s2fieldsofstudy: Biology Psychology

text:
Overnight Social Isolation in Pigs Decreases Salivary Cortisol but Does Not Impair Spatial Learning and Memory or Performance in a Decision-Making Task

Pigs in modern farming practice may be exposed to a number of stressors, including social stressors such as mixing or isolation. This may potentially affect both cognitive abilities and stress physiology of the animals. We tested the hypothesis that overnight social isolation in pigs impairs performance in a cognitive holeboard (HB) task (Experiment 1) and the Pig Gambling Task (PGT) (Experiment 2), a decision-making task inspired by the Iowa Gambling Task. In addition, we tested the effect of overnight social isolation on salivary cortisol levels. A within-subjects approach was used in which performance in the two behavioral tasks and cortisol levels were first determined during normal social housing, followed by performance and cortisol levels after experiencing stress induced by overnight social isolation. A total of 19 female pigs with a birth weight closest to their respective litter average was selected from 10 different litters and placed in two pens after weaning. Following habituation, pigs were trained in the HB task, starting at 10 weeks of age. Then, the pigs were isolated overnight, five individuals per night, at 15, 16, and 17 weeks of age. Between these three isolations, social housing and training in the HB continued. Starting 6 weeks after the end of the HB experiment, at approximately 23 weeks of age, the pigs were trained in the PGT. The effects of overnight social isolation on performance in this task were assessed once, when the pigs were 25 weeks old. Salivary cortisol was measured from samples collected 15 min after the start of isolation and at the end of the isolation period and compared to baseline values collected before the start of social isolation. Our results did not confirm the hypothesis that isolation impaired HB performance and decision-making in the PGT. Unexpectedly, overnight social isolation decreased cortisol levels below baseline values, an effect that was not associated with changes in performance of the behavioral tasks. We hypothesized that the housing and testing conditions may have prepared the animals to cope efficiently with stress.

inTrODUcTiOn
In industrial pig production systems, animals are confronted with cognitive challenges. They must learn to recognize their penmates in order to establish and maintain a stable social hierarchy and must learn to use their pen environment optimally [see in Ref. (1,2)]. In group housing systems, where automatic feeding stations are increasingly used, the pig must learn how to gain access to the food (3). Pigs are also exposed to various types of stressors, such as repeated regrouping (mixing), aggression of penmates, and unchallenging, barren environments. As social animals, pigs may experience stress if socially isolated from their penmates for a longer period of time (4,5). Stress may interfere with or disrupt spatial memory performance and may compromise pigs' cognitive abilities (2,6) and affect their welfare (7).
Results of studies addressing the effects of adverse manipulations on cognitive performance of pigs are equivocal. There is some evidence for the notion that events such as confinement and isolation cause stress and interfere with subsequent cognitive performance [e.g., Ref. (2,6)], whereas others found the effects of social isolation stress only in piglets younger than 35 days (8). Düpjan et al. (9), however, did not find effects of repeated isolation (a total of ten, 2½-h isolations during the course of seven successive days) on the performance of juvenile pigs in a cognitive bias task.
Pigs are able to discriminate between spatially distinct locations at a very young age, e.g., when developing a teat order. Teats can be considered as spatially distributed objects; once a specific teat preference is determined, the preference is preserved, even if the piglets are transferred to a foster sow (10). A broad range of tasks has been developed to test pigs' cognition [reviewed by Ref. (11,12)], such as spatial cognitive holeboard (HB) tasks (13)(14)(15)(16), and more recently, a decision-making task (17). Pigs at weaning age and slightly older are already able to learn these tasks (13,18,19).
The spatial HB task allows measuring spatial working memory (WM) and reference memory (RM) simultaneously. In this task, food can only be found in a subset of potential sites (20)(21)(22). The WM "is a short-term memory that, once used, should be forgotten or ignored" [(23), p. 701] to avoid interference with the next trial. The WM holds information that is relevant only within a specific trial, such as a list of locations that have already been visited/explored during a particular trial. This measure represents the pig's ability to avoid re-visits to baited holes during a trial (24).
The RM holds information about the solution of the spatial discrimination task, e.g., about the localization of the food, and that, once a food reward has been found and consumed, the hole will not be refilled during the trial. It also contains information about the actions necessary to get the bait (23,25), for example, a head dip into a food-containing hole. This measure provides an index for the ability of pigs to discriminate between baited and unbaited holes (24). RM thus stores the general rules of a task, whereas the WM stores information that is relevant only within a specific trial (22).
The pig HB task has been used in a number of studies. All studies confirmed that Göttingen minipigs (16,19,26) and commercial pigs of different breeds were able to acquire this task [e.g,.
Ref. (14,15,(26)(27)(28)(29)]. In Göttingen minipigs, performance in a HB task was slightly affected by a 9-and 38-day retention interval. After a 9-day retention interval, WM and RM performance was poorer than during the last trial block of the learning phase. Both WM and RM also decreased between the two memory phases (19).
In a study by Arts et al. (14) mixing, i.e., regrouping and housing of pigs with unfamiliar conspecifics, a practice that has been shown to induce stress, did not affect HB performance of well-trained pigs. Studies of HB performance of low birth weight (LBW) vs. normal birth weight (NBW) piglets have shown varying effects: either transiently reduced WM following reversal in LBW piglets (27) or improved RM performance in LBW compared to NBW piglets in both the acquisition and reversal phase of the HB task (13). In the latter study, the WM performance of the LBW was less disrupted than that of the NBW animals when switched to the reversal phase. Bolhuis et al. (15) and Grimberg-Henrici et al. (29) assessed the effects of environmental enrichment on HB performance in pigs. Bolhuis et al. (15) found that WM performance was better in enriched pigs than barren-housed piglets. In the study by Grimberg-Henrici et al. (29), the RM performance of the enriched-housed pigs was better than that of their barrenhoused littermates during acquisition. During the reversal phase, enriched-housed pigs had a better general WM performance than the barren-housed pigs as indicated by reduced revisits to holes already visited during a trial, irrespective of whether they were of the baited or the unbaited set.
The HB task has also been used to assess the effects of dietary manipulations on cognitive performance in pigs. Haagensen et al. (16) tested the effects of a high fat and cholesterol, low carbohydrate diet or a low fat, high carbohydrate, and sucrose diet on the performance of Göttingen minipigs during the acquisition, after a retention interval, and during reversal learning in a HB. Both diets impaired WM and RM, compared to the standard diet, on retention and reversal. In a recent study assessing the effects of pre-weaning iron deficiency on post-weaning cognitive performance, Antonides et al. (18) showed a lasting impairment on the RM component during acquisition and reversal of the HB task.
For testing decision-making, a recently developed simple two-choice probabilistic task, the Pig Gambling Task (PGT) (17) was used. In this task, an advantageous option yields small but frequent rewards and a disadvantageous option yields large but infrequent rewards. In the long run, i.e., over a series of successive trials, choosing the advantageous option offers greater overall gain. This task is a modification of the Iowa Gambling Task (IGT) used to assess decision-making behavior under risk in humans (30). A main modification for use in animal research is the reduction of the number of "sets": whereas human subjects can usually choose from four sets in the IGT, only two sets are presented in the animal modifications such as the PGT. This modification facilitates the performance of animals [e.g., rodents: (31); pigs: (17)] in the PGT.
Stress may affect decision-making behavior [reviewed by Ref. (32)]. For example, using the IGT, psychological studies with human subjects showed that judging and deciding are influenced by emotions and by the individual's personality (33). Subjects exposed to stress made more unfavorable, disadvantageous choices, i.e., took more high-risk decisions, compared to unstressed subjects (34). In a study comparing the decisionmaking behavior of NBW piglets and LBW piglets in the PGT, the LBW piglets started to choose the advantageous option more often that the NBW piglets in the later phase of training (17). These piglets also performed the Judgment Bias Task in a manner that can be characterized as less optimistic than the NBW pigs. Murphy and colleagues (17) interpreted these findings as evidence that LBW pigs developed different behavioral strategies with respect to decision-making.
We addressed the question whether an adverse event, overnight social isolation, would interfere with performance in two different tasks on which pigs had been trained for a large number of training trials. We hypothesized that overnight social isolation would interfere negatively with spatial learning and memory performance in a HB task (i.e., that the pigs would make more WM and/or RM errors; Experiment 1), and with decision-making behavior in the PGT (i.e., that the pigs would make more disadvantageous choices; Experiment 2). We also expected that overnight social isolation would induce a physiological stress response, measured as increased salivary cortisol [e.g., Ref. (35)]. eXPeriMenT 1: sPaTial hOleBOarD DiscriMinaTiOn TasK

Materials and Methods
All methods of the two experiments were reviewed and approved by the local ethics committee (DEC Utrecht, DierExperimentenCommissie) and were conducted in accordance with the recommendations of the EU directive 86/609/EEC. All efforts were taken to minimize the number of animals used and to avoid suffering.

Animals
Nineteen female piglets [Duroc × (Yorkshire × Finnish Landrace)] were selected at 4 weeks of age from 10 different litters. These piglets deviated <1 SD from the average birth weight of their litter. This criterion was used because we have seen in two previous studies that performance in LBW piglets differed from their normal weight littermates in the HB task [see in Ref. (13,27)]. All piglets were born at the pig-breeding farm of University Utrecht, under conventional Dutch commercial pig housing conditions (tails were docked and plastic ear tags placed during the first week after birth).
Pairs of piglets from seven different litters (i.e., 14 piglets) fulfilled the above criterion. One piglet of a pair was randomly assigned to the first pen and the other to the second pen. Unfortunately, in the eighth litter, only one of the piglets was a female. This female was included in the study. In the ninth litter with more females, only one female fulfilled the selection criterion. In addition, from the tenth litter three female piglets close to the average litter weight were included in the study. Each of these five piglets was randomly assigned to one of the two pens. This procedure yielded 1 pen housing a group of 9, the other pen housing a group of 10 piglets. The 19 selected piglets were individually marked by ear tags and by spray-painted letters on the back to facilitate identification. The timeline of both experiments is summarized in Table 1.

Housing
Starting 1 day after weaning, the piglets were housed per group in two adjacent enriched pens (4 m × 5 m), situated in a naturally ventilated stable. Each pen contained a piglet nest, with rubber mats and straw bedding that could be accessed through transparent vertical plastic blinds. In addition, heat lamps ensured a comfortable temperature in the piglet nest. Bite sticks, balls, and gunny sacks provided additional enrichment. Both pens were cleaned daily and provided with fresh straw. Tap water was available ad libitum and piglets were fed twice a day. The ambient temperature during the study ranged from −3 to 22°C. Piglet health was monitored daily. During the first 5 days, piglets were allowed to acclimatize to the pens and to their penmates.

HB Apparatus
The HB was a square arena (5.3 × 5.3 m), manufactured by Ossendrijver BV (Achterveld, The Netherlands), with a blue slatted floor and gray synthetic walls (80 cm height), with a steel bar on top of it (see Figure 1, left panel). The arena contained four guillotine doors, one at each side of the testing area, which could be opened by pulling a rope system from the outside of the test arena. The test area in the middle consisted of a 4 × 4 matrix with 16 food bowls. Beneath the food bowls there was a false bottom with four fresh M&M's ® Milk Chocolates underneath, so the piglets could not search for the rewarded bowl by scent. Each bowl was covered by a ball (Jolly Ball Dog Toy, diameter: 24 cm, weight: 400 g) to prevent piglets from searching for rewards by sight [for details, see Fig. 1, panels 3 and 4 in Ref. (13)]. The experimental workflow was controlled, and hole visits were registered automatically using custom-made software (Bling Systems, Delft, The Netherlands). When a pig lifted a ball, this was scored as a hole visit; the signal was registered by an interface (LabJack) and sent to a computer [for details see in Ref. (13,26)]. Lifting a ball was not counted as a (re)visit when it was lifted again within 10 s and no other holes were visited in between.

Isolation Pens
In a room adjacent to the pens housing the pigs, there were two rows with three isolation pens, each measuring 90 cm × 120 cm. The pens were separated by guillotine doors. Each pen measured 90 cm × 120 cm and was fitted with a drink nipple and a food bowl. The floor of the pens was covered with straw.

HB Habituation
During a habituation phase of 15 working days in three successive weeks, animals were allowed to explore the spatial HB in groups. During this phase, all holes were rewarded with chocolate M&M's ® . The piglets walked down the hallway to the waiting area (11.5 m 2 ), next to the HB apparatus. The floor of the waiting area was covered with straw and water was available ad libitum. During this period, all piglets were trained to voluntarily approach the experimenter and chew on cotton swabs (in order to habituate them to the procedure of saliva sampling; see below).  On the first 3 days of the habituation phase, all piglets of a pen were led into the HB arena once daily, followed by three daily sessions with five piglets each, followed by 3 days with three or two piglets. Then, all pigs were habituated individually to the HB on three additional days: each pig was trained to walk around the test arena through a narrow, 40 cm wide corridor, until it found the opened guillotine door and entered the HB voluntarily (see Figure 1, left panel). All pigs were habituated two times a day, and stayed in the test area for 20 min. Per habituation trial, it was determined randomly which of the four doors gave access to the HB arena.
During HB habituation and training, the pigs were fed twice a day; they received 1 /3 of the total amount of food before testing in the early morning and the remaining 2 /3 after testing, late in the afternoon.

HB Acquisition
During the acquisition phase, starting 1 week after habituation, only four bowls contained bait. Every piglet had its own configuration of rewarded holes. Piglets received at least 20 days of testing with two trials a day in close succession until they had reached the criterion of a RM performance level of 0.7, averaged across two successive sessions (i.e., four trials). If the piglet did not reach this criterion, the training schedule was continued until the criterion was met (18,28). This criterion ensured that all pigs had reached the same high performance level before we started to test the effects of overnight social isolation.
The testing area was cleaned daily with water, and the M&M's ® underneath the false bottom of the food bowls [see in Ref. (13), Fig. 1, panels 3 and 4 for details] were replaced by new ones.

Testing the Effects of Overnight Social Isolation on HB Performance
After completion of the acquisition phase, training in the HB continued with daily sessions of two trials each on working days for 3 weeks. Once per week during this 3-week period, all pigs underwent overnight social isolation, for a total of three isolations per piglet. The order in which piglets were isolated was determined randomly. The 18-h isolation period started at 15:00 in the afternoon and lasted until 9:00 in the morning of the next day. Subjects stayed in a pen individually during one night, with ad libitum access to water. During the isolation period, piglets could hear and smell the pigs in the adjacent isolation pens, but could not see them.

Collection of Saliva
During the last 3 days of the acquisition phase in the HB apparatus, saliva from all subjects was collected for determining the baseline cortisol level according to Merlot et al. (35). As cortisol levels show a circadian rhythm, baselines were determined for the two timepoints that were later tested following isolation, i.e., at 9:00 in the morning and at approximately 15:15 in the afternoon of the same day. The pigs chewed on two cotton swabs (Cotton Swabs 150 mm × 4 mm WA 2PL; Heinz Herenz, Hamburg, Germany) until they were thoroughly moistened. The first sample during overnight social isolation was taken approximately 15-30 min after the isolation started at 15:00, i.e., at a time point where a peak in the cortisol response due to stress was expected [(5); e.g., Ref. (36, 37)], and on the next day at 9:00, at the end of isolation.
After saliva collection, the swabs were placed in special centrifuge tubes with inner cases (Salivette, Sarstedt, Germany) and were rapidly centrifuged (Sigma 4K10, supplier: Salm en Kipp bv, Breukelen, The Netherlands) at around 3524 g for 10 min at 10°C to obtain the saliva. The collected saliva was stored in the tubes at −20°C until cortisol concentration was measured (35) by a Coat-a-Count radioimmunoassay, according to manufacturer's procedure (Coat-a-Count cortisol TKCO, Siemens Healthcare Diagnostics BV, The Hague, The Netherland). All samples from both experiments (HB and PGT) were assayed on the same day.

statistical analysis
For the acquisition phase and for testing the effects of overnight social isolation in the HB task, two spatial memory components, WM and RM, and three latency/duration measures were analyzed [see also in Ref. (22)]: WM was calculated as number of rewarded visits divided by the number of visits to the baited set of holes (38). RM was calculated as number of visits to the baited set of holes divided by the number of visits to all holes (38). In addition, the latency of the first hole visit, the inter-visit interval [i.e., the time (s) between first and last hole visits divided through (number of hole visits -1)], and the total trial duration (i.e., the time needed to find all food pellets, or the maximum trial duration, whatever event occurs first) were analyzed.
All analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, USA). The experimental unit was the individual pig because all effects tested are within-subjects behavioral changes or changes in cortisol levels. Normality of the untransformed variables was assessed by Shapiro-Wilk Test (SAS UNIVARIATE procedure). Variables measuring latencies or durations and the salivary cortisol data were log10-transformed to fulfill the normality requirement.

Acquisition of the HB Task
Means of blocks of four successive trials (i.e., two successive testing days) were calculated. All variables were subjected to an analysis of variance (ANOVA) with the within-subjects (repeated measures) factor Blocks of Trials (SAS GLM procedure).

Effects of Overnight Social Isolation on HB Performance
To assess the effects of overnight social isolation on HB performance, the means of the two trials of the session before isolation and the means of the two trials of the session after isolation were calculated and submitted to an ANOVA with the repeated measures factor Isolation (session before isolation vs. session following isolation), using the GLM procedure.

Effects of Overnight Social Isolation on Salivary Cortisol
The means of the three baseline afternoon samples of the baseline measurement and of the three baseline morning samples, and the means of the three isolation afternoon samples and of the three isolation morning samples were calculated and log10 transformed. Effects of isolation on salivary cortisol were analyzed using an ANOVA with the repeated measures factors Isolation (baseline vs. isolation) and Sampling time point (sampling at 15:15 vs. 9:00), using the SAS GLM procedure.

Latencies and Durations
There was a slight increase in the mean latency of the first hole visit over time for all pigs (F9,162 = 4.10, p < 0.0001). Effects of social isolation were tested once in the PGT. Note that outliers detected by Grubbs' test (http://graphpad.com/quickcalcs/grubbs1/) were excluded from statistical analysis.

Effects of Overnight Social Isolation on Cortisol Levels
Cortisol levels were lower during isolation than at baseline (F1,18 = 50.38, p < 0.0001), and in the morning, they were lower than in the afternoon (F1,18 = 6.76, p = 0.0181) (see Figure 3). There were no differential effects of isolation on this difference between

Animals and Housing
The 19 pigs from the previous experiment were used. Approximately 3 weeks after the end of the HB experiment, the pigs were transferred to new pens (each measuring 4 m × 5 m) in another section of the same stable. The new pens were highly similar to those used during Experiment 1. The pigs were allowed to habituate to the new environment for 2 weeks. The testing apparatus was adjacent to the home pens.

Testing Equipment
The apparatus for testing pigs in the PGT consisted of a start box (1.2 m 2 ), which was connected to a test chamber (3.6 m × 2.4 m) (see Figure 1, right panel). Access to the test chamber was controlled via a guillotine door, remotely operated by an experimenter. The left and right corner of the testing arena contained a "goal box" each (0.4 m wide) containing a bowl, which was covered by a red hard-plastic large ball. The pigs were trained to perform the operant response of lifting the ball that covered the food bowl. If the response was rewarded, the appropriate number of M&M's ® was delivered into the central food bowl between the two goal boxes [for technical details, see in Ref.

Habituation and Training in the PGT
Because they were already habituated to the experimenters and to performing behavioral tasks alone, the pigs were only habituated 1 day in the PGT apparatus. The pigs were allowed to explore the test setup with M&M's ® underneath the balls in the goal boxes and in the central food bowl. Then, the pigs were trained individually to lift the ball in one goal box, whereas the other goal box stayed closed. Both sides were open equally often to prevent that the pigs developed a side preference. After pushing up the ball, an M&M's ® reward was made available in the central food bowl (see Figure 1, right panel). When the pigs reliably showed the required response, the next phase started. At this point, each pig was assigned to one of three test batches, and training of the three test batches started staggered on three successive days to enable testing the effects of overnight social isolation at the end of training at staggered time points.
At the start of each training trial, the guillotine doors of both goal boxes were open. As soon as the pig had lifted the ball in one of the goal boxes, the other box was closed (to prevent the pig from choosing the other goal box). The appropriate number of M&M's ® fell through a vertical tube into the central food bowl that was covered by a transparent lid. If the animal selected the disadvantageous side, four M&M's ® were released into the food bowl [see in Ref. (17), Fig. 1C] and the reward was made accessible in 3 trials of a series of 10 trials by rising the lid that covered the food bowl [see in Ref. (17), Fig. 1D]. Two M&M's ® were released into the food bowl when the pig chose the advantageous side, and the reward was made accessible in 8 trials of a series of 10 trials.
Over 12 daily sessions with 10 trials each (total: 120 trials), pigs could choose freely between the two goal boxes. Any correct response in a goal box resulted in the delivery of reward into the central food bowl. The number of M&M's ® and the accessibility of reward were predetermined. A response in the advantageous goal box yielded a small quantity of reward (two M&M's ® ) but had a high probability (80%) that the rewards would be made accessible. A response in the disadvantageous goal box yielded higher quantities of reward (four M&M's ® ), but there was a low probability (30%) that the rewards were made accessible. In each series of 10 successive trials, the advantageous option yielded 16 accessible M&M's ® while the disadvantageous option yielded 12 accessible M&M's ® (see Figure 4).
In rewarded trials, as soon as the pig moved to the central food bowl, the lid was raised giving access to the M&M's ® . In unrewarded trials, rewards were also delivered after the pig had pushed up the ball in the goal box, but the lid stayed closed and the reward remained inaccessible (which may be experienced as "punishment" by the pigs). After each trial, pigs were allowed to return to the start box for the next trial 25 s after making a choice. The order in which the rewards were accessible and inaccessible differed daily but the probability of getting a reward remained the same within each series of 10 trials. The number of advantageous choices was recorded per pig for each of the six blocks of 20 trials.

Testing the Effects of Overnight Social Isolation on the Percent Advantageous Choices
After the 12th training day, the pigs were socially isolated overnight (from 15:00 to 9:00), using the isolation pens from the first experiment. In the morning, after isolation on day 13, the pigs were tested again. The pigs received 10 trials per session. The number of advantageous and disadvantageous choices was registered and expressed as percentage advantageous choices for each of these sessions. We calculated and analyzed the percentage of advantageous choices for comparability reasons: we considered sessions of 10 or 20 trials in the statistical analyses.

Collection of Saliva
During the first week of training on the PGT, saliva from all subjects was collected by letting the pigs chew on cotton swabs during 5 min twice, at 9:00 in the morning and at approximately 15:15 in the afternoon, two subjects at a time, to determine the baseline cortisol level according to Merlot et al. (35). Saliva was collected on three consecutive days. On the last (12th) training day in the PGT in the afternoon, the first salivary sample was collected, and on the next morning, the second saliva sample was collected. Then, the pigs were tested in the PGT for the last time (13th training day).

statistical analysis
One pig had to be excluded from analyses because it refused to enter the test arena. After checking whether the variables were normally distributed using the Shapiro-Wilk test (UNIVARIATE procedure), they were analyzed by a repeated measures ANOVA with the (within-subjects) factor Blocks.
Inspection of the individual learning curves revealed that some pigs learned the task, i.e., increased the number of advantageous choices across blocks, some pigs did not learn the task (no change of advantaged choices across blocks, but also no obvious side preferences), whereas others showed persistent side preference as seen in Figure 5B. The pigs were classified a posteriori as learners (N = 7), non-learners (N = 5), advantageous side preferring (N = 4), or disadvantageous side preferring (N = 2). Advantageous side preferring was defined as 17-20 advantageous choices, whereas disadvantageous side preferring was defined as 17-20 disadvantageous choices per block of 20 trials in at least four of the six blocks of the learning phase. Differences in learning between the four subgroups defined a posteriori were analyzed by ANOVA with the repeated measures factor Blocks and between subjects factor Subgroups.

Effects of Overnight Social Isolation on Choices in the PGT
To assess the effects of overnight social isolation on HB performance, the percent advantageous choices in the last 10 trials of the session before isolation and in the 10 trials of the session after isolation were calculated and submitted to a repeated measures ANOVA with the factor Isolation (session before isolation vs. session following isolation), using the GLM procedure.

Effects of Isolation Between Subgroups
Effects of isolation between subgroups were analyzed by ANOVA with the between subjects factor Subgroups (learners, nonlearners, advantageous side preferring, disadvantageous side preferring) and the repeated measures factor Isolation (session 12, before isolation vs. session 13, after isolation).

Comparison of Cortisol Baseline Levels of the Two Experiments
We compared the baseline cortisol levels measure in the two experiments by an ANOVA with the repeated measures factors Experiment (mean baseline cortisol of first vs. mean baseline cortisol of second experiment) and Sampling time point (sampling at 15:15 vs. 9:00), using the GLM procedure.

Effects of Overnight Isolation on Salivary Cortisol
The means of the three baseline afternoon samples and the three baseline morning samples were both calculated and log10 transformed. Effects of isolation on salivary cortisol were analyzed using an ANOVA with the repeated measures factors Isolation (mean baseline cortisol vs. cortisol during isolation) and Sampling time point (sampling at 15:15 vs. 9:00), using the GLM procedure.
results PgT

All Pigs
Averaged over all pigs, the percentage of advantageous choices increased across the six successive blocks (F5,85 = 4.53, p = 0.001) (see Figure 5A). The overnight social isolation had no effect on the pigs' choices (comparison of performance on day 12 and day 13; F1,17 = 0.36, p = 0.5547).

Four a Posteriori Subgroups
Averaged over the six successive blocks of the training phase, the four subgroups differed for the number of advantageous choices (Subgroups; F3,14 = 23.49, p < 0.0001) (see Figure 5B). A marginal Block effect (F5,70 = 2.32, p = 0.0524) and a block by subgroups interaction effect (F15,70 = 3.20, p = 0.0005) indicate that the learning curves of the four groups were indeed different. As a consequence of large differences in learning between the four subgroups, the average percent advantageous choices across the 12th (before isolation) and 13th day (after isolation) was different between subgroups (F3,14 = 6.70, p = 0.0050). Overnight social isolation did not affect the percent choices between subgroups on day 12, compared with day 13 (Isolation: F1,14 = 2.32, p = 0.1499; Isolation by Subgroups interaction: F3,14 = 2.92, p = 0.0711).

Comparison of the Baseline Cortisol Measurements
On average, the cortisol baseline values in the HB experiment (Experiment 1) were higher than those in the PGT experiment (Experiment 2) (F1,17 = 121.41, p < 0.0001) (see Figure 3). In both experiments, the levels measured at 9:00 were lower than those measured at 15:15, but the difference was larger in the PGT experiment (sampling time point, F1,17 = 42.04, p < 0.0001; Experiment by Sampling time point interaction, F1,17 = 10.61, p = 0.0046).

Effects of Overnight Social Isolation on Cortisol Levels
The data of 17 pigs were used, due to exclusion of one pig with an outlier cortisol morning measurement (see Figure 3). Cortisol levels were lower during isolation than at baseline (F1,16 = 8.13, p = 0.0115), and in the morning, they were lower than in the afternoon (F1,16 = 6.76, p = 0.0181). There was no differential effect of isolation on this difference between the two sampling time points (i.e., afternoon -morning; Isolation by Sampling time point interaction: F1,16 = 0.36, p = 0.5547).

DiscUssiOn
Stress may affect cognitive functioning and decision-making behavior [reviewed by Ref. (32)]. The present study investigated FigUre 6 | contingencies in the PgT. Pigs may choose (I) invariably either the left or right goal box (side preference, side bias; as long as the pig persists in selecting one side, it will not learn anything about the contingencies that are in effect on the other side), (II) the side that yields the larger reward (i.e., four M&M's ® ), (III) the side that yields reward with the highest probability in the long run, (IV) the side, which yields the lowest probability of punishment, i.e., non-reward in the long run, or, finally, (V) the side, which renders the largest number of M&M's ® in the long run. The green and red bars against the gray background on top of the figure show the relative contrast between the advantageous and disadvantageous choices, depending on the contingency according to which the pig chooses. It is obvious that the contrast between the advantageous and disadvantageous choices is lowest with option (V). van der Staay et al.
Overnight Social Isolation in Pigs Frontiers in Veterinary Science | www.frontiersin.org the effects of overnight social isolation in pigs on performance in a spatial HB task, in the PGT, and on salivary cortisol as physiological stress marker. We did not observe effects of overnight social isolation on either behavioral task. This lack of effect was already evident after the first overnight social isolation, and consequently, it cannot be ascribed to a habituation effect of repeated isolations. This result contrasts with findings by Laughlin et al. (6). Overnight social isolation did affect cortisol levels. However, contrary to our hypothesis, we observed decreases in cortisol levels when measured both at 15 min following the start of social isolation and after 18 h of social isolation. This decrease was observed at both periods in which social isolation was performed, at 15−17 weeks and at 25 weeks of age.

spatial learning and Memory in the hB
The pigs learned the HB task readily, corroborating earlier findings [e.g., Ref. (13,14,27)]. All piglets were trained to a RM criterion of 0.7 -this ratio measure reflects the ability of the animals to avoid re-visits to baited holes during a trial and reaches the value 1.0 in error-free trials (22). Then, they were exposed to the overnight social isolation. However, overnight social isolation did not affect the spatial WM or RM performance in the HB task, nor did it affect latencies or durations in this task.
Our results are in line with a study by Arts et al. (14), in which no differences were found in pig HB performance following a social stressor, namely regrouping of pigs (mixing). Latency and duration measures can be taken to reflect pigs' motivation. The lack of change in performance after isolation thus indicates that the isolation did not affect pigs' motivation to perform the task or work for rewards (22,24). We may conclude that neither stress induced by overnight social isolation nor by mixing affected subsequent HB performance. It is also possible that the pigs did not find overnight social isolation stressful, as discussed below in relation to the cortisol measures.

Decision-Making Behavior in the PgT
The learning curve in the PGT based on the performance averaged over all pigs (see Figure 5A), suggests that the pigs had learned the task, albeit to a moderate performance level. The level reached is comparable to that reported by Murphy et al. (17) who studied decision-making behavior in LBW and NBW piglets. However, as depicted in Figure 5B, not all pigs learned the task and increased choices of the advantageous side in the present study. Thus, the learning curve calculated across all pigs is not a relevant representation of the pigs' behavior. Based on their choice behavior during the entire training period of 120 trials, only 7 of the 18 pigs were classified a posteriori as learners; they increased the number of advantageous choices during training. Five pigs showed no learning at all, i.e., they randomly chose the advantageous or disadvantageous side. This group was classified as non-learners. Four of the 18 pigs showed directional persistence and were classified as preferring the advantageous side and two pigs were classified as preferring the disadvantageous side, i.e., the latter two groups showed a strong side preference or side bias. Persistent side preference is difficult to break in pigs [e.g., Ref. (39,40)]. Side bias prevents learning about the contingencies that are in effect at the other side (goal box). Overnight social isolation did not affect the pigs' choice behavior in any of the four subgroups. As described in the Section "introduction", gambling tasks in animal research use a reduced number of "sets" to choose from compared to human gambling tasks. This modification makes the tasks easier for animals to learn but also reduces the number of choice alternatives. A small number of discriminable contingencies and consequences underlies the advantageous and disadvantageous choices (see Figure 6). Given the increased usage of gambling tasks in animal research and the variability we have observed in performance of the task, it is worthwhile to consider the potential interpretations of performance in these types of tasks.
It remains to be demonstrated which contingencies animals detect and which ones guide their choice behavior. Moreover, Anselme (41)  When responding in the gambling task, pigs may choose (I) the preferred side (side preference, side bias), (II) the side yielding the larger reward (more M&M's ® ), (III) the side that yields reward with the highest probability, (IV) the side that yields the fewest punishments, i.e., impedes consumption of the M&M's ® in the central food bowl, or, finally, (V) pigs may choose the side that, within series of 10 trials, yields the largest number of M&M's ® (see Figure 6). It has not been established whether pigs are able to assess the probabilities of earning maximum number of reward/minimum number of punishment in the long run. It is conceivable, and perhaps more likely, that they simply choose the contingency that provides reward in the majority of trials, where the number of chocolate M&M's ® is not relevant. Based on the set of contingencies in effect in the present study, it is not possible to distinguish between probabilities III, IV, and V.
In an earlier study, we found that pigs needed a large number of trials to acquire successive and conditional learning tasks (42). In the present study, only seven pigs were able to detect the contingencies that yielded the maximum reward. It remains to be determined whether this modification of the task is suited for testing pigs, and how many trials are needed to train the pigs to consistently make advantageous choices.
One could argue that the lack of effect of social isolation on behavior in the two experiments is due to habituation to the isolation procedure. This explanation, however, is unlikely. First, even the very first isolation during the HB task did not affect cognitive performance in the session following isolation. Second, the pigs did not experience any social isolation in the period of approximately 1½ months between the HB task (Experiment 1) and PGT (Experiment 2). Because we expected to find robust effects of social isolation stress on performance in subsequent behavioral tests, and to keep the number of animals used to a minimum, we decided to use the same animals in both experiments.

Age-Associated Decrease of Baseline Cortisol
Baseline salivary cortisol measures at 19 weeks of age were considerably lower than at 14 weeks of age. This observation corroborates earlier findings by de Jong et al. (43) who reported a steep drop of salivary cortisol in pigs from the age of 15-22 weeks. However, Hillmann et al. (44) reported that the cortisol level increased with age and body mass of pigs.

Circadian Fluctuation of Cortisol
Cortisol levels show a circadian rhythm. A general finding is that cortisol levels are lower during the night than during daytime (43,45,46). We expected, based on these publications, that the baseline cortisol levels at 9:00 and 15:15 should be similar. According to Hillmann et al. (44), the circadian pattern of cortisol is more pronounced with increasing age in pigs.
Provided that cortisol levels of a treatment are compared with a pre-treatment baseline, salivary cortisol can provide a good indication of the HPA response to a stressor (35). As sampling of saliva is considered stress-free (47), it is unlikely that this procedure affected the stress state of the pigs (48), all the more because the pigs had been thoroughly trained to chew voluntarily on the cotton swabs.

Effects of Social Isolation on Salivary Cortisol Levels
In the present study, the cortisol level during isolation(s) was lower than that of the baseline measurements. Considering that the baseline measurements in the first and second experiment were separated by a 10-week time period (see Table 1; Figure 3), in which the baseline levels dropped considerably [corroborating the results of de Jong et al. (43), but not of Hillmann et al. (44)], one could hypothesize that the effects of isolation simply reflect the effects of aging. However, the period between baseline and isolation measurements was only 1 week for the first isolation in Experiment 1, and for the isolation in Experiment 2. An additional statistical comparison between baseline and first isolation in Experiment 1 (data not shown) makes this hypothesis unlikely. Therefore, we assume that the drop in cortisol levels in isolation sessions does not merely reflect effects of an age-related drop in cortisol.
The decrease in cortisol that we observed following exposure to social isolation in both tests was an unexpected finding. We assume that social isolation is a stressor, and cortisol levels generally increase following acute exposure to a stressor. Certainly our first measures, 15 min after being placed in social isolation, would be expected to reflect an acute response to the situation. Interestingly, three separate studies have shown that individually housed gilts had decreased salivary cortisol levels compared to gilts housed in groups (49)(50)(51), and that group-housed gilts showed a decrease in cortisol after transfer to individual housing in farrowing crates (50). Based on these and the present study, group housing with all of the social interactions that it entails may provide more stress than social isolation as operationalized in the present study.
The decrease in cortisol observed in the animals following social isolation may be related to the housing as it was operationalized in the present study. One hypothesis, as put forward by Geverink et al. (49), is that the decrease in cortisol may be an effect of decreased activity levels in individually housed animals, as activity and exercise are known to increase cortisol levels. The animals were not restrained during social isolation, but had much less space than during group housing and were not stimulated by conspecifics to move around in the pen. Furthermore, the isolation pens were situated next to each other. The pigs in these pens had ad libitum access to water and food, and could root in the straw that covered the floor.
All pigs could hear and smell their isolated neighbors. Notably, "social support might not require all senses but rather rely on a few senses that are important to that particular species" [(52), p. 7]. Social support can dampen stress reactivity (53), and can help animals to cope with stressful events (52). Social support most likely occurs in stable social groups like the ones in the present study. Familiarity and a social bond are considered as minimum requirements for social support to occur (52). Both the provider and the receiver of social support may benefit, i.e., stress reactivity in the isolation condition may have been dampened in all pigs, albeit through different mechanisms (53). Low and high stress levels seem to impair cognitive performance, whereas intermediate stress levels may even facilitate learning and memory (54). In the HB task, the pigs had already reached a high level of performance before they were subjected to social isolation. Consequently, it is unlikely that the (mild) stress that they experienced during social isolation improved performance post isolation. In the PGT, comparison of the subgroups, and separate analysis of the "learners" did not reveal any differential effect of social isolation. If the training itself is not very stressful, or if stress is experienced long after training, its consequences on cognitive performance are less predictable (55). In order to keep the behavioral training itself stress free, we extensively habituated the pigs to all aspects of the testing procedures and testing environment.
Our training and testing procedure itself may be considered as "cognitive enrichment" [see also in Ref. (29)]. This type of enrichment seems to be able to reduce excitement and fear in pigs (56) and may thus be able to dampen stress responses. For example, Zebunke et al. (57) trained pigs to approach a "call feeding station" approximately 30 times a day and to operate a button on a fixed ratio schedule to earn a portion of feed. They concluded that the cognitive enrichment can reduce stress, as measured by heart rate and heart rate variability. In line with this study, Siegford et al. (58) reported that training in a spatial maze task reduced the stress response of 12-day-old piglets and reduced their fear response, measured at 7 weeks of age, i.e., that maze training may reduce fear of novel persons and ameliorate cognitive deficits. Their study suggests that the exposure of young male piglets to environments that requiring spatial learning is beneficial, an effect not found in females. The enrichment effects of training and testing per se may thus interfere with the aim of the present study to demonstrate an adverse effect of the stress induced by overnight social isolation. cOnclUsiOn Overnight social isolation did not affect pigs' behavior in a HB task and had no effect on pigs' decision-making in the PGT, in line with observations by Düpjan et al. (9) and Murphy et al. (59). However, the average cortisol level during isolation was lower than the average cortisol level during baseline, indicating that the isolation affected physiological but not behavioral measures. During overnight social isolation, the pigs could still smell and hear their penmates. They had straw as bedding and could move around. These conditions might have reduced the averseness of the overnight social isolation to a level that effects on performance during behavioral testing were not detectable, whereas the salivary cortisol even decreased during social isolation.

aUThOr cOnTriBUTiOns
FS and RN designed the study and wrote the final version of the manuscript. AS and BS performed the study, collected the data, and wrote the two BSc theses on which this manuscript is based. FS performed the data analysis. All authors approved the final manuscript.

acKnOWleDgMenTs
We would like to thank Jan van Mourik, Zias Lukasse, and Dirk van der Heide for taking care of our animals, and Elly Zeinstra and Christine Oei for their guidance and advice in the laboratory and their help with measuring salivary cortisol.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Engineering

text:
Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli

Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW) rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

Introduction
Bacterium Escherichia coli (E. coli) remains a predominant host for the expression of heterologous proteins. Like other organisms, E. coli uses 61 available amino acid codons for mRNA production. However, not all 61 mRNA codons are used equally [1,2]. The so-called 'major' codons occur in highly expressed genes, whereas 'rare' codons are present in low expressing genes [1,2,3]. The frequency of the codon usage in mRNAs also reflects the abundance of their cognate tRNAs in the cells. When the codon usage of the overexpressed heterologous protein differs considerably from the normal codon usage of the expression host, protein synthesis can be inhibited due to the depletion of rare tRNAs cellular pool [4]. Viral proteins are encoded by genes that contain codons rarely used by E. coli. For instance, genes of HIV-1 proteins contain 8.21% (in gene encoding Nef protein) up to 23.17% (in gene encoding Vpu protein) codons that are rarely used by E. coli ( Table 1). These genes express poorly in E. coli and as a result, little and/or poor quality protein is produced [4,5]. To alleviate codon bias-associated problems, one option is to optimize the gene sequence by changing rare codons into more frequently used codons [6]. Alternatively, specialized E. coli strains such as BL21-CodonPlus (Stratagene) and Rosetta2(DE3) (EMD Millipore) can be used. These strains harbor ColE1-compatible, rare tRNA expressing helper plasmids, which are maintained under chloramphenicol selective pressure [7].
High level expression of numerous heterologous proteins has been achieved by using either of the two above mentioned strategies. However, there are some issues associated with these approaches. 1. Codon optimization via gene synthesis can be costly and time-consuming especially for genes longer than 500bp. Moreover, codon changes can affect secondary structure of mRNA with unknown consequences [8,9]. 2. Maintenance of tRNA-expressing helper plasmids together with expression vectors results in additional metabolic pressure because the bacteria constitutively express two antibiotic resistance genes [10]. 3. It complicates expression strategies where multiple vectors are employed for co-expression of protein subunits [11]. 4. Certain engineered strains such as those containing pLysS (to reduce background expression levels) cannot be transformed with rare tRNA vectors that contain p15A ori and constitutively express chloramphenicol acetyltransferase gene for selection [12].
To address abovementioned limitations, we engineered an expression vector that would express both the heterologous protein of interest, and rare tRNA genes in E. coli. We started off with cloning HIV-1 nef gene in an expression vector pSA-HP24-6His, which we have previously used for high level expression of HIV-1 p24 [13]. We expressed HIV-1 Nef because it has gained increased interest as a new therapeutic target for HIV/AIDS treatment in recent years [14,15,16,17,18,19] and we are engineering cell internalizing antibodies to target this pathogenic factor. We then modified the backbone of the resulting pSA-HNef-6His vector by replacing a non-essential DNA segment between lacI gene and T7 promoter with rare tRNA genes argU, ileY, and leuW. We call this vector pSA-HNef-6His-RIL. In order to further validate the utility of this rare tRNA gene vector, we replaced nef with HIV-1 p24 and vif genes and the resulting plasmids were designated as pSA-HP24-6His-RIL and pSA-HVif-6His-RIL respectively, which were then used to express P24 and Vif proteins. To facilitate clone manipulation, we substituted the nef gene with a multiple cloning site (MCS) and the resulting plasmids called pSA-C6His-RIL.

Materials and Methods
Bacterial culture conditions

Construction of pSA-C6His-RIL
Vector pSA-C6His-RIL was constructed by replacing nef gene with a multiple cloning site (MCS). The MCS introducing BglII, NheI, EcoRI, NruI, SacII, SalI, and NotI restriction sites was constructed by annealing two oligonucleotides, MCS-F and MCS-R ( Table 3). The oligonucleotides anneal to create NdeI and SacI cohesive ends for ligation into a pSA-6His-RIL vector restricted with 5 0 NdeI and 3 0 SacI sites, maintaining the correct frame for the C-terminal 6His tag and stop codon. The MCS-F and MCS-R oligonucleotides were resuspended in annealing buffer (10mM Tris, pH 7.5-8.0, 50mM NaCl, 1 mM EDTA) and mixed in equimolar concentrations (200μM each) in 50μl reaction. The annealing reaction was incubated for 5 min in a hot block at 90°C. The block was removed from the heat source and the reaction was left to gradually cool to room temperature for 45 min and stored at -20°C. The pSA-HNef-6His-RIL vector (5μg) was restricted with NdeI/ SacI at 37°C for 4h and 5.837kb vector backbone (pSA-6His-RIL) was purified from 1% agarose gel. Annealed oligonucleotides were diluted 1:10 with nuclease-free water and ligated with NdeI/ SacI-restricted vector in a 4:3 molar ratio. Ligation reaction was transformed into DH5α E. coli cells, and selected on ampicillin-containing LB-agar plates after 18h incubation at 30°C. Randomly selected bacterial colonies were used to prepare plasmid minipreps, which were subjected to DNA restriction and sequence analyses.

Expression of proteins in NiCo21(DE3) transformed with pACYC-RIL, pRARE2, and pLysSRARE2
NiCo21(DE3) bacteria were individually transformed with pACYC-RIL, pRARE2, and pLysS-RARE2 vectors and the transformants selected on chloramphenicol-containing LB agar plates. Transformants were grown and competent cells were prepared according to the method of Inoue [20]. The pACYC-RIL, pRARE2, and pLysSRARE2-containing NiCo21(DE3) were then transformed with pSA-HNef/P24/Vif-6His vectors and the transformants selected on LB agar plates containing both ampicillin and chloramphenicol. Cultures were expanded and the expression protocol given above was essentially followed.

Bacterial cell lysis and protein extraction
To every gram of bacterial cell pellet, 4ml of B-PER extraction reagent (Thermo Scientific, #78248) supplemented with DNAse I (Thermo Scientific, #90083) and protease cocktail (Thermo Scientific, #87785) was added. The suspension was incubated at 22°C for 60min with gentle shaking. Soluble and insoluble proteins were partitioned by centrifuging bacterial cell lysate at 15,000xg for 10 min at 4°C. Clear supernatant containing soluble proteins was passed through a 0.45μm membrane (Millipore, #HPWP04700) and used for immobilized-metal affinity chromatography (IMAC). Insoluble lysed bacterial biomass was resuspended to its original volume with Tris-buffered saline (TBS). Both, soluble and insoluble fractions were stored in small aliquots at -80°C for further analysis.

Purification of recombinant proteins
Recombinant HIV-1 Nef protein was purified in two steps. First, the soluble protein fraction was pre-adsorbed onto chitin resin (NEB, #S6651) to capture bacterial Histidine-rich proteins. An appropriate amount of Chitin beads was added into Econo-Pac Chromatography Column (BioRad, #732-1010) and equilibrated in two resin-bed volumes of Equilibration/Wash Buffer (50mM sodium phosphate, 500mM sodium chloride and 10mM imidazole, pH 7.4). Soluble protein fraction was mixed with an equal volume of Equilibration/ Wash Buffer and added to the equilibrated chitin beads (1 ml of chitin resin for each volume of lysate corresponding to 1 gram of NiCo21(DE3) cell pellet). The column was then placed on an end-over-end rotator and revolved for 30min at 4°C. Void volume containing target protein was eluted by gravity flow and used for IMAC purification step. For IMAC, HisPur Cobalt resin (Thermo Scientific, #89965) was used following the manufacturer's protocol. Briefly, an appropriate amount of cobalt resin was added in a 15ml centrifuge tube and washed with two resin-bed volumes of Equilibration/ Wash Buffer. The chitin bead pre-adsorbed soluble protein fraction was combined with equilibrated cobalt resin and mixed on an end-over-end rotator for 60min at 4°C. The resin was then washed with Equilibration/Wash Buffer until the absorbance at 280nm reached to the baseline. Bound protein was eluted using one resin-bed volume of Elution Buffer (50mM sodium phosphate, 500mM sodium chloride, 150mM imidazole, pH 7.4). This step was repeated 2-3 times while saving individual fractions. Fractions were analyzed by SDS-PAGE/Western blot, dialyzed against phosphate buffered saline (PBS) using Slide-A-Lyzer Dialysis Cassettes, 7K MWCO (Thermo Scientific, #66710) and stored at -80°C in small aliquots.

Results
Construction of a plasmid expressing HIV-1 Nef pSA-HNef-6His vector (Fig 1) used to produce HIV-1 Nef protein was constructed by modifying the pSA-Hp24-6His vector [13]. The HIV-1 p24 coding sequence was removed by restricting the plasmid with NdeI and SacI, and replaced with HIV-1 nef coding sequence at the same sites. The resulting 6.517kb vector was verified by DNA restriction and sequence analyses (data not shown). The mRNA sequence encoding the nef gene is transcribed from an inducible T7 promoter and the resulting protein has a C-terminal 6His tag for IMAC-mediated purification.
Expression of Nef in pSA-HNef-6His-transformed NiCo21(DE3) E. coli E. coli NiCo21(DE3) was used to express 6His-tagged HIV-1 Nef using shaker flask culture conditions for expression. Plasmid-borne retroviral sequences are unstable in E. coli when grown at 37°C [22]. Therefore, the cultures of pSA-HNef-6His-transformed NiCo21(DE3) were grown at 30°C until the OD 600 reached 0.5-0.6. Cultures were then induced with 0.4mM IPTG and incubated for another 6h at 30°C. When subjected to SDS-PAGE analysis, the expressed Nef protein appeared as an approximately 27kDa band in the IPTG-induced fractions (Fig 2A). Overexpressed Nef protein was mainly present in the soluble fraction. However, immunoblot analysis with the anti-Nef antibody revealed that some Nef was also present in the insoluble fraction (Fig 2B).

Optimization of IPTG concentration and induction temperature
In order to optimize Nef expression, we tested ranges of IPTG concentrations (0-0.4mM) and incubation temperatures (30, 22, and 18°C). Expression of HIV-1 Nef was optimal at the lowest tested concentration of IPTG i.e. 0.05mM as determined by the western blot analysis (Fig 3A). HIV-1 Nef production was similar in 0.05mM IPTG-induced cultures when grown at 30°C and 22°C, but lower in 18°C-grown cultures (Fig 3B). However, neither IPTG concentration nor various incubation temperatures enhanced overall Nef production in E. coli NiCo21(DE3) cells. Effect of rare tRNA supplementation on Nef expression in NiCo21(DE3) E. coli Over-expression of heterologous proteins in E. coli may be considerably circumscribed by the presence of "rare" codons in the foreign mRNA that are seldom used by E. coli [23,24]. When subjected to 'rare' codon analysis, the 618bp coding sequence for HIV-1 Nef was found to contain (a) three rare codons (AGG, AGA, CGA) for arginine at positions 17,19,21,22,35,77,105,106,134,178,184,194, and (b) one rare codon (CTA) for leucine at positions 37, 58, 100, 145, and 189 ( Fig 4A). To investigate whether Nef expression could be improved by expressing nef together with rare tRNA genes, NiCo21(DE3) were transformed with rare tRNA expressing pACYC-RIL, pRARE2, and pLysSRARE2 helper plasmids. The pACYC-RIL plasmid supplies tRNA for four rare codons (AUA, AGG, AGA, CUA), whereas pRARE2 supplies those for seven rare codons (AUA, AGG, AGA, CUA, CCC, CGG, and GGA). In addition to seven rare codons, transformation with pLysSRARE2 also results in lower background expression due to the expression of T7 lysozyme. When expressed in the presence of rare tRNA supplying helper plasmids, Nef expression strikingly improved as shown in Fig 4B. However, this also lead some Nef protein to end up in the insoluble fractions, suggesting that bacterial protein folding machinery was saturated by the enhanced expression. There was no significant change in Nef expression between the NiCo21(DE3) containing pACYC-RIL and pRARE2/ pLysSRARE2, suggesting that supplementation with four rare codons was sufficient to optimize Nef expression. In a side-by-side comparison, NiCo21(DE3)/pACYC-RIL showed relatively higher production of soluble Nef (Fig 4B).
In attempts to shift the relative soluble/insoluble protein production, protein expression was tested at 18°C, but it did not alter the distribution of recombinant protein between soluble and insoluble fractions, nor did the production of soluble Nef improve further (data not shown). Another modification was tested by the addition of 2-3% ethanol to the culture medium [25,26] and incubation at 42°C [27]. These conditions lead to overexpression of bacterial chaperones, which may result in improved folding and enhanced solubility of overexpressed recombinant proteins. However these approaches failed to generate more soluble Nef with our production system (data not shown). Expression of Nef protein in the presence or absence of rare tRNA genes had minimal effect on the bacterial growth as determined by the total biomass yield (~12 g/L and 12.5 g/L for NiCo21(DE3) transformed with pSA-HNef-6His, and pSA-HNef-6His + pACYC-RIL respectively, when grown in LB broth.

Construction of pSA-HNef-6His-RIL for concomitant expression of Nef and rare tRNA genes
In order to alleviate issues associated with coexistence of tRNA helper plasmids with expression plasmids, we modified pSA-HNef-6His to express rare tRNA genes in concomitant with nef gene. The modified pSA-HNef-6His-RIL plasmid was constructed by exchanging 936bp nonessential DNA fragment between the lacI gene and T7 promoter with 874bp DNA fragment containing argU, ileY, and leuW rare tRNA genes. Blunt-end ligation of rare tRNA gene fragment into the vector resulted in a plasmid with tRNA gene array arranged in either clockwise (CW) or counter-clockwise (CCW) orientation relative to the T7 promoter (Fig 5A and 5B).

Expression of Nef in NiCo21(DE3) transformed with pSA-HNef-6His-RIL
We expressed Nef in E. coli transformed with pSA-HNef-6His-RIL(CW)/(CCW) and compared Nef production with E. coli transformed with pSA-HNef-6His (served as negative control), and pSA-HNef-6His together with pACYC-RIL (served as positive control). Expression experiments were carried out using shaker flask culture conditions for expression as described above. When subjected to SDS-PAGE analysis, Nef expression in bacteria containing pSA-H-Nef-6His-RIL was comparable with those that contained both pSA-HNef-6His and pACY-C-RIL. Little Nef produced in bacteria that harbored pSA-HNef-6His vector (Fig 6A). There was noticeable background expression in un-induced cultures from pSA-HNef-6His-RIL (CW) compared to pSA-HNef-6His-RIL (CCW) (green arrow, Fig 6A and 6B). This suggests that in addition to T7 promoter, Nef was also expressed under the control of some promoter upstream of T7 promoter, most likely of ileY (Fig 5B). As a result, the pSA-HNef-6His-RIL (CCW) was not used in subsequent expression experiments. Samples from pSA-HNef-6His+-pACYC-RIL showed a prominent 25kDa band present in both un-induced, and IPTG-induced cultures (red arrows, Fig 6A). This band most probably represents chloramphenicol acetyltransferase monomer expressed from pACYC-RIL vector. Proteins were also subjected to immunoblot analysis (Fig 6B) using anti-Nef MAb as primary antibody and relative band intensities were determined and plotted as shown in Fig 6C. Together, these experiments show that recombinant protein can be produced from a single vector concomitantly expressing rare tRNA and a heterologous gene, and in amounts comparable (or better) to traditional two-vector system.

Construction of pSA-HP24-6His-RIL for concomitant expression of P24/ CA and rare tRNA genes
In order to further confirm the hypothesis that recombinant protein(s) can be produced in similar or better amounts from a single vector concomitantly expressing rare tRNA and a heterologous gene compared to traditional two-vector system, we expressed HIV-1 p24 gene.  Rare tRNA Gene-Containing E. coli Expression Vector replaced the nef gene in pSA-HNef-6His-RIL with p24 and compared the expression profile of P24/CA from pSA-HP24-6His, pSA-HP24-6His together with pACYC-RIL, and pSA-HP24-6His-RIL (Fig 7). The helper plasmids pRARE2/pRARE2 lysS were omitted because in the previous work they were found not to offer any advantage over pACYC-RIL helper plasmid [13]. As anticipated, almost 2 fold more P24/CA was expressed in cultures containing pSA-HP24-6His+pACYC-RIL and pSA-HP24-6His-RIL. The SDS-PAGE (Fig 8A), Western Blot ( Fig  8B), and densitometric ( Fig 8C) analyses showed that relatively more p24/CA was produced from the single plasmid (pSA-HP24-6His-RIL) compared to traditional two plasmid (pSA-HP24-6His+pACYC-RIL) system. Expression of Nef from pSA-HNef-6His vectors with or without rare tRNA genes array. To evaluate the effect of the introduction of rare tRNA genes array in pSA-HNef-6His vector, the NiCo21 E. coli were individually transformed with pSA-HNef-6His, pSA-HNef-6His-RIL(CW), and pSA-HNef-6His-RIL(CCW) and Nef expression was compared with NiCo21 E. coli co-transformed with pSA-HNef-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HNef-6His or pSA-HNef-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HNef-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (OD 600~0 .5-0.6), at which point IPTG was added to a final concentration of 0.05 mM. The induced cells were grown for another 12 h at 22°C and stored on ice. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 15% SDS-PAGE gel and analyzed by (A) Coomassie staining, (B) immunoblotting and (C) band densitometry. Nef expression in NiCo21 transformed with pSA-HNef-6His-RIL (CW/CCW) was comparable with NiCo21 cotransformed with pSA-HNef-6His and pACYC-RIL. A prominent band running at 25 kDa (red arrows) appears in NiCo21 harboring pSA-HNef-6His/ pACYC-RIL. This is most probably chloramphenicol acetyltransferase monomer. Higher baseline expression for un-induced cultures were noted in NiCo21 harboring pSA-HNef-6His-RIL(CCW) (see green arrows). This suggests that ileY tRNA gene most probably doesn't contain a transcription terminator downstream and the baseline expression of Nef is a result of transcriptional read-through of mRNA synthesis from ileY promoter. Expression of Vif was compared between the cultures that contained pSA-HVif-6His, pSA-HVif-6His together with pACYC-RIL, and pSA-HVif-6His-RIL (Fig 7). As shown in Fig  9A, 9B and 9C, around two fold more Vif was produced in cultures containing pSA-HVif-6His+pACYC-RIL and pSA-HVif-6His-RIL compared to pSA-HVif-6His. Consistent with previous observations for Nef and P24, relatively more Vif was produced from the vector that expresses both the heterologous gene and rare tRNA genes. However, unlike Nef and P24, Vif expressed as insoluble protein and optimizing the IPTG concentration and temperature did not yield any soluble Vif (data not shown).
Growth profile, expression, and purification of HIV-1 Nef and P24 in NiCo21(DE3) transformed with pSA-HNef-6His-RIL and pSA-HVif-6His RIL By expressing three rare codon -contain HIV-1 genes i.e. nef, p24, and vif, we showed the suitability of our proposed vector that concomitantly expresses the gene of interest and rare tRNA genes. Here we compared the growth profile, expression, and purification HIV-1 Nef and P24 between traditional two-plasmid system with that of single expression plasmid system. Because HIV-1 Vif failed to express as soluble protein, we have omitted it from this set of experiments.
Bacterial cultures were grown using optimized conditions i.e. super broth (SB) as culture medium, 0.05mM IPTG as inducer, and cultivation at 22°C for 12h. Cells were harvested and optical densities at 600nm and total biomass yield were determined. Lysed bacterial pellets were used to purify Nef and P24/CA as described in Materials and Methods. Growth curves were similar between the cultures expressing Nef or P24/CA from pSA-HNef/P24-6His-RIL and pSA-HNef/P24-6His/pACYC-RIL. Cultures expressing Nef or P24 from pSA-HNef/P24-6His exhibited a lag in growth (Fig 10A and 10C) suggestive of Nef and P24 toxicity towards bacteria when overexpressed in the absence of rare tRNAs. Highly purified Nef and P24/CA were obtained from cultures expressing nef and p24 in presence of rare tRNAs (Fig 10B and  10D). Using the optimized conditions, we obtained~5-5.5 mg highly pure (up to 84%) recombinant HV-1 Nef and P24/CA protein from each 1g biomass of pSA-HNef/P24-6His-RIL or pSA-HNef/P24-6His + pACYC-RIL -transformed NiCo21(DE3) bacteria when propagated in To evaluate the effect of the introduction of rare tRNA genes array in pSA-HP24-6His vector, the NiCo21(DE3) E. coli were individually transformed with pSA-HP24-6His, and pSA-HP24-6His-RIL and P24 expression was compared with NiCo21(DE3) E.coli co-transformed with pSA-Hp24-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HP24-6His or pSA-HP24-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HP24-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (A600~0.5-0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C and stored on ice. Three microliters of samples were mixed with 4X loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining (A), immuno-blotting (B), and band densitometery (C). The P24 expression in NiCo21 transformed with pSA-HP24-6His-RIL was comparable with NiCo21 co-transformed with pSA-HP24-6His and pACYC-RIL.

Discussion
We have engineered an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. We started off with cloning HIV-1 nef gene in an expression vector pSA-HP24-6His, which has previously been used for high level expression of HIV-1 P24 [13]. Nef is an accessory protein of HIV that is responsible for a number of viral pathogenic effects. Progression to AIDS is delayed and in some well-documented cases abolished on infection with naturally occurring HIV strains that contain truncated or mutated nef sequences in their genomes [28,29]. It has been proposed that specific Nef inhibitors could potentially abrogate or slow down gradual deterioration of immune system in chronically infected patients in absence of functional cure [14,15,16,17,18,19]. We are engineering To evaluate the effect of the introduction of rare tRNA genes array in pSA-HVif-6His vector, the NiCo21(DE3) E. coli were individually transformed with pSA-HVif-6His, and pSA-HVif-6His-RIL and Vif expression was compared with NiCo21(DE3) E.coli co-transformed with pSA-HVif-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HVif-6His or pSA-HVif-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HVif-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (A600~0.5-0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C and stored on ice. Three microliters of samples were mixed with 4X loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining (A), immuno-blotting (B), and band densitometery (C). The Vif expression in NiCo21(DE3) transformed with pSA-HVif-6His-RIL was comparable with NiCo21 co-transformed with pSA-HVif-6His and pACYC-RIL. therapeutic antibodies against HIV-1 Nef, and therefore need large amounts of highly purified recombinant protein. Production and purification of HIV-1 Nef and P24. NiCo21(DE3) transformed with pSA-HNef/P24-6His, pSA-HNef/P24-6His/pACYC-RIL, and pSA-HNef/P24-6His-RIL were grown overnight from a single colony at 30°C in super broth (SB) containing appropriate antibiotic(s). The cultures were then diluted to 0.05 OD 600 in the same medium and appropriate antibiotics(s). Cultures were grown (at 30°C) to mid-log phase (A600~0.5-0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C. A small aliquot (100μL) of culture was removed every hour, diluted 10 fold in the same medium and cell density was measured at OD 600 and plotted. A. Growth kinetics of cultures expressing HIV-1 Nef from various expression vectors/combinations. B. Comparison of Nef production/purification from cultures expressing Nef from pSA-HNef-6His/ pACYC-RIL with those expressing Nef from pSA-HNef-6His-RIL vector. C. Growth kinetics of cultures expressing HIV-1 P24 from various expression vectors/combinations. D. Comparison of P24 production/purification from cultures expressing P24 from pSA-HP24-6His/pACYC-RIL with those expressing P24 from pSA-HP24-6His-RIL vector. doi:10.1371/journal.pone.0130446.g010 Rare tRNA Gene-Containing E. coli Expression Vector Little Nef was produced when expressed in NiCo21(DE3) E. coli, and optimization of expression conditions had no effect on the final yield. We then analyzed nef gene for the presence of codons rarely used in E. coli, and found that nef contained 8.21% of such codons. We therefore anticipated that co-expression of rare tRNA genes from a helper plasmid could help with high level Nef expression. Indeed, almost 8-fold increase in expression was achieved when Nef was produced in the presence of a rare tRNA-expressing helper plasmid (pACYC-RIL). Nef expression is toxic towards expression hosts such as bacteria and yeast [30,31], though the exact mechanism of this toxicity is not known. We observed that Nef expression was toxic towards E. coli but only when it was expressed in the absence of rare tRNA genes. We anticipate that this could be due to the misincorporation or omission of one or several rare tRNA-coded arginine and/or lysines, which results in the production of mutant Nef protein(s) that is toxic towards the expression host. It is also possible that rare tRNAs get sequestered by ribosomes engaged in the translation of rare codon -containing heterologous gene that may affect the cell's ability to produce essential endogenous protein needed for optimal cell growth.
In order to construct a vector that could concomitantly express the nef and the rare tRNA genes, we modified the backbone of the resulting pSA-HNef-6His vector by replacing a nonessential DNA segment between lacI gene and T7 promoter with argU, ileY, and leuW tRNA genes. The resultant vector was called pSA-HNef-6His-RIL, which was then used to produce high levels of recombinant HIV-1 Nef protein. In addition to nef gene, we also cloned HIV-1 p24 and vif, two genes that contain 10.77 and 14.5% rare codons respectively. In side-by-side experiments we showed that the p24 and vif genes expressed from single pSA-P24/Vif-6His-RIL vector produced similar or better levels of recombinant proteins compared to traditional two vector system in which rare tRNA genes are expressed from a ColE1 -compatible helper plasmid such as pACYC-RIL.
It would be interesting to note that protein expression in presence of rare tRNA genes yielded two fold more P24 and Vif compared to eight fold more Nef, whereas the p27 (nef) gene contains fewer rare codons (8.25% of total codons) compared to p24 (10.77% of total codons) and vif (14.5%) genes. Moreover, HIV-1 p27(nef) gene contains rare codons for two amino acids arginine and leucine, whereas p24 and vif genes contain rare codons for four amino acids arginine, leucine, isoleucine, and proline. These observations suggest that neither total number of rare codons nor all the rare codons affect the heterologous protein expression in E. coli. Upon close examination we noticed four rare codons (the last two as tandem repeat) arranged in a cluster in the 5' of p27 (nef) gene encoding arginine (highlighted region in S1A Fig). There are another two rare codons for arginine also arranged as tandem repeat in the middle of the nef gene (highlighted region in S1A Fig). We did not notice similar clustering of arginine -encoding rare codons in either p24 or vif genes (S1B and S1C Fig). These observations are in agreement with previous findings that not all rare codons affect the heterologous protein expression and it is the priority among those rare codons (for instance those encode for arginine), their position in the gene, and tandem arrangement that affects the protein expression in E. coli [32].
To facilitate clone manipulation, we substituted nef gene with a multiple cloning site (MCS) clustered with eight unique restriction enzyme sites (Fig 11). Any two of the restriction enzyme sites can be chosen to clone gene of interest into the pSA-C6His-RIL vector. However it will be desirable to clone the gene of interest between NdeI and SacI, in order to avoid the addition of extra amino acids at N-and/or C-terminals of the resultant recombinant protein. Another possibility is to introduce desired restriction enzyme sites to the plasmid using whole-plasmid PCR. Several high fidelity polymerases suitable for whole-plasmid PCR are now available at reasonable cost. Whenever possible, we amplify the entire plasmid backbone with a set of primers containing desired restriction enzyme sites using Phusion or Q5 High-Fidelity DNA Polymerases. By using 50-100 ng plasmid vector as template, and using 18-25 PCR cycles, it is possible to obtain sufficient amount of PCR amplified plasmid, which can be used to clone the gene of interest with compatible restriction enzymes sites. To reduce the background, we add 10U of DpnI restriction enzymes and 1U of alkaline phosphatase in the reaction. This treatment effectively eliminates the bacterially-produced methylated-plasmid template, and dephosphorylates the ends of the restricted PCR amplified plasmid vector.
Eschenfeldt and co-workers have recently described the construction of LIC expression vectors containing rare tRNA genes [33]. These vectors contain tRNA genes covering rare codons for arginine (AGG/AGA) and isoleucine (AUA), and accept heterologous genes via LIC (ligation independent cloning). While these vectors are useful, they are limited in their utility due to the presence of only two rare tRNA genes. Furthermore, albeit of its advantages over restriction enzyme -based cloning, LIC is not a widely used method in various labs. The expression vector described in the present study contains three rare tRNA genes and therefore suitable for the expression of an assortment of heterologous genes in E. coli. We understand that cloning and expression of heterologous genes in the described rare tRNA-containing expression vector will save both time, and cost, and prove a useful addition to the existing tools/reagents available to scientific community.