, reducing hemocyte number, encapsulation ability and lytic activity in both males and females, but increasing phenoloxidase (PO) activity in females (Fedorka et al., Reference Fedorka, Zuk and Mousseau2004). Therefore, these data suggest that immune defense is involved in trade-offs with multiple life-history traits including reproduction and this trade-off is shaped by many ecological and evolutionary factors (Schmid-Hempel, Reference Schmid-Hempel2003; Short et al., Reference Short, Wolfner and Lazzaro2012). The majority of reports on the patterns of post-mating immune regulation in insects have mostly focused on females, but little is known on the males.
To address the above questions, we first characterized the immune response of B. dorsalis across the different life stages by measuring the PO activity and the humoral antibacterial capability. PO and its activation system are an ultimate and conserved component of the immune system of insects and other arthropods (Cerenius et al., Reference Cerenius, Lee and Söderhäll2008). Several lines of evidence showed that PO generated reactive compounds, such as melanin and 5,6-dihydroxyindole, which have broad-spectrum antibacterial and antifungal activity (Zhao et al., Reference Zhao, Lu, Strand and Jiang2011). Consequently, PO activity is often employed as one of the major index by ecologists to evaluate insect immunocompetence (Boughton et al., Reference Boughton, Joop and Armitage2011; Moreno-García et al., Reference Moreno-García, Córdoba-Aguilar, Condé and Lanz-Mendoza2012). Second, we investigated possible sex differences in the immunocompetence of B. dorsalis and the potential effect of mating on the immunocompetence of male and female adults by controlling their mating access in the laboratory. Immune defense is vital for the survival of insects, so our study will shed light on the determination of the life stage that can be easily targeted to improve the strategy of biological control for this pest species.
Materials and methods
Insect rearing and manipulation
All flies used in this study were from an established laboratory colony that has been cultured in the laboratory for at least 13 generations. The colony was maintained in cubical screen cages (0.3 × 0.3 × 0.3 m) and provided with a filter paper (9 cm in diameter) containing with artificial food (sugar: yeast extract powder in a 3:1 ratio by weight) and a dish plate (9 cm in diameter) which was firstly filled with a wet cotton ball. When adults become sexually mature, fresh bananas in which small holes had been made were placed in the cage as the oviposition substrate. The eggs hatched in the bananas. Three days later, the bananas were transferred to a plastic container filled with wet sand, and the larvae jumped into the wet sand and pupated after 1–2 days (Lin et al., Reference Lin, Zeng, Lu, Liang and Xu2004). They were kept at 28 °C and 70–80% RH and received both natural and artificial light in a 12 h day/night cycle. New larvae (late 1st-instar larvae), old larvae (3rd-instar larvae) and 2-day-old pupae and adults (15 days after emergence) were used for hemolymph collection. In this study, ten individuals were treated as one replicate for the following experimental tests and each treatment included three replications.
In the experiments in which access to mating was manipulated, virgin females and males within 3 days after emergence were transferred in two separate cages. At least 80 females and 80 males were transferred per time. 15 days later, 20 sexually mature females and males were transferred to two other new cages as the controls, but the remaining females and males were placed together in another new screen cage at 6:00 PM. Because the copulation of B. dorsalis often occurred at dusk, copulation in the cage was checked every 30 min from 7:00 to 11:00 PM. The mating pair was captured and let their copulation continued in a long glass tube (18 mm × 18 cm), with one mating pair per tube. 12 h after copulation, these adults were used to collect hemolymph according to the procedure of Gliksman & Yuval (Reference Gliksman and Yuval2010). Totally, 60 pairs of mated adults were gotten and treated as above in the following experiments. These mated pairs were randomly assigned to the PO activity assay and antibacterial activity assay, and each assay included 30 pairs of mated adults.
Bacteria challenge to adults
To evaluate the magnitude of the immune response in B. dorsalis at different life stages, Escherichia coli DH5α and a Staphylococcus aureus strain were used as antigens to challenge the B. dorsalis individuals. The two bacteria species were cultured overnight with OD600 = 1 in Luria-Bertani's (LB) medium (1000 ml distilled water, 10 g tryptone, 5 g yeast extract, 10 g NaCl and pH 7.2). Insects were individually pricked with an entomological pin which has been dipped in a suspension of the combined bacteria (Dang et al., Reference Dang, Tian, Yi, Wang, Zheng, Li, Cao and Wen2006) in physiological saline (130 mM NaCl, 5 mM KCl, 1 mM CaCl2 and pH 6.0). The untreated and sterilized entomological pin-pricked insects represented the negative and positive controls, respectively. The hemolymph was collected as describe by Gliksman & Yuval (Reference Gliksman and Yuval2010).
The insects were washed with distilled water, sterilized with 75% ethanol and immobilized on ice (2–3 min). The hemolymph in the following experiments was obtained by perfusing the abdomen with 100 μl cold phosphate buffer saline (500 ml distilled water, 4 g NaCl, 100 mg KCl, 720 mg Na2HPO4, 720 mg K2HPO4 and pH 7.2) and bled into a 0.5 ml clean Eppendorf tube. All hemolymph samples were stored at −70 °C until analysis.
PO activity assay
For each sample, PO activity was measured using a spectrophotometric assay (Shi & Sun, Reference Shi and Sun2010). After 10 min of centrifugation (4 °C, 10,000 rpm, Sigma, Osterode am Harz, Germany, 1-15PK Centrifuge), 30 μl of the supernatant was mixed with 110 μl ultrapure water plus 30 μl phosphate buffer saline (500 ml distilled water, 4 g NaCl, 100 mg KCl, 720 mg Na2HPO4, 720 mg K2HPO4 and pH 7.2) and 30 μl L-Dopa (Acros Organic, Morris Plains, NJ, USA, 4 mg ml−1 ultrapure water) as a substrate. The reaction was allowed to proceed at 30 °C in a SpectraMax reader (Molecular Devices Corp., California, USA) for 90 min. Readings were taken every 30 s at 490 nm. The enzyme activity was determined by quantifying the slope of the linear phase of the reaction. For each individual, we performed two independent measurements and determined an average V max (the slope value of the reaction curve) for the two reactions to measure the enzyme activity.
Antibacterial activity assay
The antibacterial activity of the B. dorsalis hemolymph was assayed against E. coli which was cultured overnight as described above. The hemolymph samples were taken out from the −80 °C refrigerator and dissolved on ice. Antibacterial activity assays were performed in sterile 96-well plates with a final volume of 140 μl. An aliquot of 100 μl of bacteria culture, or sterilized (Phosphate buffer saline, NaCl 137mM, KCl 2.7mM, Na2HPO4 10 mM, K2HPO4 2 mM, pH 7.2) or tetracycline solution (1 mg ml−1) was added to 40 μl of hemolymph samples. The two latter treatments served as the negative and positive controls, respectively. Subsequently, plates were incubated at 30 °C for 12 h. Growth of bacteria was measured as the cell concentration which was determined by the absorbance value at the wave length of 600 nm using a SpectraMax reader (Molecular Devices Corp., USA).
Statistical analysis
One-sample Kolmogorov–Smirnov test was used to test if the distribution of our collected data was normal. If it was not normal, all the data were transformed by the function square root. ANOVA was employed to reveal the effect of life stage on the immunocompetence index, and a general linear model (GLM) was used to determine the effect of adult sex and mating status on the humoral immunocompetence. In the analysis of GLM, mating status and adult sex were assigned as the fixed factor and covariate, respectively. Least-significant difference (LSD) or Tamhane's T2, determined by the results of the test of homogeneity of variances, was used as the multiple comparison procedure to determine cohort group differences. A confidence level of 95% (P < 0.05) was maintained for all tests. All statistical analyses were performed with SPSS 12.0 for Windows (SPSS Inc.: Chicago, IL, USA, 2003).
Results
PO activity
Our data indicated that the PO activity of B. dorsalis varied significantly across the different life stages (ANOVA: F 3, 126 = 18.474, P < 0.001). Old larvae and pupae had the greatest PO activity, followed by adults and newly hatched larvae (fig. 1). These results showed that the PO activity of B. dorsalis increased with the fly's development but decreased markedly when the adults emerged.
PO activity of B. dorsalis at different life stages. The results are presented as the mean ± S.E. Different letters above the bars indicate significant differences which are determined by Tamhane's T2.
GLM analysis uncovered that the PO activity of B. dorsalis was markedly affected by mating access, but not by the adult sex (table 1). As shown in fig. 2, the level of phenoloxidase did not differ significantly between the virgin males and females. Both the PO activity of the mated males and mated females were significantly higher than that of the virgin males and virgin females, respectively (fig. 2).
PO activity in male and female adult B. dorsalis before and after copulation. The results are presented as the mean ± S.E. Different letters above the bars indicate significant differences which are detected by LSD.
The effects of adult sex and mating status on the PO activity in the hemolymph of B. dorsalis were determined by the general linear method univariate.
1 r 2 = 0.150 (adjusted r 2 = 0.134).
* Significance was detected at P < 0.05.
Antibacterial activity
The antibacterial activity of hemolymph from the adults of B. dorsalis without bacteria challenge differed significantly across the life stages (ANOVA: F 3, 164 = 4.011, P < 0.01). Pupae and old larvae had the highest antibacterial activity, followed by adults and new larvae (fig. 3). After the immune challenge, the antibacterial activity of the samples, excluding the new larvae, improved markedly. Significant differences in antibacterial activity were also found among B. dorsalis across different life stages (ANOVA: F 3, 164 = 17.225, P < 0.001). The hemolymph from new larvae after the immune challenge showed the lowest ability to inhibit the growth of E. coli (fig. 3).
Antibacterial activity in the hemolymph of new larvae, old larvae, pupae and adults of unchallenged and challenged B. dorsalis. The results are presented as the mean ± S.E. Different letters above the bars indicate significant differences which are determined by LSD.
As shown in fig. 4, no significant differences were detected in the cell concentration of E. coli solution 12 h after being incubated with the hemolymph from virgin males and females, respectively. This was also detected in the concentration of bacteria cell after being incubated with that from copulated males and females. These data indicated that male and female adults of B. dorsalis performed the similar antibacterial capability. After copulation, both the antibacterial ability of the hemolymph from males and females significantly increased (GLM: F 3, 112 = 13.083, P < 0.001, fig. 4).
Antibacterial activity in the hemolymph of male and female adult B. dorsalis before and after copulation which are detected by LSD.
Discussion
Results showed that the immunocompetence of B. dorsalis increased with its development from new larvae to pupae. As an insect develops from a larva to an adult, many distinct physiological changes occur, especially in holometabolous insects. For fruit flies such as B. dorsalis and Ceratitis capitata Weidemann, the development from eggs to larvae was often accompanied with the proliferation of bacteria in the rotting fruits (Behar et al., Reference Behar, Jurkevitch and Yuval2008). The infection risk for B. dorsalis also increased with rapid deterioration of fruits. The old larvae often jumped into the soil, another environment where accommodate diverse species of microbe, to pupate. Moreover, B. dorsalis adults often feed on the plant honeydew which contains fewer microbes compared with that of its larvae. Therefore, we predicted that B. dorsalis should increase its investment in immune defense to confront the deteriorated conditions of the living environment. As our data illustrated, old larvae and pupae had the highest levels of immunocompetence as indicated by PO activity and induced antibacterial activity in contrast with new larvae and adults. On the other hand, our data also help to explain why superparasitism by braconid parasitoids is often needed on the large tephritid larvae because they often have a stronger immune response level as demonstrated here (Benelli et al., Reference Benelli, Gennari and Canale2013).
After emerged, B. dorsalis adults have a hard exoskeleton. This exoskeleton is often considered as the first line of defense against pathogens (Schmid-Hempel, Reference Schmid-Hempel2005; Moret & Moreau, Reference Moret and Moreau2012). Furthermore, adults often exhibit high dispersal ability. It has been suggested that higher dispersal rates reduce the risk of pathogen infection (Suhonen et al., Reference Suhonen, Honkavaara and Rantala2010). Unlike larvae and pupae, adults should balance the investment of resources in survival with investment in reproduction. It is probable that the trade-off between adult immunocompetence and reproductive processes, confirmed by an increasing number of studies, explains our observation here (Schmid-Hempel, Reference Schmid-Hempel2005). The adults’ hard exoskeleton and high dispersal ability allowed a greater investment of limited resources into reproduction-related processes, and fewer resources were invested in immunocompetence. Collectively, our data show that B. dorsalis has a flexible strategy of investment in immunocompetence to meet the specific needs of each developmental stage. To the best of our knowledge, life-stage-specific variation in immune function has also been detected in other insect species, including the wax moth Galleria mellonella Miller (Meylaers et al., Reference Meylaers, Freitak and Schoofs2007), the bark beetle Dendroctonus valens LeConte (Shi & Sun, Reference Shi and Sun2010), the honeybee Apis mellifera L. (Wilson-Rich et al., Reference Wilson-Rich, Dres and Starks2008; Laughton et al., Reference Laughton, Boots and Siva-Jothy2011), the burying beetle Nicrophorus vespilloides Herbst (Urbański et al., Reference Urbański, Czarniewska, Baraniak and Rosński2014) and the ground beetle Carabus lefebvrei Dejean (Giglio & Giulianini, Reference Giglio and Giulianini2014), but the mechanisms that contribute to this gating remain unknown. Interestingly, different patterns of immune system activity were observed in the above insect species. These data indicate that immune system activity during the different developmental stages of B. dorsalis is shaped by the endo- and exogenous factors such as their distinct food source and living environments (Schmid-Hempel, Reference Schmid-Hempel2003).
Mating and immunity are linked to fitness. Furthermore, immunity is considered costly, and it is thus expected to be included in trade-offs involving other energy-demanding aspects of the life history, such as growth and reproduction (Rolff & Siva-Jothy, Reference Rolff and Siva-Jothy2003). A mating-induced downregulation of immunocompetence has been found in some previous studies on invertebrates (Rolff & Siva-Jothy, Reference Rolff and Siva-Jothy2002; Fedorka et al., Reference Fedorka, Zuk and Mousseau2004). The results of our study showed that PO activity and antibacterial activity in the male and female adults of B. dorsalis was significantly upregulated after copulation. This finding is in accordance with the results of Valtonen et al. (2010), which uncovered that mating enhanced resistance against fungal infection and that the effect was stronger in males than in females. The similar post-mating immune regulation pattern was also detected in C. capitata (Gliksman & Yuval, Reference Gliksman and Yuval2010). Mating is fraught with danger. In addition to the fitness costs associated with finding sexual partners, copulation and offspring production, mating increases the risk of acquiring sexually transmitted infections (Knell and Webber, Reference Knell and Webberley2004). Thus our finding of mating-induced upregulation in immune function has important evolutionary and ecological consequences. Enhanced immunocompetence in mated adults of B. dorsalis could be an adaptive response to reduce the risk of sexually transmitted diseases and other microbes (McGraw et al., Reference McGraw, Gibson, Clark and Wolfner2004). After copulation, B. dorsalis females often fly to search for suitable oviposition sites in new situations, but the males should remain in place to gain increased mating access (Benelli et al., Reference Benelli, Daane, Canale, Niu, Messing and Vargas2014). Consequently, the mating-induced upregulation in immunity would reward male adults of B. dorsalis from an evolutionary perspective. For the females of B. dorsalis, the increased immunocompetence would help them to effectively fight against the potential infection risk in the fresh oviposition sites. In Drosophila, the upregulation of the antimicrobial peptides in females after copulation was caused by a major seminal component sex peptide (Peng et al., Reference Peng, Zipperlen and Kubli2005). Furthermore, one member of the Turandot family of immune and stress response genes, Turandot M, promotes immunity of Drosophila females against sexually transmitted fungal infection (Zhong et al., Reference Zhong, McClure, Evans, Mlynski, Immonen, Ritchie and Priest2014). Interestingly, we also determined that both PO activity and antibacterial activity in the hemolymph of B. dorsalis males were increased after copulation. However, the mechanism for this upregulation of immunocompetence in B. dorsalis remains elusive. This question is reserved for intensive investigation in the future.
In conclusion, we documented that significant stage-specific differences occurred in B. dorsalis. The level of immunocompetence was the highest in pupae and old larvae, followed by adults and new larvae. Mating could significantly enhance the PO activity and antibacterial activity of the male and female adults. Our observations imply that the immune defenses of B. dorsalis are conditionally expressed to maximize its fitness under different circumstances. Additionally, our findings also provide an important support to the view that the parameters such as the host/parasitoid ratio and host exposure time should be properly set to induce the heavily superparasitized larvae to improve the output of parasitoid offspring and its control efficiency when they are used as biological agents (Benelli et al., Reference Benelli, Gennari and Canale2013).
Acknowledgements
This study is granted by Chinese Natural Science Foundation (31201522), the earmarked fund for Modern Agro-industry Technology Research System (no. CARS-27), the Special Fund for Agro-scientific Research in the Public Interest (no. 200903047) and China Postdoctoral Science Foundation Supported Project (no. 2012M511628).
