extfieldsofstudy: Medicine Biology

text:
Performance of the Species-Typical Alarm Response in Young Workers of the Ant Myrmica sabuleti (Hymenoptera: Formicidae) Is Induced by Interactions with Mature Workers

Abstract Young workers of the ant Myrmica sabuleti (Hymenoptera: Formicidae) Meinert 1861 perceived nestmate alarm pheromone but did not display normal alarm behavior (orientation toward the source of emission, increased running speed). They changed their initial behavior when in the presence of older nestmates exhibiting normal alarm behavior. Four days later, the young ants exhibited an imperfect version of normal alarm behavior. This change of behavior did not occur in young ants, which were not exposed to older ants reacting to alarm pheromone. Queen ants perceived the alarm pheromone and, after a few seconds, moved toward its source. Thus, the ants’ ability to sense the alarm pheromone and to identify it as an alarm signal is native, while the adult alarm reaction is acquired over time (= age based polyethism) by young ants. It is possible that the change in behavior observed in young ants could be initiated and/or enhanced (via experience-induced developmental plasticity, learning, and/or other mechanisms) by older ants exhibiting alarm behavior.

In eusocial insects, each individual is able to perform many social tasks and can respond to many different sources of information. In ants, workers generally use multiple information sources such as nest odor (Cammaerts and Cammaerts 2000), one or several nest entrance and vicinity odors (Cammaerts and Cammaerts 1999), nestmate odors (Hamidi et al. 2004), a foraging area odor (Cammaerts-Tricot et al. 1977), trail pheromones (Evershed et al. 1981, Hölldobler andWilson 1990), a recruiting odor (Cammaerts 1978, Hölldobler et al. 2001, visual and odorous cues to help in navigation (Philippides et al. 2011, Cammaerts and Rachidi 2009, Cammaerts et al. 2012b, acoustic signals (Roces andHölldobler 1996, Casacci et al. 2013), and antennal contacts (Bonavita-Cougourdan andMorel 1984, Czaczkes et al. 2013). Ant workers perform numerous social tasks such as caring for the brood, foraging, collecting food, recruiting nestmates, performing trophallaxis, and nest defence. Young ants, <6 months old, have been observed to be unable to perform such social tasks and to correctly respond to their specific social signals (Cammaerts 2013a).
Previous studies have investigated the ontogenesis of ants' cognitive abilities. It has been shown that, in Myrmica rubra (L.) 1758, young workers (not yet fully pigmented, about 4 months old) learn the odor of their nest entrance via imprinting (Cammaerts 2013b), the visual characteristics of their nest entrance via operant conditioning (Cammaerts 2013b), become imprinted with their foraging area odor , and recognize their trail pheromone and begin to efficiently follow a trail when in the presence of older trail following nestmates (Cammaerts 2013c). Here, I report similar research on the acquisition of the response to alarm pheromone.
Alarm behavior is one of the most spectacular reactions of ants, and many alarm pheromones have been chemically identified (Passera and Aron 2005). Although long studied, alarm behavior and the chemical composition of alarm pheromones are still under investigation (e.g., Amoore et al. 1969, Mizunani et al. 2010. Myrmica ants possess an alarm pheromone produced by the workers' mandibular glands. It is essentially a mixture of 3-octanone and 3-octanol ) and it induces a typical alarm reaction consisting of a true attraction, an increased walking speed (¼ positive orthokinesis) and a decreased path sinuosity (¼ negative klinokinesis) (Cammaerts-Tricot 1973). Using Myrmica sabuleti Meinert 1861 colonies maintained in the laboratory, I addressed the following questions. 1) Do young ants, <4 months old, recognize their specific alarm pheromone and respond to it in a manner similar to that of older workers (walking toward the emission source and rapidly circling it)? 2) If the response of young workers is different, what causes workers to eventually display a normal alarm response? Do worker ants simply display the normal alarm response as the mature, or is exposure to mature ants displaying normal alarm behavior necessary for young ants to begin displaying normal alarm behavior themselves?

Materials and Methods
Collection and Maintenance of Ants. Three colonies of M. sabuleti were used. Two colonies were collected in March at Marchin (Condroz, Belgium) and one was collected in July at Olloy/Viroin (Ardenne, Belgium). Each colony contained one or two queens, brood, and about 500 workers. From March to May, several workers emerged in the two colonies collected from Marchin and were about 4 months old in June, when experiments 1-4 were undertaken. From July to September, callows emerged in the third colony and were about 4 months old at the end of October when a supplementary experiment (5) was performed. Callow ants are lighter in color than older ones and are expected to live for nearly 3 years (Cammaerts 1977). I also performed a sixth experiment on the queens of two previously used colonies and two other smaller ones, collected at Marchin (Condroz, Belgium).
The colonies were maintained in the laboratory in artificial nests made of one to three glass tubes half-filled with water, with a cottonplug separating the ants from the water. The glass tubes were deposited in trays (42 Â 27 Â 7 cm), the sides of which were covered with talc to prevent the ants from escaping. The trays served as foraging areas in which the ants were fed with sugar-water provided ad libitum in a small glass tube plugged with cotton, and with cut Tenebrio molitor larvae (Linnaeus 1758) provided twice a week on a glass slide. Temperature was maintained between 18 and 22 C, and humidity at about 80%, with these conditions remaining constant over the course of the study. Lighting was at a constant intensity of 330 lux when caring for the ants (e.g., providing food, renewing nesting tubes) and during testing. During other time periods, the lighting was dimmed to 110 lux. The electromagnetic field had an intensity of 2-3 mW/m 2 .
Obtaining, Recording, and Analyzing the Ants' Alarm Reaction. In order to test differently aged workers and queens separately, without disturbing the colonies, 40 individuals per assay were transferred to an experimental tray (28 Â 15 Â 3.5 cm), the borders of which were covered with talc. A few minutes after the transfer, either a piece of blank white strong paper (1 cm 2 ) or a piece of this paper supporting a worker's isolated head was deposited in the middle of the experimental tray. The species alarm pheromone is produced by the workers' mandibular gland ). An isolated worker's head, with widely open mandibles, is an ideal experimental source of alarm pheromone identical to that produced by an alarmed worker, in terms of the dimensions of the emitting source (the mandibular glands opening) and the quantity of pheromone emitted. When presented with an isolated nestmate worker head, surrounding ants present the species' typical alarm reaction (Cammaerts-Tricot 1973). The behavior of the individuals located at about 6 cm from the presented stimulus was then observed for 10 min ( Fig. 1A and B) and 2 Â 30 (30 for assessing the orientation of ants approaching the source of emission, and 30 for assessing the linear and angular speed of ants having approached the source of emission, see below) of their trajectories were recorded and analyzed as follows. Trajectories were recorded manually, using a water-proof marker pen, on a glass slide placed on the top of the experimental tray, set horizontally 3 cm above the area where the tested individuals were moving. A metronome set at 1 s was used as a timer for assessing the total time of each trajectory (not for entering the trajectories in the assessing system, see below). Each trajectory was recorded until the ant reached the stimulus or walked for about 6 cm. All the trajectories were then traced (copied) with a water-proof marker pen onto transparent polyvinyl sheets (Fig. 1C). These sheets could then be affixed to a PC monitor screen. The trajectories were then analyzed using specifically designed software (Cammaerts et al. 2012a and figures therein). Briefly, each trajectory was defined in the software by clicking as many points as needed with the mouse. Then, the total time of the trajectory (assessed using the metronome) was entered, and feature of the trajectory could be measured (orientation, linear speed, angular speed). I have often used this method for studying ants' locomotion (e.g., Cammaerts et al. 2013).
The three variables used to characterize the recorded trajectories were defined as follows: The orientation (*) of an animal toward a given point (here an empty piece of paper or an ant's head) is the sum of the angles, measured at each successive point of the registered trajectory, made by the segment "point i of the trajectory ! given point" and the segment "point i ! point i þ 1" divided by the number of measured angles. This variable was measured in angular degrees. When such a variable (*) equals 0 , the observed animal perfectly orients itself toward the reference point; when O equals 180 , the animal fully avoids the reference point; when * is lower than 90 , the animal has a tendency to orient itself toward the point; when * is larger than 90 , the animal has a tendency to avoid the point. The linear speed (V) of an animal is the length of its trajectory divided by the time spent moving along this trajectory. It was measured in mm/s. The angular speed (S) (i.e., the sinuosity) of an animal's trajectory is the sum of the angles, measured at each successive point of the trajectory, made by the segment "point i ! point i À1" and the segment "point i ! point i þ 1", divided by the length of the trajectory. This variable was measured in angular degrees/cm.
In total, 30 trajectories were recorded and analyzed so as to quantify the orientation toward the presented stimulus of individuals approaching the stimulus, and 30 other trajectories were recorded and analyzed for assessing the linear and angular speed of individuals having approached the stimulus and then moving in its vicinity. Each distribution of 30 variables were characterized by their median and their quartiles (since they were not Gaussian) (Table 1) and were compared with one another using the nonparametric Chi-square test (Siegel and Castellan 1989, pp. 111-116) as well as the nonparametric Mann-Whitney test (Siegel and Castellan 1989, pp. 128-137). Two distributions were considered statistically different at P < 0.05. After having been tested, ants were carefully returned to their colony.
Marking of the Ants. During experiment 3, old and young ants were assayed together but their trajectories had to be separately recorded. Though old ants are darker than young ones, it was difficult to tell them apart when they were in motion. Thus, old ants were marked with a small spot of blue paint (enamel, Airfix) deposited on the first tergite of the gaster, using an entomology pin. she is oriented toward the head; she is moving toward that source of alarm pheromone.
Remarks. Due to the complexity of the experimental protocol, the specifics of each experiment are provided alongside their results (see "Results" section). (Table 1, Line 1). Foragers (with dark pigmentation, at least 18 months old) did not walk toward or avoid a blank piece of paper: their median orientation value did not equal 30-50 , and was not more than 90 . In the vicinity of such a control stimulus, they went on moving as usually. The obtained numerical values of orientation, linear, and angular speed were similar to those usually obtained while making control experiments on M. sabuleti (e.g., Cammaerts et al. 1981Cammaerts et al. , 2013. By contrast, they obviously perceived, from a distance of about 5-8 cm, the specific alarm pheromone released from the isolated worker heads during experimental treatments and immediately oriented themselves toward these sources (Fig. 1A): their median orientation value was 38.3 (v 2 ¼ 13.46, df ¼ 2, P ¼ 0.001; z ¼ 3.80, P < 0.001). They returned many times to the source of the alarm pheromone, and circled it, displaying an increase in linear speed (v 2 ¼ 10.56, df ¼ 2, P < 0.01; z ¼ À 2.92, P % 0.003) and a decrease in angular speed (v 2 ¼ 18.03, df ¼ 2, P < 0.001; z ¼ 3.23, P % 0.001). They thus presented the specific alarm reaction of Mymica ants (Cammaerts-Tricot 1973).

Experiment 1. Foragers' Alarm Reaction
Experiment 2. Response of Naïve Young Ants to Their Alarm Pheromone (Table 1, Line 2). Young ants, which had not been experimentally exposed to alarm pheromone, moved more slowly than their older congeners (v 2 ¼ 17.5, df ¼ 2, P < 0.001) and were slightly, nonsignificantly, affected by the presence of a blank paper (median O ¼ 94.4 vs. 84.5 ; v 2 ¼ 2.36; df ¼ 2; P % 0.30; NS). They appeared to perceive, from a distance of about 3-6 cm, the specific alarm pheromone released from the isolated worker heads during experimental treatments but did not orient themselves toward such a source ( Fig. 1B  and C; v 2 ¼ 1.63, df ¼ 2, P > 0.30, NS; and z ¼ À 0.47, NS). In fact, young ants' orientation toward an isolated head (103 ) was higher than that of older ants (38.3 ) but also higher than older ants' control one (84.5 ). So, young ants not only did not orient themselves toward an isolated head but also somewhat avoided it. They also neither exhibited increased linear speed (8.1 vs. 8.0 mm/s; NS with the two kinds of test) nor decreased path sinuosity (126 vs. 139 /cm; NS with the two kinds of test) as their older congeners did when presented with a source of alarm pheromone. They thus recognize their alarm pheromone, but present a different reaction than that exhibited by older ants.
Experiment 3. Mixed Marked Old Foragers' and Unmarked Young Ants' Reaction to the Alarm Pheromone (Table 1, Line 3). In the presence of young nestmates, older foragers continued to perceive a source of alarm pheromone when exposed to an isolated worker head and to react to it by a true attraction, an increase of linear speed, and a slight decrease of angular speed (v 2 ¼ 22.13, 14.04, 11.06, respectively; df ¼ 2, P < 0.001, < 0.001, < 0.01, respectively; z ¼ 4.45, À 3.78, 1.29, respectively; P < 0.001, < 0.001, NS, respectively). Their reaction was even slightly, but not significantly, stronger than that exhibited in the absence of younger nestmates (O: 36 vs. 38 ; V: 13.8 vs. 13.0 mm/s; NS).
When young ants perceived a source of alarm pheromone in the presence of older nestmates, they no longer avoided such a source; however, they also did not orient themselves toward it. Indeed, their orientation averaged 82.5 , showing a slight decreasing trend compared with 103.9 (v 2 ¼ 5.03, df ¼ 2, 0.05 < P < 0.10; NS; z ¼ 2.33, P ¼ 0.02). However, the orientation values were nonetheless far from values associated with true attraction (30-50 ). They moved more rapidly (10.4 vs. 8.1 mm/s; v 2 ¼ 15.09, df ¼ 2, P < 0.001; z ¼ À3.57, P < 0.001), showed hesitations, and displayed a large increase of path sinuosity (200 vs. 126 /cm; v 2 ¼ 20.58, df ¼ 2, P < 0.001; z ¼ À5.06, P ¼ 0.0000). Thus, the behavior of young naïve ants in the presence of older ants differed from that of young naïve ants in the absence of older ants, but was yet different from the behavior of older ants. In fact, they The ants' locomotion was characterized by their orientation toward the presented stimulus, their linear and angular speed in the vicinity of the stimulus. The table gives the median and quartiles (in brackets) of the distributions of 30 values collected for each variable and experiment. Details of the method and statistical results are given in the text.
ceased avoiding the source of the alarm pheromone. They did not yet present the true attraction and the increase of linear speed commonly exhibited by older ants perceiving alarm pheromone. On the contrary, they appeared to perform pronounced klinokinesis (see "Discussion" section).
The young ants tested were carefully replaced inside their nest tubes at the end of the experiment. Experiment 4. "Alarm Response" of Young Ants Previously Tested With Responding Older Foragers (Table 1, Line 4). Three and an half days after the experiment described above (note that workers remain young for 4-6 months), young ants were seen moving in the colonies' foraging area, not only in the vicinity of the nest entrances but also further afield. These young ants were those previously tested in the presence of older ones because no emergence occurred in the intervening 3 1 =2 d. All of these young ants were removed and retested, as previously, in the experimental tray, in the absence of older congeners, first with a blank paper and then with an older ant's isolated head. First, the young ants were no longer affected by the presence of a blank piece of paper: their control orientation (95.1 ) was similar to that of older workers (84.5 ) (v 2 ¼ 2.2, df ¼ 2, 0.30 < P < 0.50; z ¼ À0.51, NS). Second, unexpectedly, they oriented themselves toward the presented isolated head (45.1 vs. 95.1 ; v 2 ¼ 17.16, df ¼ 2, P < 0.001; z ¼ 4.02, P < 0.001), moved more quickly in its surroundings (10.3 vs. 8.9 mm/ s; v 2 ¼ 12.99, df ¼ 2, P < 0.01; and z ¼ À3.14, P % 0.001) (though not as quickly as older ants-13.0 mm/s) and somewhat less sinuously (141 vs. 173 /cm; v 2 ¼ 8.81, df ¼ 2, P < 0.02; and z ¼ 1.62, P ¼ 0.10) (though more sinuously than older ants-104 /cm). Thus, they exhibited alarm behavior qualitatively similar to that of older nestmates (but not yet fully quantitatively). These young ants thus seem to have acquired the specific alarm reaction while being in the presence of their older nestmates. However, this experiment did not rule out the possibility that young ants could acquire the species' alarm reaction simply by being repeatedly exposed to the alarm signal, whether or not they were grouped with older reacting nestmates. The possibility that previous experience with older nestmates is necessary for the acquisition of a mature alarm response was tested by performing a supplementary experiment (5) on naive callows, which were about 4 months old, and belonged to a separate colony (see "Material and Methods").
Experiment 5. "Alarm Response" of Young Ants Having Never Met Older Responding Ants (Table 1, Line 5). In total, 40 callows from the colony collected at Olloy/Viroin were transferred into the experimental tray and tested in the presence of a blank paper, and then a worker's isolated head. They were then kept in the tray for three days, and provided with water and a red cover. They were retested in front of a worker's head, all the experiments having been performed blind. First, even if issued from two different localities, young ants of all three colonies studied behaved similarly during initial testing. Second, when tested for the first time, young ants did not orient themselves toward the isolated head (102.8 vs. 96.6 ; v 2 ¼ 2.15, df ¼ 2, 0.30 < P < 0.50; and z ¼ À1.67, NS), did not move more rapidly (7.7 vs. 7.6 mm/s; v 2 ¼ 3.27, df ¼ 2, P % 0.20; and z ¼ 1.27, NS) nor obviously less sinuously (140 vs. 133 /cm; v 2 ¼ 1.25, df ¼ 2, P % 0.50; and z ¼ À2.92, P < 0.01) like old ants would have done in the same situation. When tested for the second time, the callow ants behaved like they did during the first experiment: they displayed no true attraction (98.5 vs. 96.6 ; v 2 ¼ 3.28, df ¼ 2, P % 0.20; and z ¼ À1.32, NS), no increase in walking speed (8.3 vs. 7.6 mm/s; v 2 ¼ 3.27, df ¼ 2, P % 0.20; and z ¼ À0.93, NS), and no decrease in path sinuosity (139 vs. 133 /cm; v 2 ¼ 2.44, df ¼ 2, P % 0.30; and z ¼ À0.73, NS). They thus did not begin to react like older ants faced with an alarm signal in the absence of older nestmates.
Experiment 6. Queens' Response to Their Specific Alarm Pheromone (Table 1, Line 6). After performing the experiments described above, I asked whether queens (founder queens, the mothers of all the workers and sexuals) could recognize the species' alarm pheromone and react to it. I tested the queen of four colonies confronted to a blank piece of paper, and then an old worker's isolated head exactly as I tested foragers and young ants. All the four queens behaved similarly. For the first few seconds, the queens appeared not to perceive the alarm pheromone. After that, they generally stopped, moved their antennae, turned around, and apparently detected the presented source of alarm pheromone. They oriented themselves toward the presented isolated head (Fig. 1D, 38.2 vs. 100 ; v 2 ¼ 21.93, df ¼ 2, P < 0.001; and z ¼ 3.91, P < 0.001), moved more quickly (13.1 vs. 11.5 mm/s; v 2 ¼ 12.53, df ¼ 2, P < 0.01; and z ¼ À3.26, P ¼ 0.001), and moved less sinuously (90 vs. 117 /cm; v 2 ¼ 9.62, df ¼ 2, < P < 0.01; and z ¼ 2.27, P < 0.05). They thus recognized the alarm pheromone and apparently reacted to it in a similar manner to old ants. However, as for the statistical significance of the results, it must be noted that the 30 values of orientation, linear speed, and angular speed were obtained by testing four queens (the queen of four different colonies), which reacted several times, and not by using 30 different queens. Thus, the statistical analysis suffered from nonindependence and is intended for illustrative purposes only.

Discussion
Ants' cognitive abilities are well documented, involving navigation, recruitment, communication, food exchange, and brood care (Passera andAron 2005, Cammaerts and. However, there is little information about the ontogenesis of such behaviors. Here, I examined the acquisition of a species' cognitive ability. Using M. sabuleti as a model, I examined how young ants acquire the species' specific reaction to alarm pheromone. I found that young ants recognize the pheromone (¼ innate behavior) but react to it in a different manner to older ants: they do not walk toward the alarm signal, and even slightly avoid it, and do not run as quickly as old ants. Their behavior seems adaptive to their young age: by avoiding alarm signals, they may avoid performing risky tasks such as defense or foraging. Such temporal polyethism has previously been reported by other authors, e.g., Hölldobler and Wilson (1990) on Green tree ants, Muscedere et al. (2009) in Pheidole dentata, and Vieira et al. (2010) in Ectatomma vizottoi. I also found that young ants (about 4 months old) change their behavior and begin to respond to an alarm signal when in the presence of older responding ants. They may eventually acquire the specific alarm reaction (orientation toward the source of alarm, quickly moving) by themselves, but their acquisition may be more rapid if they encounter older reacting ants. In other words, as a potential explanation of our results, it is possible that alarm behavior acquisition may be initiated and/or enhanced by older responding ants. Such an acquisition via interactions with older reacting congeners may result from different mechanisms. Some form of imitation may occur Nemeghaire 2012, Cammaerts 2013d). Some kind of task allocation may exist: individuals are then more likely to perform given behaviors after having encountered congeners performing such behaviors (Gordon and Mehdiabadi 1999). Older ants may emit some primer pheromone initiating the development of alarm response (¼ experience-dependent developmental plasticity). Older ants may mediate the learning of the alarm response through classical conditioning. Note that, under natural conditions, young ants will almost always perceive the alarm signal for the first time while in presence of older reacting ants, except for the very first young workers emerging in founding colony: how these first workers acquire the species' behaviors has yet to be investigated. Note also that the visual perception ability of Myrmica workers allows them to see congeners moving rapidly toward an alarm signal (Cammaerts and Cammaerts Biologia 2014). Whatever the precise mechanism, young ants must be old enough to begin behaving like older congeners. In nature, age-based polyethism and task allocation on one hand, and imitation and subsequent learning (see below) on the other hand, could be nonmutually exclusive mechanisms by which workers develop the ability to respond appropriately to alarm pheromone. During their initial alarm reaction acquisition, young ants seem not to be able to orient themselves toward the alarm signal using efficient true taxis; they turn left and right, and do not walk directly toward the source of emission. They seem to resort using pronounced kinesis, as evidenced by a high sinuosity of movement. In other words, it is possible that, being yet unable to perform efficient taxis, they performed a pronounced klinokinesis, a simpler behavior which allows them to progressively approach the alarm signal (Fraenkel and Gunn 1961). It is also possible that, after their first "alarm" experience, young ants will continue improving their alarm reaction by some operant conditioning and/or true learning, through individual experience, in the course of exposure to further alarm situations. Indeed, middle-aged ants (12-24 months old) perfectly orient themselves toward an alarm signal, move very quickly, and are ready to behave in a risky manner (Cammaerts-Tricot 1973). The present work also revealed that founder queens recognize the alarm pheromone and move toward its source of emission. Inside the nest, queens seldom have the opportunity to do so. However, the alarm pheromone is a species-specific signal which indicates the presence of specific (perhaps even nestmate) workers. It is thus logical that queens, deprived of their workers, move toward an alarm signal, a specific odor probably emitted by nest-mate workers.
In conclusion, M. sabuleti workers' specific response to their alarm pheromone results from innate behavior (the pheromone is natively recognized), age-based polyethism (very young ants avoid the alarm signal source and so potential danger), eventual imitation (young ants imitate older reacting ants) and task allocation (young ants are inclined to behave like encountered older workers), experience-dependant developmental plasticity or classical conditioning mediated by older workers, and, presumably, from improvement acquired through individual experience during exposures to alarm situations.
Young individuals' acquisition of their species' behavioral patterns is well documented. Two aspects of such acquisition are age-based polyethism (already largely examined, Hölldobler and Wilson 1990 and references therein) and task allocation (also somewhat studied, for instance by Gordon and Mehdiabadi 1999). On the other hand, from the numerous studies concerning the ontogenesis of behavior in invertebrates (among others, cuttlefish, spiders, damselflies, ants, stingless bees, bees) and in vertebrates (fishes, birds, squirrels, and monkeys) (Caillere 1974, Jaisson 1975, Livecche 1979, Ferron 1980, Wehner and Ghering 1999, Pompignac-Poisson 2000, Tarnaud 2003, Poirier 2004, Delcourt 2008, Reichle et al. 2013, it can be concluded that an individual's behavior is acquired via genetically stored information, early social experience, and experience gained in the course of the individual's life.
In the present study, performed on ants and their alarm behavior, all these elements seem to exist (see above). However, these elements do not explain the ontogenesis of every behavior. Several cognitive abilities are, at least partly, acquired thanks to imprinting, habituation, and to classical conditioning. In ants, the situation can be summarized as follows. Soliciting of food, as well as trying to leave the nest and re-enter it, is apparently innate behavior (personal observation). Nest odor and brood recognition are acquired via imprinting and/or habituation (Jaisson 1975, Boss andd'Ettorre 2012). Odor of the nest entrance and foraging area odor are "learned" via imprinting (Cammaerts 2013b. The visual aspect of congeners is "learned" at emergence via imprinting (Cammaerts and Gosset in press). The trail following behavior and the alarm reaction are acquired via innate behavior, imitation and/or task allocation, and operant conditioning and/or true learning (Cammaerts 2013c. The use of olfactory and visual cues for navigation is acquired thanks to operant conditioning (Schatz et al. 1999, Cammaerts and Rachidi 2009, Cammaerts et al. 2012b, Steck et al. 2011, Wolf et al. 2000, Wystrach et al. 2011.
Our understanding of the ontogenesis of ant behavior would be greatly improved by future studies focusing on the acquired abilities of naïve callow ants, removed from their colonies as pupae, and maintained either in a group or in isolation. Preliminary results suggested that such young ants are unable to collect food, recruit nestmates, and relocate their nest.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Corynebacterium pseudotuberculosis cp09 mutant and cp40 recombinant protein partially protect mice against caseous lymphadenitis

Background Caseous lymphadenitis (CLA) is an infectious disease that affects small ruminants and is caused by Corynebacterium pseudotuberculosis. This disease is responsible for high economic losses due to condemnation and trim of infected carcasses, decreased leather and wool yield, loss of sales of breeding stock and deaths from internal involvement. Treatment is costly and ineffective; the most cost-effective strategy is timely immunisation. Various vaccine strategies have been tested, and recombinant vaccines are a promising alternative. Thus, in this study, different vaccine formulations using a recombinant protein (rCP40) and the CP09 live recombinant strain were evaluated. Five groups of 10 mice each were immunised with saline (G1), rCP40 (G2), CP09 (G3), a combination of CP09 and rCP40 (G4) and a heterologous prime-boost strategy (G5). Mice received two immunisations within 15 days. On day 30 after primary immunisation, all groups were challenged with a C. pseudotuberculosis virulent strain. Mice were monitored and mortality was recorded for 30 days after challenge. Results The G2, G4 and G5 groups showed high levels of IgG1 and IgG2a; G2 presented significant IgG2a production after virulent challenge in the absence of IgG1 and IgG3 induction. Thirty days after challenge, the mice survival rates were 20 (G1), 90 (G2), 50 (G3), 70 (G4) and 60% (G5). Conclusions rCP40 is a promising target in the development of vaccines against caseous lymphadenitis.

Background
Caseous lymphadenitis (CLA) is an infectious, chronic and subclinical disease of sheep and goats with a worldwide occurrence [1,2]. CLA is characterised by the formation of subcutaneous abscesses in lymph nodes or as a subclinical infection in internal organs, such as the lungs, liver and kidneys [3]. The etiologic agent of CLA is Corynebacterium pseudotuberculosis, a gram-positive, non-spore-forming, aerobic and facultative intracellular parasite of macrophages [3]. CLA is responsible for significant economic losses in the sheep and goat industries due to its high incidence, causing condemnation of carcasses, devaluation of leather due to scars left by abscesses, decreased production of meat or milk, and occasional death of affected animals [4,5].
CLA commercial vaccines are based on attenuated strains of C. pseudotuberculosis and do not provide satisfactory protection for goats. Additionally, annual boosters are needed, which makes prophylaxis more costly [6]. Thus, the search for an ideal CLA vaccine has been prioritised, with the aim of obtaining long-lasting protection with few side effects. Additionally, the vaccine should allow the differentiation between vaccinated and infected animals [1].
Pursuing this purpose, different vaccine strategies have been tested, and live attenuated vaccines and recombinant proteins are promising options. Through a process of random mutagenesis, it was identified 34 live recombinant strains of C. pseudotuberculosis [7], one of which (CZ171049, now called CP09) presented promising results in pilot trials, where inoculated mice presented no clinical signs of caseous lymphadenitis and produced a significant amount of specific IgG.
The serine protease CP40, encoded by the cp40 gene of C. pseudotuberculosis, was tested in infected animals and demonstrated immunogenic properties, showing a strong humoral response on immunoblots and a 82% reduction in the proportion of infected sheep and a 98% reduction in lung lesions [8]; these protection levels were similar to those found with other immunogens, as inactivated bacteria and genetically detoxified phospholipase D [9,10].
Considering the need for new immunogens for caseous lymphadenitis prophylaxis and the promising results already found with the mutant CP09 strain and the recombinant CP40 protein (rCP40), the aim of this work is to perform a comparative evaluation of four vaccine formulations containing these immunogens alone or in combination, and determine their ability to induce immunoprotection against caseous lymphadenitis in a murine model.

C. pseudotuberculosis strains and culture conditions
The previously obtained C. pseudotuberculosis CP09 mutant strain [7], the T1 pathogenic wild-type parental strain [11], and the caprine-pathogenic strain MIC-6 from the Laboratory of Genetics and Control of Microorganisms (Belo Horizonte, Brazil) bacterial collection were employed in this work. Strains were aerobically grown in "Brain Heart Infusion" broth (BHI, Oxoid) at 37°C. Kanamycin (kanamycin sulphate 25 μg/mL; Sigma-Aldrich, USA) was added to the mutant growth media. T1 and MIC-6 strains were isolated from caseous lesions of goats and have previously been employed in vaccination trials using goats and mice [11,12].

Bacterial interaction assays with murine J774 cells
Pre-macrophagic J774 cells from murine lymphomas were cultivated in Dulbecco's modified Eagle's essential medium (DMEM, Sigma-Aldrich, USA) supplemented with 5% foetal bovine serum, 50 μg/mL gentamicin and 2.5 μg/mL fungizone at 37°C in a 5% CO 2 atmosphere. The CP09 mutant strain and T1 wild-type strain were grown for 48 hours at 37°C and washed three times with PBS, resuspended in DMEM to a concentration of 10 6 CFU/mL, and used to infect J774 cells (multiplicity of infection [MOI]: 10 bacteria: 1 cell) grown to approximately 95% confluence in 24-well tissue culture plates.
For determination of intracellular viable bacteria, after 1, 3 and 6 hours of incubation, infected J774 monolayers were washed six times with PBS and treated with 150 μg/mL gentamicin sulphate (Sigma-Aldrich, USA) diluted in DMEM for 1 h. The number of intracellular bacteria was determined by viable counts after lysis of monolayers with 0.5 mL of 0.1% TritonX-100 (Sigma-Aldrich, USA) in PBS.

DNA extraction and PCR reaction
C. pseudotuberculosis genomic DNA was obtained from the T1 strain as previously described [13]. Briefly, 5.0 mL of the bacteria cultured in BHI broth was centrifuged and resuspended in 50 mM EDTA with 10 mg/mL lysozyme, and the Wizard® Genomic DNA Purification Kit (Promega, EUA) was used following the manufacturer's instructions. After DNA extraction, the material was resolved by electrophoresis on a 1.0% agarose gel and quantified. Eluted DNA was stored at −20°C pending PCR amplification.
PCR was performed using the Go Taq® Green Master Mix Kit (Promega, EUA), with the objective to amplify the cp40 gene (accession number NC_014329.1NCBI). The primers used in the PCR assay were CP40F (5Ć GCGGATCCATGCATAATTCTCCTCGATCAG 3') and CP40R (5' CGGGAATTCTTATCTAG AACCAGTTGGC TTTC 3'), which contain BamHI and EcoRI restriction sites, respectively. Each PCR reaction had a final volume of 50 μL, with 25 μL from the kit, 1 μL of each primer (100 μM), 22 μL of ultra-purified water and 1 μL of genomic DNA from C. pseudotuberculosis strain T1. The amplification reaction consisted of an initial denaturation step at 94°C for 2 min, 35 cycles of denaturation for 1 min at 94°C, primer hybridisation for 1 min at 55°C and extension at 72°C for 1.5 min, followed by a final extension at 72°C for 10 min. The reaction was conducted in a thermocycler (Mastercycler Gradient, Eppendorf, Germany), and the final product was analysed by electrophoresis on a 1% agarose gel with GelRed™ (Biotium, USA) staining.

Cloning and recombinant protein production and purification
Cloning, recombinant protein production and protein purification were performed as previously described [13]. The pAE vector [14] and the amplified cp40 gene were digested using BamHI and EcoRI restriction enzymes (Fermentas, USA). After that, the cp40 gene was inserted into the pAE vector using the T4 DNA ligase enzyme (Fermentas, USA), and electrocompetent TOP 10 E. coli cells were transformed by electroporation. The transformed bacteria were cultivated in Luria-Bertani (LB) broth with 100 μg/mL ampicillin for 16 h, and the recombinant plasmid was purified using a MiniPrep Kit (Qiagen, USA). The pAE/CP40 recombinant plasmid was transformed by thermal shock in E. coli BL21, and the recombinant bacteria were cultivated in LB broth containing 1 mM IPTG for 3 h at 37°C in an orbital shaker.
For recombinant protein purification, the bacteria resulting from a large-scale culture (500 mL) were pelleted by centrifugation and resuspended in a washing buffer (200 mM NaH 2 PO 4 ; 500 mM NaCl, 5 mM Imidazole; 8 M Urea pH 8.0) with 100 mg/mL lysozyme, sonicated five times for fifteen seconds (20 KHz) and maintained under agitation at 4°C for 16 h. The recombinant protein was purified using the HisTrap TM HP (GE Healthcare, USA) affinity chromatography column followed by dialysis in a cellulose tube-based system (Sigma, USA).

Animal model
Six-to eight-week-old BALB/c mice, a strain which is susceptible to C. pseudotuberculosis infection [1], were employed in this assay. For immunity tests, fifty mice were kept in a vivarium under suitable conditions of temperature and humidity with a 12 h light/dark cycle; food and water were provided ad libitum. The study was approved by the Ethics Committee for Animal Experimentation of the Tiradentes University (protocol number 010413).

Immunisation protocol, challenge and determination of the protection level
The rCP40 recombinant protein and CP09 live recombinant strain were used in vaccine formulations. Animals were divided into five groups (n = 10 each) and immunised intraperitoneally (i.p.) and/or subcutaneously (s.c.) on days 0 and 15. The administration routes followed what was previously described for recombinant proteins and mutant strains immunisation protocols [7,8,10]. The negative control group (G1) was inoculated i.p. with 100 μL of saline solution (0.9% NaCl). The G2 group was inoculated i.p. with 200 μL of a solution containing rCP40 (50 μg) and saponin (7.5 μg) in sterile saline solution. G3 was inoculated s.c. with 100 μL of the CP09 recombinant live strain (10 6 CFU). G4 was inoculated i.p. with 200 μL of a solution containing rCP40 (50 μg) and saponin (7.5 μg) and s.c. with 100 μL of the recombinant live CP09 strain (10 6 CFU). Group G5 used a heterologous primeboost strategy and was first inoculated i.p. with 200 μL of rCP40 (50 μg) and saponin (7.5 μg) in solution followed by an s.c. booster dose of 100 μL of the live recombinant strain CP09 (10 6 CFU) 15 days after the first vaccination.
The previously immunised animals were challenged with 10 4 CFU/mL (i.p.) of the MIC-6 virulent strain thirty days after the last immunisation. The protection conferred was assessed according to the survival rate of the animals immunised with different vaccine formulations. The animals were observed daily, and mortality was recorded over four weeks following the challenge.

Blood collection and ELISA for specific IgG determination
Animals were bled at days 0, 15, 30, 45 and 60 postimmunisation to determine the antibody levels. Blood was collected from the retro-orbital sinus with Pasteur pipettes. All blood collections were performed under anaesthesia with ketamine 10% (1 mL/kg) and xylazine 2% (0.1 mg/kg) (Agener União Saúde Animal, Brazil), and all efforts were made to minimise suffering. After coagulation, blood was centrifuged at 1,500 g for 10 min. Serum was removed and stored at −20°C.
Sera samples from the immunised mice were used in immunoassays (ELISA) for detection of specific IgG1, IgG2a and IgG3 antibodies. For this purpose, polystyrene 96-well plates (Greiner, USA) were coated with 100 μL of C. pseudotuberculosis T1 strain supernatant diluted 1:100 [15] (for G3 sera samples) or 0.125 ug/mL of rCP40 (for G2 sera samples) or a combination of both antigens (for G4 and G5 sera samples). All antigen dilutions were made in bicarbonate-carbonate buffer (pH 9.6). Plates were incubated at 4°C for 18 h. After this period, plates were washed twice with PBS-T (1X PBS, pH 7.4, 0.05% Tween 20) and blocked with 200 μL/well of 5% defatted milk in PBS-T for 2 h at 37°C. After this blocking step, the wells were washed five times with PBS-T and then 100 μL/well of sera samples from mice diluted 1:100 in PBS-T were added in duplicate. After one hour of incubation at 37°C and five washes with PBS-T, 50 μL/well of an anti-mouse antibody against IgG1, IgG2a or IgG3 (Invitrogen Life Technologies, USA) diluted 1:5000 was added. The plates were again incubated for 45 min at 37°C and then washed five times with PBS-T. After the washes, 50 μL/well of TMB developing solution (10 mL citricphosphate buffer pH 5.1, 4 mg of orthophenylenediamine, 4 μL of H 2 O 2 ) was added. The reaction was stopped by adding 25 μL of 4 N H 2 SO 4. The absorbance was measured at 450 nm using an ELISA reader (Thermo Plate TP Reader, Thermo, USA).

Statistical analysis
Statistical analyses were performed using GraphPad Prism version 6.0 for Windows (GraphPad Software, USA). The results of intracellular viability and specific immunoglobulins production were expressed as the means ± standard deviations. Differences between IgG production in the different groups were calculated by one-way ANOVA followed by the Tukey post-test test. The data were considered statistically significant when p < 0.05.

Bacterial interaction assays with murine J774 cells
Analysing the results presented in Figure 1, it can be observed that the mutation harboured by the CP09 mutant had a major influence on the ability of this strain to adhere to J774 monolayers. After 6 hours of interaction, the rate of CP09 cell invasion in J774 cells was only 20% of the rate of cellular invasion achieved by the T1 parental strain at the same time point.

Evaluation of the IgG levels
Immunisations with the combination of rCP40 and CP09 induced the production of IgG1 in all immunised groups, as shown in Figure 2a. The results demonstrated that all groups on days 0 and 15 post-immunisation (p.i.) showed no significant IgG1 production (p > 0.05). On day 30, the G2, G4 and G5 groups showed significantly higher levels of IgG1 than groups G1 and G3 (p < 0.05). On days 45 and 60, after the challenge with the virulent strain, G4 and G5 groups showed significantly higher concentrations of IgG1 than the control and G3 groups (p < 0.05). No significant IgG1 production was observed in the G2 group after challenge. Figure 2b shows the results obtained from the ELISA assay for the analysis of IgG2a production. On day 30 p.i., G2, G4 and G5 groups showed significantly higher concentrations of IgG2a (p <0.05) than groups G1 and G3. The same trend was observed in these groups after the MIC-6 strain challenge at 45 and 60 days p.i..
No significant IgG3 production was observed in any group, either before or after the virulent challenge (Figure 2c).

Determination of protection levels
The survival curve of mice immunised with vaccine formulations and challenged with C. pseudotuberculosis strains is presented in Figure 3. The animals were evaluated for thirty days after the challenge. In these trials, 10 mice in each group were monitored daily, and mortality events were recorded. Animals from the G2, G3, G4, and G5 groups showed, respectively, 90, 50, 70 and 60% survival 30 days after infection with the C. pseudotuberculosis MIC-6 wild strain.

Discussion
Recombinant proteins and live attenuated vaccines are considered promising tools for the development of new vaccine models. In this regard, Dorella et al. [7], using a reporter transposon-based system, identified 34 out of 1,500 mutants that presented detectable alkaline phosphatase (PhoZ) activity. This feature was associated with 21 C. pseudotuberculosis loci that encode fimbrial and transport proteins. One of these mutants, CZ171049 (now called CP09) showed promising protective results in pilot trials and was suggested for further investigation in attenuation and immunisation-challenge trials.
In this study, we conducted a cellular adhesion experiment to observe how the mutation in the CP09 strain affected its capacity to adhere and infect murine macrophage cells. The results showed a significantly reduced capacity for cell adhesion and invasion, corroborating the genetic fimbrial mutant profile previously observed by Dorella et al. [7]. Clayton et al. [16] observed that mutations in Salmonella enterica fimbrial subunits genes led to reduced intestinal colonisation in chickens. Additionally, work by van der Velden et al. [17] showed that multiple fimbrial adhesions are required for a fully virulent profile in a murine model of Salmonella typhimurium infection. This attenuated cellular adhesion profile was confirmed for the C. pseudotuberculosis CP09 strain, and other experiments were conducted with the objective of determining the immunogenicity and protective potential of this mutant in a caseous lymphadenitis model in mice.
The development of cellular and humoral immune responses depends on the set of cytokines produced by various cells, including CD4 + and CD8 + T cells. When a naive T helper (Th) cell is activated by dendritic cells, it can differentiate into one of three distinct types of effector Th cells called T helper 1 (Th1), 2 (Th2) and 17 (Th17). Th1 cells are mainly involved in immunity against intracellular pathogens, and aid in the activation of macrophages and cytotoxic T cells and the production of opsonising and complement activator immunoglobulin isotypes, such as IgG2a in mice. Th2 cells are involved primarily in immunity against extracellular pathogens, such as multicellular parasites, and assist B cells in producing neutralising antibodies (IgG1 and IgG3) [18,19].
Based on the results presented herein, the G2 (rCP40), G4 (rCP40 + CP09) and G5 (prime-boost) groups had a very significant induction of IgG1, mainly from day 30 after the start of the trail. The G2 group did not presented presented significant IgG1 levels after the challenge (45 and 60 days after primary immunisation). These results suggest that the groups treated with inoculation solutions containing the mutant strain presented an immune response mediated by Th2 lymphocytes, as can be indicated by significant IgG1 production [20].
Although the protective role of IgG2a in this disease model is largely unknown, this IgG subtype is thought to modulate the immune response by inducing phagocytosis Figure 2 Total specific IgG isotype levels in immunised mice with different vaccine formulations. The sera of immunised mice were collected 0, 15, 30, 45, and 60 days post-immunisation and tested by ELISA for the presence of specific IgG1 (A), IgG2a (B) and IgG3 (C). The results are presented as the means and standard deviation (bars) for each experiment with ten animals per group. Significant differences between IgG production in the different groups was calculated employing one-way ANOVA followed by the Tukey post-test. D,d: groups significantly different on day 30 (p < 0.05); E,e: groups significantly different on day 45 (p < 0.05); F,f: groups significantly different on day 60 (p < 0.05). Arrows correspond to the two immunisations (days 0 and 15), and stars indicate the challenge with the virulent strain (Day 30). All bleedings were performed before immunisations and challenge. by neutrophils and macrophages and activating the complement system and cytotoxicity mediated by antibodydependent cells. IgG2a production requires the induction of IFN-gamma by Th1 and NK cells [21][22][23]. Our results indicate that G2, G4 and G5 groups presented significant IgG2a production thirty days after the first immunisation, with a perceptible boost in its concentration after challenge with the recombinant strain; the group inoculated with the CP09 mutant strain alone was not able to produce specific IgG2a. Thus, significant IgG2a production in the above groups demonstrates the ability of the rCP40saponin formulation to generate a Th1 adaptive immune response and memory. The Th1 immune response is responsible for the elimination of intracellular pathogens such as C. pseudotuberculosis [24], suggesting that the type of response induced in animals inoculated with the recombinant protein is consistent with the immune activity responsible for the control of intracellular bacteria.
According to Marini et al. [25], immune responses with high levels of IgG1, IgG2a and IgG3 in the serum are important mechanisms that help eliminate microorganisms. This indicates that our results are promising for the development of an effective vaccine against CLA. Vaccine formulations used in G2 (rCP40), G4 (rCP40 + CP09) and G5 (heterologous prime-boost) groups showed more significant results with respect to the level of specific immunoglobulins generated against rCP40, a recombinant protein that is considered to be an important C. pseudotuberculosis virulence factor in genomic and bioinformatics surveys [26].
A 90% protection rate was obtained in animals immunised with rCP40 alone, which is consistent with earlier work from Walker et al. [8]. These authors used the native semi-purified CP40 to evaluate antibody levels generated before and after infection in sheep and observed that this native 40 KDa protein provided an 82% protection rate, significantly reducing lung lesions. The protection in animals immunised only with CP09 mutant strain resulted in 50% survival after virulent challenge, which is in agreement with previously published results [7]. Ribeiro et al. [12] described a high protection rate (80%) in a murine model when using an iron acquisition-deficient mutant obtained with the same methodology employed to develop the CP09 mutant, showing that C. pseudotuberculosis live attenuated mutants can be promising tools for protection against caseous lymphadenitis.
Animals in groups G4 (CP09 + rCP40) and G5 (primeboost) had survival rates of 70 and 60%, respectively. These two vaccine formulations had not been previously tested, so there is no work in the literature describing aspects of survival. After evaluating the results, it was observed that the survival rates of these groups were intermediate compared with those found in the G2 and G3 groups, which were vaccinated with only rCP40 (90% survival) and CP09 (50% survival), respectively.
The fact that CP40 recombinant protein alone led to a protection rate of 90% whereas the combination of rCP40 and CP09 mutant showed only a 70% protection can be explained by a differential complexation of the CP40 protein with the saponin adjuvant in the formulation using only the recombinant protein, when compared to the CP09 mutant-CP40 protein immunogen, which can lead to a different availability of the antigen to the immune system cells. A similar situation was seen by Keyburn et al. [27,28] when working with vaccines to Clostridium perfringens; they described that the protection rate achieved with the recombinant NetB protein alone was different from the one observed with the combination of the protein with the C. perfringens toxoid.
Taken together, these results suggest that vaccine formulations using the recombinant protein CP40 alone or in combination with the live recombinant strain CP09, or both antigens used in a prime-boost strategy, result in satisfactory survival percentages and antibody production. However, only the groups that had rCP40 in the inoculation solution presented an IgG2a response, and the group inoculated only with rCP40 was not able to induce an IgG-based humoral response, even after the challenge. These results indicate that rCP40 was able to induce a Th1 immune response, and the higher levels of protection in the G2 group can be observed as a result of this type of cellular immune response. All the groups stimulated with the combination of rCP40 with CP09 had a significant IgG1 response, indicating a Th2 response. The finding that the marked Th2 response elicited a low protection rate in those groups can be explained by the bacterial attenuation profile, which showed a reduced capacity of the mutant to adhere and invade murine cells. This reduced invasion can result in a defective presentation of bacterial antigens through a Major Histocompatibility Class (MHC) I pathway [21] because these antigens are not present in sufficient levels in the intracellular compartment. In this way, a Th2 response, represented by significant IgG1 production, prevailed, and the amount of Th1 cytokines was potentially insufficient to induce an adequate cellular response against the virulent C. pseudotuberculosis strain.

Conclusions
The C. pseudotuberculosis CP09 live attenuated strain was not able to induce a humoral immune response when inoculated alone in mice; IgG production could only be observed when the mutant strain was combined in a vaccine formulation with the CP40 recombinant protein. The protection level was higher when the vaccine solutions included only the recombinant protein and was associated with a Th1 response in the absence of a Th2 profile after challenge, as seen by the IgG isotype production in immunised mice. Thus, rCP40 associated with a saponin adjuvant is a promising vaccine formulation in the control of caseous lymphadenitis.


The protective effect of Nigella sativa against liver injury: a review

Nigella sativa (Family Ranunculaceae) is a widely used medicinal plant throughout the world. N. sativa is referred in the Middle East as a part of an overall holistic approach to health. Pharmacological properties of N. sativa including immune stimulant, hypotensive, anti-inflammatory, anti-cancer, antioxidant, hypoglycemic, spasmolytic and bronchodilator have been shown. Reactive oxygen species (ROS) and oxidative stress are known as the major causes of many diseases such as liver injury and many substances and drugs can induce oxidative damage by generation of ROS in the body. Many pharmacological properties of N. sativa are known to be attributed to the presence of thymoquinone and its antioxidant effects. Thymoquinone protects liver from injury via different mechanisms including inhibition of iron-dependent lipid peroxidation, elevation in total thiol content and glutathione level, radical scavengering, increasing the activity of quinone reductase, catalase, superoxide dismutase and glutathione transferase, inhibition of NF-κB activity and inhibition of both cyclooxygenase and lipoxygenase. Therefore, this review aimed to highlight the roles of ROS in liver diseases and the mechanisms of N. sativa in prevention of liver injury.

Introduction
Despite all the considerable improvement in modern medicine, traditional herbal medical profession has always been practiced (1). Plants have organized the basis of sophisticated traditional medicine systems which have given rise to some important drugs that are still in use today (2). Many plants are in use today, but their potential mechanism of action, medicinal properties, toxicological studies and safety evaluation are not fully known to us (3).

Pharmacognostical characteristics
N. sativa is a 20-90 cm tall plant with finely divided leaves and the leaf segments narrowly linear to threadlike. Flowers are pale, blue on solitary long peduncles. N. sativa is a bisexual and forms a fruit capsule which consists of many white trigonal seeds. The fruit is a large and inflated capsule composed of 3-7 united follicles (3). Matured fruit capsule opens up and the seeds contained within are exposed to the air, becoming black in color (black seeds). The seeds are trigonous and black in color (25).

Chemical composition of N. sativa seeds
Seeds of N. sativa contain numerous esters of structurally unusual unsaturated fatty acids with terpene alcohols (7%). Furthermore, traces of alkaloids are found which belong to two different types: isochinoline alkaloids and pyrazol alkaloids (see Table 1 for additional information) (26).
The essential oil contains significant (10%) amounts of fatty acid ethyl esters. On storage, thymoquinone turns into the dithymoquinonene and higher oligocondensation products. Fatty oil rich in unsaturated fatty acids, such as linoleic acid (50 60%), oleic acid (20%), eicodadienoic acid (3%) and dihomolinoleic acid (10%) are also find in the seeds. Saturated fatty acids (stearic acid and palmitic acid) amount is approximately 30% or less. Furthermore contain parts of the essential oil, mostly thymoquinone that is responsible for an aromatic flavor. The oil contains d -limonene, carvone, and a carbonyl compound, nigellone (26).
N. sativa seeds contain 36-38% fixed oils, saponin, proteins, alkaloids and 0.4-2.5% essential oil. By high performance liquid chromatography analysis of N. sativa essential oil, thymoquinone, thymohydroquinone, dithymoquinone, and thymol are considered the main active ingredients. The most prominent activity of thymoquinone is its antioxidant effect (26).

Traditional uses of N. sativa
N. sativa seeds are used as a stimulant, aromatic, carminative, anthelmintic, diuretic, galactagogue and diaphoretic. They are used as a spice in curries. A tincture provided from the seeds is useful in diarrhea, indigestion, dropsy, loss of appetite, dysmenorrhea and amenorrhea and in the treatment of worms and skin eruptions. To arrest vomiting, seeds are roasted and given internally (26)(27)(28). Externally, the oil is used as an antiseptic (27). N. sativa has been used as an antitussive for many years (29).

Reactive oxygen species and the liver diseases
Oxidative stress is one of the main causes of liver injury that depletes the antioxidant enzymes sources and decreases the ability of cells in functioning against injury (30). Nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidases, nitric oxide synthases (NOS), and myeloperoxidases are three main enzymes that produce ROS in the body. ROS, including super-oxide anion radical (O2 -), hydrogen peroxide (H2O2), peroxynitrite (ONOO), hydroxyl radical (OH -) and hypochlorous acid (HOCl -) are by-products of the normal metabolic processes in the cells. Beneficial effects of ROS disrupt when the balance between amount of ROS and antioxidants is diminished. This imbalance state defines as oxidative stress. Polyunsaturated lipids and sulfhydryl groups of proteins are the main constituents of cells which have essential roles in the functions of the cells. Disruption of their structural properties by free radicals may cause cellular functions aborted (30).
The liver is a very important organ with high rate of metabolism in the body. It is very sensitive to oxidative stress and damages caused by free radicals. Preventing the production of free radicals, scavenging free radicals or enhancement in antioxidant defense can minimize the adverse effects of them (31).
The majors liver damages mediated by oxidative stress are including: a) alcoholic liver disease, b) fibrosis/cirrhosis, c) hepatic stellate cells, d) hepatocellular carcinoma, e) ischemic/reperfusion liver injury, f) paracetamol-induced liver damage, g) viral hepatitis, h) fatty liver and i) chemical pollutant-induced liver damage (30).

a) Alcoholic liver disease
Ethanol is a potent inducer of ROS production in the body. This characteristic of ethanol can lead to oxidative stress and the liver is the main organ that is affected by that. Lipid peroxidation, depletion of glutathione and formation of lipid radicals can induce liver damages (31). Administration of antioxidants,
The role of free radicals in hepatic injury induced by alcohol and the protective effects of antioxidant therapy in reducing the toxic effects showed that oxidative stress is the main cause in ethanol-induce liver damages (32).

b) Fibrosis/cirrhosis
Production of ROS and lipid peroxidation is associated with liver fibrosis and cirrhosis (33). This effect can lead to the death of hepatocytes and many researchers have shown that oxidative stress has a critical role in fibrosis and cirrhosis of liver (34). Free radicals initiate cell damage by binding to the membrane proteins. These effects are partially prevented by antioxidants (33).

c) Hepatic stellate cells
The source of hepatic stellate cells (HSC) in liver injury is not completely understood but many investigations have shown that ROS are involved in necrosis and apoptosis of hepatocytes and HSC activation (35,36). ROS induces the production of platelet-derived growth factor that is the most potent mitogen of HSC and is, therefore, likely to be a major mediator during liver fibrogenesis. The usage of antioxidant was able to reduce of these states (37,38).

d) Hepatocellular carcinoma
Oxidative stress and chronic inflammation are the common mechanisms for hepatocellular carcinoma (39). ROS interacts directly with DNA and changes expression of specific genes which are responsive for cell proliferation and apoptosis (40). ROS can directly stimulate the growth of cancer cells. Chronic inflammation can worsen this state. The formation of 5-hydroxylmethyluracil, 8-hydroxydeoxyguanosine, thymine and thymidine glycol in this state is a main evidence that ROS is a cause of hepatocellular carcinoma (41).

e) Ischemic/reperfusion (I/R) liver injury
Periods of ischemia can occur in hepatic surgeries. Reperfusion increases the damage induced during the ischemic period when the flow of oxygen and blood is re-established. In this state ATP depletion, decline in calcium homeostasis with decline in cytoprotective compounds such as prostacycline, nitric oxide (NO), and elevation of ROS are present (42,43). ROS is responsible for many processes in I/R (formation of xanthine oxidase, induction of NADPH oxidase and NO formation). At this state lipid peroxidation, inactivation of heme group and nitrosylation of iron-sulfur group induced by ROS worsen I/R conditions (44,45). According to initiate mechanism of I/R the use of antioxidant can ameliorates the adverse effect of this condition.
Administration of antioxidants, especially in the early stages of reperfusion, may significantly diminish I/R injury in transplanted livers (30).

f) Paracetamol-induced liver damage
Paracetamol-induced liver damage is the most prominent drug toxicity in the world. The reactive metabolite of paracetamol (N-acetyl-p-benzoquinone imine) can bind to cellular proteins and cause hepatocyte death. The formation of peroxynitrite and depletion of glutathione resources can lead to liver damages. Oxidant stress of mitochondria triggers the mitochondrial membrane permeability transition pore, depletion of ATP and release of cytokines that are responsible for DNA fragmentation (30).
Taking antioxidant or compounds that restore glutathione resources in the body can protect the liver from toxicity of paracetamol overdoses (46).

g) Viral hepatitis
Hepatitis C virus (HCV) and oxidative stress are parallel to each other. Lipid peroxidation and antioxidant levels are increased in patients with HCV (47). Decreased content of glutathione in the blood, liver, and lymphatic system causes increase in GSSG level, indicating a high glutathione turnover (48).
Inflammatory cytokines and oxidative stress play important roles in the mediation of hepatic injury in HCV. The expressions of cyclooxygenase-2 and iNOS which can increase ROS are increased in HCV patients (30). Use of antioxidants with current treatments of HCV can enhance the beneficial outcomes in patients with HCV (49).

h) Fatty liver
Accumulation of fat in the liver can result in steatosis and steatohepatitis. This condition can progress to cirrhosis. Up to 10% of cirrhotic fatty liver develop hepatocellular carcinoma. Free fatty acids are the main sources of production of free radicals. With progression of stage of illness, oxidative stress level is increased and biomarker (malondialdehyde) is rises in the body. Elevated plasma malondialdehyde and tumor necrosis factor α (TNFα) reflected an increase of oxidative stress and inflammation, respectively (50).

i) Chemical pollutant-induced liver damage
Industrialization in today's world increases the environmental pollutant sources, such as mercury, carbon tetrachloride, arsenic, cadmium, thioacetamide etc. Mechanism of these chemicals is peroxidation of the hepatocyte lipids causing destruction of the cells and their intracellular organelles and covalent binding to the membrane proteins (51). Many researches have shown that chemical pollutants increase oxidative stress and then the effects of this condition on the body organelles such as liver (52)(53). Anti-inflammatory TQ inhibited both cyclooxygenase and lipoxygenase 62 TQ increased the ratio of helper to suppressor T cells, enhanced natural killer cell activity, enhanced production of IL-3 and had a stimulatory effect on macrophages 69 TQ inhibited of NF-Kβ reduction of cytochrome c production 75 TQ inhibition PG E2 formation 72 Mechanisms mention above revealed that many liver damage causes are mediates by oxidative stress and production of ROS. Thus, the enhancement of antioxidant defense and increase of the glutathione resources in the body can attenuates oxidative damages to hepatocytes.

Mechanisms of hepatoprotection of N. sativa
Studies on N. sativa have been confirmed the potential therapeutic effects of it. One of the most important effects is hepatoprotective which is shown in many research projects (54)(55).
In this article we summarized the mechanisms of hepatoprotection of N. sativa, and its main constituents, such as thymoquinone (Table 3).

Antioxidant properties
Antioxidant properties of N. sativa have shown in many studies (57)(58)(59)(60). Thymoquinone has the ability to inhibit iron-dependent lipid peroxidation in concentration-dependent manner (59). It is a potent O -2 scavenger activity (60)(61)(62). With this characteristic, thymoquinone can decrease oxidative stress and increase antioxidant defense in the body. Decrease in malondialdehyde and other biomarkers of oxidative stress in parallel with increase in total thiol content and glutathione level are the results of thymoquinone treatment (48,61,62). The content of glutathione in the liver is found particularly in high concentration in the liver and is known to have key functions in cellular protective mechanisms. Depletion in total thiol content caused by oxidative stress can result in protein inactivation, protein oxidation, lipid peroxidation, perturbation in calcium homeostasis and resultful loss of cell viability (62). Depletion in free radicals with thymoquinone can decrease the risk of them attacking to DNA and decrease the risk of cancers (63,64). Thymoquinone inhibits the activity of hepatic CYP1A1/A2 isozymes involved in biotransformation of many xenobiotics into reactive genotoxic radical derivatives (64).
The antioxidant properties of thymoquinone are responsible for the antischistosomi characteristic of thymoquinone and reduce in liver injury caused by parasites (65). The antioxidant properties of thymoquinone can reduce the adverse effects of ROS that produced in I/R state. Thymoquinone increased catalase (CAT) activity, and this is consistent with its protective effect on liver tissue against I/R injury (66). In addition, thymoquinone can protect against renal I/R induced damage through an antioxidant mechanism as well as the decrease of CYP3A1 and SSAT gene expression. CYP3A1 mRNA expression was induced significantly by I/R in both liver and kidney tissues. I/R caused induction of mRNA expression of spermidine/spermine N-1-acetyl-transferase (SSAT), a catabolic enzyme that partakes in polyamine alteration, in kidney and liver tissues. Thymoquinone reduces SSAT mRNA expression significantly in liver and markedly in kidney (67). Oral administration of thymoquinone is a promising prophylactic agent against chemical carcinogenesis and toxicity in liver tissues by increasing the activities of quinone reductase and glutathione transferase (68).
Exposure of isolated rat hepatocytes to oxidative stress induced by tert-butyl hydroperoxide, as an oxidative agent, lead to rapid depletion in intracellular glutathione due to its oxidation by glutathione peroxidase (GSHPx), and ultimately oxidation of pyridine nucleotides, which has been associated with the impairment of calcium sequestration by endoplasmic reticulum and mitochondria. This is followed by formation of surface blebs in the plasma membrane as well as the leakage of cytosolic enzymes. Thymoquinone can inhibit formation of blebs and it can preserve the integrity of cell membrane of hepatocyte (69).
Thymoquinone can inhibit the expression of iNOS, that is participated in oxidative stress state and can increase the expression of antioxidant enzymes such as GSHPx and SOD (50). Thymoquinone may be able to reduce NADH, thereby reducing the NADH-NAD + changes, which lead to inhibition lipogenesis in the hepatocytes (70).
Many studies have evaluated antioxidant properties of thymoquinone that mentioned before.

Anti-inflammatory properties
The use of thymoquinone has also been shown to have anti-inflammatory effects in several inflammatory diseases (71)(72)(73). Inflammatory cytokines in hepatocytes can promote signaling pathways that induce cell injury. Thymoquinone is a potent inhibitor of eicosanoid generation namely thromboxane B2 and leukotriene B4, by inhibiting both cyclooxygenase and lipoxygenase enzymes, respectively. The roles of these mediators are formation of bleb in cell membrane of hepatocytes and activation of free radicals production (62).
Antioxidant and anti-inflammatory actions of thymoquinone are two main mechanisms that parallel to each other preserve hepatocytes from injury (46). Thymoquinone increased in the ratio of helper to suppressor T cells, enhanced natural killer cell activity, enhanced the production of IL-3 and had a stimulatory effect on macrophages (69). Inflammatory responses and activated neutrophils can increase myeloperoxidase activity in the liver tissue. Myeloperoxidase increases lipid peroxidation and free radicals formation. This state worsens the liver injury (74).
Thymoquinone has a designation in reducing inflammation by decreasing malondialdehyde and lipid peroxidation products, reduction in amount of cytokines via inhibiting activity of NF-κB and to reducing cytochrome c production from mitochondria via inhibition of generates ROS in the liver (75).

Anticancer properties
Oxidative stress, nitrosative stress and inflammation are three major concepts in cancer. With two mechanisms thymoquinone preserve cells from cancer: 1) with antioxidant and antiinflammatory effect thymoquinone decreases oxidative stress and preserves the activity and expression of antioxidant enzymes (68,76) and 2) induces apoptosis (77).
Thymoquinone inhibits the ROS-induced oxidative DNA damage and therefore reduces the risk of cancer. Oxidative DNA cleavages reactions were mediated by polyphenolic antioxidants in the presence of copper (Cu) ions. Cancer cells have higher content of copper in comparison to normal cells. Thymoquinone may generate superoxide anion radicals. It also exhibit prooxidant DNA-damaging properties. Thymoquinone is capable of binding to DNA and operates thymoquinone -Cu (II)-mediated DNA cleavage (76).
Thymoquinone causes a time-and dosedependent formation of cytoplasmic vacuoles. Upon exposure to cytotoxic compounds cells will attempt to sequester the compounds into vacuoles to protect themselves. Thymoquinone causes lysosome membrane permeability and lead to leakage of lysosomal proteases, such as cathepsin B and D that induce apoptotic cell death (77).

Animal studies
Sixty days treatment with carbon tetrachloride (CCl4) followed by sixty days treating with N. sativa, in fifty-six healthy male Wistar albino rats, was done. CCl4 increased the lipid peroxidation and liver enzymes, and reduced the antioxidant enzyme levels. N. sativa treatment decreased the elevated levels of lipid peroxidation and reduced the liver enzyme levels, also increased antioxidant enzyme levels (33).
According to mechanism of paracetamol-induced hepatotoxicity Yesmin and co-workers described hepatoprotection of N. sativa with antioxidant and anti-inflammatory properties against paracetamol toxicity. N. sativa improved histopathological changes such as centrilobular necrosis, pyknosis of hepatocytes and neutrophils infiltration that were induced by oxidative stress in the liver in rats (46).
The effect of N. sativa on inflammatory fatty liver was shown by Al-Okby and co-workers. Induction of inflammatory fatty liver in rats with feeding high fructose diet exhibited significant dyslipidemia, high plasma TNF-α and malondialdehyde along with significant high liver triglycerides and cholesterol and liver dysfunction. N. sativa produced significant improvement of all parameters (50).
Thioacetamide is an experimental hepatotoxic agent. A study was conducted to evaluate its hepatotoxicity, in this research intraperitoneal injection of thioacetamide at a concentration of 20 mg/kg body weight for a period of eight week, in male albino rat leads to cirrhosis. N. sativa oil (10 ml/kg body weight) improved the altered levels of albumin, bilirubin, total protein, γ-glutamyltransferase, alanine transaminase and alkaline phosphatase also significant improvement in SOD, CAT, GSHPx, and reduced glutathione and reduction in thiobarbituric acid reactive substances was seen, as well as the histological tissue damage of liver decreased in N. sativa treated group (53). In another study, after 24 hr of CCl4 administration (0.625 ml/kg IP), for 2 weeks the rats were treated with N. sativa, then sacrificed and alkaline phosphatase, Alanine aminotransferase, and aspartate aminotransferase in serum of rats was measured, the changes that produced by CCl4 on hepatic cells and enzymes revised with thymoquinone treatment (62).
The effect of N. sativa on ischemia/reperfusion injury on liver in rat was performed, after 45 min hepatic ischemia followed by 60 min reperfusion. In the group that received intraperitoneal N. sativa (0.2 ml/kg) prior to ischemia and before reperfusion, liver enzymes were significantly lower than control group which received 0.9% saline solution. Total antioxidant capacity was higher in N. sativa group compared to control group, myeloperoxidase, total oxidative status, and oxidative stress index in hepatic tissue were significantly lower in N. sativa group than in the control group. The histological tissue damage in the N. sativa group was milder compared to control group (66).
Diethylnitrosamine is a hepatocarcinogen. In liver tissue diethylnitrosamine induced severe histopathological lesions and lead to increase the level of total nitrate/nitrite, total bilirubin, thiobarbituric acid reactive substances, alanine transaminase, alkaline phosphatase and reduced CAT, GSHPx, glutathione, glutathione-s-transferase (GST) as well as diminished the gene expression of GST, GSHPx and CAT, These changes completely corrected by thymoquinone supplementation (68).
One study was performed to evaluate the hepatotoxicity of N. sativa, the result showed that up to the dose of 1 g/kg, no significant change in serum alanine aminotransferase and aspartate aminotransferase in comparison between the N. sativa treated and non-treatment groups in rat seen. Histopathological examination showed mild and very low changes in fatty degeneration in non-treatment group and treated with high doses of N. sativa also there was no inflammation and necrosis (78).
Industrialization in today's world increases the environmental pollutant exposures that they can generate ROS. Benzene, toluene and heavy metals are the leaders of them. They can deplete glutathione contents and destroy intracellular proteins especially in the liver. Salman Ashraf and coworkers showed that injection of toluene with dose 250 mg/kg body weight in dimethyl sulfoxide could significantly deplete hepatic glutathione content and could increase protein carbonyl formation in hepatocytes. The use of 100 μl, oral administration of black seed extract 1 hr after toluene injection could significantly elevate hepatic glutathione content. The application of different concentration of N. sativa extracts showed that protection against oxidative damage and elevate of hepatic glutathione was dependent to thymoquinone concentration and the method of extraction. Studies with partial purification and fractionation of N. sativa oil showed that although fractions rich in thymoquinone were most potent in terms of their antioxidant capacity (79).
Suddek showed that the hepatotoxicity induced by tamoxifen resulted in elevated serum levels of liver enzymes such as alanine transaminase, gamma glutamyl transferase, lactate dehydrogenase, alkaline phosphatase, and total bilirubin, plus diminution of reduced glutathione in the liver, reduced super oxide dismutase activity and accumulation of lipid peroxides. Pretreatment with thymoquinone significantly inhibited tamoxifen-induced hepatic glutathione depletion and normalized the activity of SOD, inhibited the rise in TNFα (80).

Human studies
Marked induction of ROS in infected cells leading to oxidative stress is responsive for induced HCVrelated fibrosis, cirrhosis and liver failure. Abdel-Moneim and co-workers showed that N. sativa in HCV patients exhibited potential therapeutic benefits via decreasing viral load and alleviating the altered liver function (47).
The most common childhood malignancy is acute lymphoblastic leukemia, and the choice drug for treatment in children is methotrexate. Long-term treatment with methotrexate may cause hepatotoxicity. N. sativa oil administered orally prevented liver damage that induced by methotrexate in leukemic children as well as improved the survival of this malignancy (81).

Conclusion
Antioxidant and anti-inflammatory properties of N. sativa are the main features of preventing and protecting liver from injury. Several studies have shown the protective effects of N. sativa against liver injury produced by ROS with its free radical scavenger properties and enhance antioxidant defenses in body. Thymoquinone is the main active ingredients of N. sativa responsible for it. Also, none of the studies reported that the use of thymoquinone in moderate doses had significant toxic effects. The efficacy of N. sativa to postpone progression in chronic liver diseases should be considered as preventive medicine in patients with hepatic disorders.