Previous military studies have shown an energy deficit during a strenuous field training course (TC). This study aimed to determine the effects of energy bar supplementation on energy balance, physical activity (PA), physical performance and well-being and to evaluate ad libitum fluid intake during wintertime 8-day strenuous TC.−1. Energy (EI) and water intake was recorded. Fat-free mass and water loss were measured with deuterium dilution and elimination, respectively. The energy expenditure was calculated using the intake/balance method and energy availability as . PA was monitored using an accelerometer. Physical performance was measured and questionnaires of upper respiratory tract infections (URTI), hunger and mood state were recorded before, during and after TC.Twenty-six men (age 20±1 yr.) were randomly divided into two groups: The control group (n = 12) had traditional field rations and the experimental (Ebar) group (n = 14) field rations plus energy bars of 4.1 MJ•dayEbar had a higher EI and energy availability than the controls. However, decreases in body mass and fat mass were similar in both groups representing an energy deficit. No differences were observed between the groups in PA, water balance, URTI symptoms and changes in physical performance and fat-free mass. Ebar felt less hunger after TC than the controls and they had improved positive mood state during the latter part of TC while controls did not. Water deficit associated to higher PA. Furthermore, URTI symptoms and negative mood state associated negatively with energy availability and PA.An easy-to-use protein-rich energy bars did not prevent energy deficit nor influence PA during an 8-day TC. The high content of protein in the bars might have induced satiation decreasing energy intake from field rations. PA and energy intake seems to be primarily affected by other factors than energy supplementation such as mood state. Ready-to-eat meal rations are commonly used during the military field training Energy intake (EI) itself has been found to have an influence on voluntary physical activity, increasing with high-energy intake −1 on energy balance, physical activity, body composition, physical performance and subjective experiences of hunger, URTI, mood state and overall well-being during the 8-day military training course (TC) in a cold environment. In the present study, the aim was not to cause energy restriction and neither force the conscripts to eat the all provided food. Instead of that the aim was to observe the effect of military training in terms of self-selected diet. An additional purpose was to define whether an ad libitum fluid intake is sufficient during winter training. We hypothesized that energy supplement of 4.1 MJ·d−1 may increase the total energy intake, improve ability to maintain physical training load and sustain FFM during TC. Furthermore, we hypothesized that an energy supplement will sustain physical performance, enhance mood state and overall well-being and decrease symptoms of URTI.There are few published energy supplements studies during military field training in purpose to assure energy balance All participants were fully informed of the experimental protocol and gave their written consent to participate in the study. They were also advised of their right to withdraw from the investigation at any time. The study protocol was approved by the Finnish Defence Forces, and the Ethical Committees of the University of Jyväskylä and the Kainuu region of Finland.−1. The two groups did not differ according to the baseline characteristics (Twenty-six men (aged 20±1 yr.) volunteered to participate in the present study during their 8-day field training course (TC). The participants were randomly divided into two groups: the control group (n = 12) were allowed to eat only traditional field rations and the experimental (Ebar) group (n = 14) field rations plus energy bars of 4.1 MJ•dayeristics .−1 during the 8-day TC. Water intake was ad-libitum. However, participants were not permitted to use any other extra nutritional supplements throughout the study. Physical performance was studied before , during (MID) and after  TC. The measurements included a 3 km combat loaded field running, vertical jump, anaerobic power and handgrip strength. The participants have already done intensive military training for six months and were familiar with the stressors during TC. In addition, they were familiarized with the performance tests, and the jump and hand-grip strength tests were performed twice before (-6 PRE and PRE) the training course for minimizing the changes due to learning. In addition, mood state, body composition and physical activity were studied together with energy and water intake recordings. The experimental protocol is presented in The study took place during the winter in Finland, when daily outdoor temperatures ranged from −16°C to −1°C, with an average of −4°C . During the study, the participants completed 8-days field training, which consisted of a variety of military-relevant tasks and skills training, sustained aerobic exercise from skiing and patrolling activities. During TC, participants carried combat gear weighing 60–70 kg including their clothes and food rations. The participants were randomly divided into two groups: a control group (N  = 12) and an energy supplement group  of which the latter received an additional energy supplement of 4.1 MJ•day2H2O. Participants consumed no foods or fluids for 10 hours after dose administration, during the overnight equilibration of the isotope with the body water. Subsequent urine samples were collected from the second and third voiding in the morning of day 1. On study days 9 and 10 the process was repeated. TBW was calculated as the 2H dilution space divided by 1.04, correcting for exchange of the 2H label with nonaqueous H+ of body solids .Body mass (BM) was measured with an accuracy of 0.01 kg . The measurements were performed between 6∶00 and 7∶00 a.m. after an overnight fast and after voiding with no exercise for 12 hours prior to the test commencing, except the measurements in MID of TC. The participants were barefoot and they wore T-shirts and shorts. The BMmean  was used for calculation energy intake variables per BM. Body height was measured to the nearest 0.5 cm using a wall-mounted stadiometer. Fat-free mass (FFM) was calculated from total body water (TBW) as follows: FFM  =  TBW/0.732 −1 with the average macronutrient composition in energy of 14 E% protein, 58 E% carbohydrate and 28 E% fat. The participants in the Ebar group were advised to eat five commercial energy bars per day totaling 4.1 MJ·d−1 extra energy. The energy bars weighed 55 g/bar with the average macronutrient composition in energy of 32 E% protein, 46 E% carbohydrate and 22 E% fat. The total provided energy for the Ebar group was 22.0 MJ·d−1 . For studying energy intake (EI) and water intake, the participants kept daily pre-filled ration diaries. The pre-filled food diary included information about the ration served, and thus the participants were only required to record the amount of food and fluid that they consumed. The participants had completed these diaries before the study to ensure that they were familiar with reporting food and water intake. In addition, they were advised to be as accurate as possible in recording the amount food and fluid consumed. Furthermore, all the food wraps and rations that were not consumed were collected. Daily nutritional consumption was quantified from manufacturer-supplied ingredient labels and by the computer programme Nutrica®software  using the information from ration diaries, wraps and returned rations.The average daily energy from a field ration was 17.9 MJ·dTotal water intake was calculated from reported food and water intakes and metabolic water as follows: Total water intake  =  fluid intake + water content of food + metabolic water. The amount of metabolic water was estimated from protein, fat and carbohydrate intake and from the change in BM. Oxidation water is 0.41 mL•g−1 for protein, 1.07 mL•g−1 for fat and 0.6 mL•g−1 for carbohydrate Water loss over TC was measured using the deuterium elimination method Water balance was calculated as follows: Water balance  =  total water intake – water loss.−1, respectively Total energy expenditure (EE) was calculated by a method of intake/balance using EI and changes in body energy stores (ΔES); EE  =  EI – ΔES Energy balance  =  EI – EE. To differentiate the need of energy intake for subjects with a different body mass, energy availability was calculated as follows: EI/BMR, where BMR  =  basal metabolic rate estimated by the equation of Schofield et al. Energy balance was calculated as follows: −2Physical activity (PA) was measured using a customized version of the Polar AW200 Activity monitor that was worn on the non-dominant wrist. AW200 has been found useful and accurate for the measurement of EE during long term exercise The vertical jump test, anaerobic power test and handgrip strength were performed -6d PRE, PRE, MID and POST the TC. Prior to these tests each participant was required to complete a standardized warm-up of 5-min. After warm-up, the participant performed either hand-grip or jump test with 5 min rest between the tests. The follow-up tests for each participant were always performed in the same order and at the same time of day. A standardized warm-up of 1 km walking for 3-km running test started immediately after completion the hand-grip and jump tests in PRE and POST having consumed a similar diet before each testing session.Leg explosive power was assessed with a static vertical jump test using a contact mat . Jump test has been found a sensitive indicator for detecting decrements in physical performance after short anaerobic power was assessed during a 15-s all-out counter movement jump test on the contact mat. Power was calculated as follows:−1)  =  ((g2) x (Tf x 15))/(4n x ((15- Tf)), where g  =  acceleration of the gravity (9.81 m·s−2), Tf  =  total flight time, n  =  number of the jumps performed in 15 s Power . Three maximum isometric efforts with one minute rest was performed and maintained for about 5 s with the right and left hand with shoulder adducted and neutrally rotated, elbow flexed to 90 degrees and forearm in a neutral position. No other body movement was allowed. The handle of the dynamometer was individually adjusted for each participant. The participants were strongly encouraged to perform with maximal effort. The hand-grip results are presented as the mean of the right and left hand maximum.The 3-km running test with maximal effort was performed on an outdoor track PRE and POST TC while carrying a 20 kg backpack. All participants were instructed to complete the test in the shortest possible time. Heart rate (HR) was recorded continuously using hear rate monitors . Blood lactate was determined before and 1 minute after completion of the exercise from a fingertip blood sample using a lactate analyzer .The participants rated how they experienced their feelings utilizing five different aspects: 1) hunger on an eight-point Likert scale, 2) physical performance on a seven-point Likert scale and 3) positive and negative mood states on a four-point Likert scale, which included six positive and nine negative items, and the means of both items were calculated Post hoc analysis was used to identify any significant differences. In addition, the effect of group and TC interaction was calculated, and 95% confidence intervals (CI) were determined. If 95% confidence interval included zero, it was concluded that there was no significant effect. Pearson product–moment correlations were used to observe associations between variables. All statistical analyses were performed with PASW Statistics software .All data is presented as mean ± SD. The level of statistical significance was set at p<0.05. If assumptions for normality were not met, data were log-transformed before statistical analysis. The untransformed values are shown in the text, tables and figures for giving more meaningful values. Responses to TC, and differences between and within the controls and Ebar participants, were assessed using the mixed-design factorial ANOVA [group (control vs. Ebar) x TC . Bonferroni as the −1, P = 0.23 vs. control 10.9±1.7 MJ·d−1). However, the estimated total energy intake and energy availability were higher  in Ebar compared to the controls  and estimated energy expenditure  did not differ between the groups. BM and FM decreased in both groups  during TC while FFM  and total water intake were similar in both groups, and there was no significant water deficit , sitting 5∶05±0∶46 h:min (3∶32–6∶53 h:min), standing 9∶10±0∶44 h:min (7∶54–10∶31 h:min), moderate activity 5∶32±1∶09 h:min (3∶46–7∶34 h:min), vigorous activity 0∶06±0∶03 h:min (0∶00–0∶14 h:min) and active time 5∶38±1∶11 h:min (3∶50–7∶45 h:min) per day. However, a main effect of TC (P<0.001) was observed for rest, sitting, and moderate activity as well as for standing and active time (P  = 0.027). Rest and sitting increased during days 5–8 compared to the first days 1–5. On the contrary, standing, moderate activity and active time decreased.−1, POST 12.1 mmol·L−1; control PRE 10.6 mmol·L−1, POST 11.9 mmol·L−1), and vertical jump remained the same in both groups. While the Ebar group improved anaerobic power before the TC , the relative change was higher than in the controls (P  = 0.007). Nevertheless, Ebar had higher anaerobic power than the controls in PRE (P  = 0.009), MID (P  = 0.046) and POST (P<0.028), there were no differences between the groups in its change during TC from PRE to POST.Relative changes in physical performance are presented in Questionnaire data analysis  revealedWhile there were no differences between the groups in physical activity, energy availability and water balance, the groups were pooled for correlation analysis. Correlations between physical activity and energy availability, water balance, hand-grip and anaerobic power are presented in −1 could not maintain energy balance might be that Ebar also had higher energy expenditure, although the difference between the groups was not significant. Energy expenditure was calculated by a method of intake/balance, which also takes into account the increased thermic effect of food of a higher protein intake and the carried combat gear of from 60 to 70 kg. Energy expenditure of physical activity was also increased by overall stress of TC The present study showed that additional easy-to-use protein-rich energy bars increased total energy intake and energy availability during a demanding wintertime military training course. Despite a higher energy supply, energy intake was insufficient in the Ebar group similarly to the control group. The participants in both groups consumed, however, only 57±13% of the provided energy during TC. The difference of this study to the previously published energy supplements studies during short-term military field training −1 , which was lower than rations offered from 17.6 to 21.8 MJ·d−1  −1  but they did not consume all of the 15.1 MJ·d−1  that they had −1 . Both of these groups lost weight  In this study, increased somatic and URTI symptoms and negative mood state already before TC was related to lower energy availability during the training, which may indicate the lack of motivation for eating. In addition, increased somatic symptoms in the middle of TC were related to lower energy availability. Although Lieberman et al. In the present study, association between physical activity and energy availability was not observed. This is in line with other studies among soldiers, where all subjects were expected to have a substantial energy deficit −1 intake of protein has been recommended −1. An easy-to-use protein-rich energy bar was thought to increase energy intake and thus decrease loss of FFM during strenuous TC. Ebar had a higher energy intake and energy availability compared to the controls. However, there were no differences between the groups in changes in FFM, energy deficit and physical activity, even Ebar had a trend for an increase in FFM, rather than a decrease. Among firefighters, eat-on-move snacks increased not only energy intake but also physical activity during a 2-d experimental period compared to ready-to-eat meal days −1 higher energy intake attenuated decrease FMM in energy deficit state in the presence of sleep deprivation, psychological stress and high physical activity. Previously, a protein intake of 1.8 g·kg−1 has been found to be ineffective in preventing a decline in FFM during the energy deficit of 4.2 MJ caused by increased energy expenditure −1) to maintain the lean body mass during the 40% energy restriction compared to the control group (∼1.0 g·kg−1) protein intake While the energy expenditure is extremely high, 1.8 g·kg−1 has been shown to have a beneficial role during recovery after exercise by attenuating impairments in endurance performance Increased protein intake has been found to produce satiation It would be assumed that the high activity level over 5 hours per day with only 4 hours rest would have induced decrements in physical performance. An improvement in the 3 km running time in both groups could be partly due to weight loss −1) or high (∼2.3 g·kg−1) protein intake has no influence on changes in performance tests after hypoenergetic weight loss −1 was lower than recommended for endurance athletes −1 carbohydrate intake has been recommended to maintain anaerobic performance Also the present results are in line with finding that low (∼1.0 g·kg−1 to 2.8 L·d−1) and water balance (from −1.7 L·d−1 to 0.8 L·d−1) was wide, water balance did not correlate with physical performance after the training course nor with change in FFM. In this study water balance was calculated by the differences between estimated water intake and water loss measured by deuterium elimination method. While 73% of the fat-free mass is water , it could assume that the change in body mass loss is trough water deficit. However, in this study changes in TBW and FFM were nonsignificant and water deficit did not exist. Thus, there was no body mass loss through a water deficit.The recommendation for fluid intake during military training in cold weather is 2 liters per day −1 did not prevent energy deficit or influence on PA during an 8-day strenuous military TC. A satiation effect of the high content of protein in the bars might have induced a decreased energy intake from field ration, since the Ebar group felt themselves less hungry after TC than the controls. However, negative mood state and URTI symptoms affected energy intake already before the training course. Thus mood state and health status are important factors in preventing undereating. In addition, during strenuous TC, PA seems to be primarily affected by factors other than energy supplementation such as mood state. The outcome and also a limitation of this study was the tremendous undereating. For evaluation the effects of increased energy intake on PA and performance, we should have had a third group which was forced to eat all provided food. Secondly to avoid biases, the subjects should be absolutely unaware of the group at they were included during the whole intervention period. In future studies, a special attention should be paid for the taste and variability of the food as well to maintain positive mood state in order to enhance energy intake during prolonged training.Based on the present study, an easy-to-use energy bar supplement of 4 MJ•day


Drosophila, cuticular hydrocarbons (CHCs) are involved in species and sexual identification, and have long been thought to act as stimulatory pheromones as well. However, a previous study reported that D. melanogaster males were more attracted to females that were lacking CHCs. This surprising result is consistent with several evolutionary hypotheses but is at odds with other work demonstrating that female CHCs are attractive to males. Here, we investigated natural variation in male preferences for female pheromones using transgenic flies that cannot produce CHCs. By perfuming females with CHCs and performing mate choice tests, we found that some male genotypes prefer females with pheromones, some have no apparent preference, and at least one male genotype prefers females without pheromones. This variation provides an excellent opportunity to further investigate the mechanistic causes and evolutionary implications of divergent pheromone preferences in D. melanogaster males.Many animal species communicate using chemical signals. In  Drosophila, cuticular hydrocarbons (CHCs) are critical communication signals both within and between species. CHCs are involved in species identification, and play key roles in sexual isolation between Drosophila species Chemical signaling is an important form of communication for many animal species D. melanogaster are still unclear. A previous study by Billeter et al. D. melanogaster males preferred to mate with virgin females lacking CHCs compared to those normally expressing CHCs. Although counter-intuitive, this finding is consistent with several evolutionary hypotheses. It has been previously suggested that inhibitory pheromones may be advantageous to females because they slow mating attempts by males, allowing females time to assess male quality and/or species identity cis-vaccenyl acetate (cVA) to females to deter future mating attempts by other males D. simulansDespite considerable investigation, the roles that CHCs play in mating preferences in Drosophila species, including D. serrataD. mojavensisvirilis and D. lummeiD. melanogasterDrosophila, we have attempted to address these discrepancies here. Our goals were to: i) replicate the results of Billeter et al. ii) determine if their findings were a result of inadequate controls, and iii) test for natural variation in the strength and direction of male preferences for female CHCs in D. melanogaster. Though we are able to replicate the results of Billeter et al. D. melanogaster males in the strength and direction of their preferences for female CHCs. This variation provides an excellent opportunity to investigate the causes and consequences of divergent pheromone preferences at an incipient stage.Although it seems plausible that selection could favor female pheromones that are unattractive to males of their own species, this finding is at odds with previous research. Male mating preferences for female CHCs have been documented in several Unless otherwise stated, all fly strains were maintained in 20 mm vials on standard cornmeal/molasses/yeast medium on a 12 h: 12 h light/dark cycle at 25°C. Under these conditions, non-overlapping two-week lifecycles were established as follows: male and female adult flies were transferred into fresh vials containing food media for 1–3 days before being discarded. 14 days later , flies were transferred into fresh food vials for 1–3 days to begin the next generation.− flies”) using the protocol described by Billeter et al. − flies were created by crossing “+ : UAS-StingerII, UAS-hid/CyO; +” to “+ : tsPromE(800)-Gal4, tubP:Gal80; +”. Progeny were reared at 18°C and virgin oe− males and females were collected within 6 h of eclosion under light CO2 anesthetization at room temperature. Adults were held at 25°C for 24 h before being subjected to three consecutive overnight heat treatments (12 h at 30°C). Flies were returned to 25°C for 12 h between heat treatments. This heat treatment was necessary to ensure the destruction of the oenocytes. Before experiments began, the effectiveness of this treatment was verified using a fluorescence microscope to ensure that ∼100 oe− females and ∼50 oe− males lacked GFP-labeled oenocytes. We then screened a subset of oe− flies (∼30) for each experiment (or replicate) to ensure the heat shock protocol had effectively ablated oenocytes. Flies were allowed to recover from this heat shock treatment for 24 h at 25°C before experiments, which were conducted when the flies were 6 days old.We created flies without oenocytes (“oetsPromE(800)-Gal4, tubP:Gal80; +” to “+ : UAS-StingerII; +”. Progeny from this cross were collected and treated identically to oe− flies but did not experience oenocyte destruction during the heat treatment, allowing them to express CHCs. Though Billeter et al. referred to these flies as “controls”, will refer to them as “oe+” to differentiate them from our additional control: oe− females that were perfumed with CHCs (see below). All stocks used to make oe− and oe+ flies were provided by J. D. Levine (University of Toronto at Mississauga), as was the D. simulans stock used below.We also created “control” flies as per Billeter et al. D. melanogaster females from an outbred, laboratory-adapted population that were homozygous for the recessive white marker, causing them to be white-eyed . We received this white population from W. R. Rice .For our perfuming protocol, we used D. melanogaster and 11 of the 15 inbred “founder” lines of the Drosophila Synthetic Population Resource ry506 and T.0) were excluded because they are weak stocks with males that were often unsuccessful at mating, and the fourth founder line was not included because it is a replicate of Canton-S. Canton-S was obtained from the Bloomington Drosophila Stock Center in 2011 and the founder lines were obtained from S. J. Macdonald (University of Kansas) in November 2011. All lines have since been maintained on two-week cycles as described above.To test for genetic variation in male attraction to female CHCs, we used the wild-type Canton-S strain of D. melanogaster collected in Raleigh, North Carolina, and by crossing them together we can approximately recreate the outbred population from this area. At the time of our experiments, this population had undergone 43 generations of outbreeding.We also used the “allRAL” population described in detail elsewhere − and oe+ males as virgins and transferred them individually into small vials containing food media for the heat treatment described above. Courtship observations were conducted within 2 h of lights-on, and used standard vials (20 mm in diameter) containing a small amount of food media with a foam plug pushed into the vial to allow for an interaction space 20 mm in diameter by ∼5 mm in height. Pairs of males were gently aspirated into the vials to create the following three treatments: 2 oe− males, 2 oe+ males, and 1 oe− male with 1 oe+ male. Aspirator tips were changed between oe− and oe+ males to prevent CHC contamination.We collected oeObservers who were blind to the treatments watched male pairs for 30 min and recorded courtship behaviors  in 1-minute intervals. We calculated a courtship index for each pair as the proportion of time spent courting by either male during a 10 minute period that began when courtship was initiated white population as virgins and held them in groups of 20 in vials containing food media for 6–7 days before the experiments began. At this point, we transferred 60 white females into an empty vial (containing no food medium) and added 10 oe− females. We transferred CHCs from the white females to the oe− females by subjecting them to three medium vortex pulses lasting 20 seconds, with a 20 second break between pulses . This ensures that females with and without CHCs were held under identical conditions before the experiments began.To control for the perfuming procedure, we “sham-perfumed” oeD. simulans males when paired with 5 different types of females: D. simulans females, D. melanogaster oe+ females (expressing CHCs), oe− females (lacking CHCs), perfumed oe− females, and sham-perfumed oe− females. All experimental flies were collected as virgins under light CO2 anesthetization and held in vials containing food media for 6 days . During this time, D. simulans males and females were held at 25°C, and all oe− and oe+ females were subjected to the heat treatment described above.We observed the courtship behavior of − females were vortexed as described above and transferred in groups of 10 to vials containing food media to recover for 1 h. At this time, we gently aspirated individual pairs of flies  into the Petri dishes. Aspirator tips were changed between different fly types to avoid CHC contamination.We performed courtship observations within the first 4 h after lights-on using 35 mm×10 mm plastic Petri dishes with a small amount of food media coating the bottom. Perfumed and sham-perfumed oeObservers who were blind to the treatments watched the pairs for 1 h and recorded courtship behaviors  in 1-minute intervals. We used this information to calculate a courtship index for each male as the proportion of time spent courting over a 10 minute period starting with the initiation of courtship We first performed mate choice trials using the Canton-S strain to replicate the results of Billeter et al. − and oe+ females under light CO2 anesthetization and held them in groups of 10 during the heat treatment described above. We also collected experimental males as virgins under light CO2 anesthetization and held them in groups of 20 at 25°C for 6 days. We conducted mate choice trials within 4 h of lights-on using 20 mm vials containing a small amount of food media, with a foam plug pushed into the vial to create a space 20 mm in diameter by ∼5 mm in height. Males and females from the same holding vial were distributed across treatments to avoid pseudoreplication. We observed these vials for 1 h; when a mating occurred, the unmated female was aspirated out of the vial for identification (see below).We collected virgin oe− and oe+ females, we gently aspirated one oe− and one oe+ female into a vial before adding a single male. Aspirator tips were changed between oe− and oe+ females to avoid CHC contamination. We were able to determine the genotype of the unmated female using a dissecting microscope equipped with fluorescent illumination, as the oenocytes of the oe+ females are GFP-positive and the oe− females are GFP-negative.For mate choice trials using oe− females, we differentiated females from the two treatments by making small holes in either their left or right wings using a dissecting pin. This procedure was completed the day after the females were collected as virgins, before they were subjected to their first heat treatment. There was no additional mortality observed in the oe− females that had their wings modified. To begin the mate choice trials, oe− females were either perfumed or sham-perfumed using the vortex treatment described above. After vortexing, we briefly (∼30 s) anesthetized females using light CO2 and set up experimental vials  with a single perfumed female and a single sham-perfumed female. These females were allowed to recover from the procedure for ∼1 h (but no more than 90 minutes) before experiments began.For mate choice trials using perfumed oe− females − (or sham-perfumed) females minus the number of males that mated with oe+ (or perfumed) females divided by the total number of trials. This value ranges from −1 to +1, with positive values indicating a preference for females lacking CHCs, and negative values indicating a preference for females with CHCs.For each male genotype, we calculated a preference index to measure the strength of the preference for oe−, oe+, perfumed oe− and white females that were 6 days old (as in our experiments). For CHC analysis, flies were gently aspirated into individual glass microvials containing 100 µL of hexane supplemented with 10 ng/mL each of octadecane (C18) and hexacosene (C26) as internal standards. Flies soaked in hexane solution for 3 minutes, were then gently agitated with a vortex mixer for 1 minute and finally were removed using fine forceps. CHCs were analyzed using a Shimadzu GC-2014 with Flame Ionization  fitted with a 15 m×0.25 mm Restek SHRXI-5MS fused silica capillary column with a film thickness of 0.25 µm. Carrier gases were helium and compressed air with a linear velocity flow control mode set at 73.2 cm/s and a column flow rate of 3.0 mL/min. A 2 µL sample of hexane solution was loaded into the GC using a splitless injection mode. The column temperature was held at 55°C for 2 minutes before being heated to 150°C at a rate of 66.6°C/min, then increased to 280°C at a rate of 7°C/min and was held at 280°C for 8 minutes.We measured cuticular hydrocarbons for virgin oeD. simulans courtship trials and the D. melanogaster male-male courtship trials using Wilcoxon tests followed by posthoc analysis with sequential Bonferroni tests − females mated  using a Chi-square test, and determined whether the preference indices calculated for each male genotype were significantly different from 0 using binomial tests . All analyses were completed using JMP 9.We compared courtship indices for the −” flies) and therefore lacking CHCs. These transgenic flies were generously shared with us, and we first used them to replicate previous findings. We successfully replicated the results of Billeter et al. − males or one oe− with one oe+ male induced significantly higher courtship indices (CIs) than the combination of two oe+ males . Likewise, the combination of two oe− males spent a higher proportion of time (in minutes) engaged in “head-to-head” courtship interactions than either of the other two combinations .Billeter et al. D. simulans males courted oe− females more intensely than oe+ females . Importantly, we found no significant difference in the courtship index of D. simulans males when paired with oe+D. melanogaster females  and when paired with our perfumed oe− females, demonstrating that our perfuming technique was successful at transferring CHCs to oe− females. We also saw that D. simulans males courted our sham-perfumed oe− females just as vigorously as regular (not vortexed) oe− females . In contrast to Canton-S, males from other genotypes demonstrated either no significant preference between perfumed and oe− females (7/11 genotypes), or a strong preference for perfumed females . D. simulans males courted our sham-perfumed females much more strongly than our perfumed females , and oe− females perfumed with the CHCs of white females. Internal standards are marked on the chromatograms with a “*”. Note that the y-axis is scaled differently for the white females.(TIFF)Click here for additional data file.Figure S2D. melanogaster− and oe+ females. male preferences when paired with oe Preference indices for D. melanogaster males from 12 inbred genotypes and one outbred population  when allowed to choose between oe+ females (expressing CHCs) and oe− females (lacking CHCs). The preference index is the relative advantage of oe− females over oe+ females, such that positive values indicate a preference for females lacking CHCs. Asterisks above the columns show preference indices that are significantly different from 0 . N (Canton-S)  = 71, all other n = 23–54.(TIFF)Click here for additional data file.


The innate host response to virus infection is largely dominated by the production of type I interferon and interferon stimulated genes. In particular, fibroblasts respond robustly to viral infection and to recognition of viral signatures such as dsRNA with the rapid production of type I interferon; subsequently, fibroblasts are a key cell type in antiviral protection. We recently found, however, that primary fibroblasts deficient for the production of interferon, interferon stimulated genes, and other cytokines and chemokines mount a robust antiviral response against both DNA and RNA viruses following stimulation with dsRNA. Nitric oxide is a chemical compound with pleiotropic functions; its production by phagocytes in response to interferon-γ is associated with antimicrobial activity. Here we show that in response to dsRNA, nitric oxide is rapidly produced in primary fibroblasts. In the presence of an intact interferon system, nitric oxide plays a minor but significant role in antiviral protection. However, in the absence of an interferon system, nitric oxide is critical for the protection against DNA viruses. In primary fibroblasts, NF-κB and interferon regulatory factor 1 participate in the induction of inducible nitric oxide synthase expression, which subsequently produces nitric oxide. As large DNA viruses encode multiple and diverse immune modulators to disable the interferon system, it appears that the nitric oxide pathway serves as a secondary strategy to protect the host against viral infection in key cell types, such as fibroblasts, that largely rely on the type I interferon system for antiviral protection. A central theme today in medical research is understanding the delicate balance of interactions between a pathogen and its host. These interactions dictate the pathological consequences of infections. It is well recognized that the innate immune response against pathogens focuses on detection of highly conserved pathogen-associated molecular patterns (PAMPs) that are distinct from the host. Host pathogen recognition receptors have evolved to recognize patterns such as nucleic acids from pathogens including bacteria and viruses As all viruses are thought to make double-stranded RNA (dsRNA) as a by-product of their replication cycles, this molecule is a potent producer of type I IFN and as such, is commonly used to study innate immune responses to virus infection. DsRNA can be recognized by three different families of pathogen recognition receptors, the toll-like receptors (TLRs), the retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) and the nucleotide oligomerization domain-like receptors (NLRs); these bind dsRNA and initiate cellular signaling pathways In the prototypic antiviral response, IRF3 and early production of type I IFN and ISGs are arguably the most important events in combating infection. In humans, inborn errors that impair the production of, or responsiveness to, either of the three classes of IFN increase susceptibility to mycobacterial and viral infection To ensure their success, however, viruses such as the highly successful human herpes simplex virus (HSV)-1 inactivate IRF3 We have previously observed a protective response against both DNA and RNA viruses in the absence of IRF3 and IRF9 in primary MEFs Given the importance of the type I IFN system as the primary antiviral defence mechanism in non-hematopoietic cells, it is unknown how these cells protect themselves in the absence of this system. There are other innate molecules induced by viral infection that play a role in the host antiviral response. One such molecule is nitric oxide. Endogenously produced nitric oxide has many biological functions including smooth muscle relaxation and neurotransmission −/−9−/−MEFs, which fail to make or respond to type I IFN, induce an antiviral response against HSV-1 and VSV following treatment with poly I∶C. The antiviral response against HSV-1 in these cells was found to occur earlier and was more potent than the response against VSV −/−9−/−MEFs, treatment with poly I∶C subsequently decreases HSV-1 infectious virus production by ∼15 fold (data not shown). To confirm that residual poly I∶C was not responsible for the protective effects, we measured the level of poly I∶C in the supernatants, relative to a standard curve of poly I∶C in medium  and IRF3poly I∶C .−/−9−/− MEFs decreased initiation of replication of HSV-1, varying concentrations of nitric oxide were added to untreated cells with the use of the nitric oxide donor, diethylenetriamineNONOate (DETA-NO). DETA-NO and the control compound diethylenetriamine (DETA) were added to cells at concentrations ranging from 0–200 µM. It was determined by Griess assay that DETA-NO used between 50–100 µM produced nitric oxide to levels similar to those observed in MEFs after treatment with 8.5 nM poly I∶C for 5 hours . Transcript levels of iNOS increased in both WT and IRF3−/−9−/−MEFs upon treatment with poly I∶C within 2 hours  was used to determine the involvement of iNOS in the antiviral response observed against HSV-1 in IRF3 −/−9−/−  MEFs was −/−9−/−  or fully −/−9−/−  restoredin vitro. Thus, to determine whether nitric oxide or a component(s) of the nitric oxide pathway is responsible for protection of naïve monolayers following supernatant transfer, we transferred supernatants from cells treated with dsRNA in the presence or absence of the iNOS inhibitor L-NIL. We observed partial  alone. Although treatment with poly I∶C increased levels of IRF1, these levels were significantly decreased upon addition of IRF1-targeting siRNA.To assess the importance of IRF1 in iNOS induction and the antiviral response in the absence of IRF3 and IFN, an IRF1-targeting siRNA was generated. The control siRNA was a scrambled sequence of the IRF1 siRNA used to account for nonspecific effects. The efficacy of siRNA on IRF1 transcript levels was assessed by qRT-PCR . IRF1 tr−/−9−/− MEFs in which IRF1 levels were decreased following siRNA treatment , suggesting that the level of nitric oxide production that is sufficient to block virus replication is not linked to cytotoxicity. While it is well known that NO has antiviral activity, particularly against DNA viruses such as HSV and vaccinia virus Previous studies have shown that HSV-1 is susceptible to the effects of nitric oxide We have found in this study the importance of iNOS as the enzyme by which nitric oxide is synthesized. This is not surprising, as iNOS induction in response to virus infection, as well as viral components, is well known The iNOS gene locus has low homology between human, rat and mouse sequences. As a result, the transcription factors involved in iNOS induction are species and cell type dependent Overall, our data show that components of the nitric oxide pathway serve as alternative antiviral factors in the absence of the IRF3- and IFN-mediated signaling pathway, and that they also contribute to the antiviral response in WT cells. While we conclude that the nitric oxide pathway is important, we cannot rule out involvement of other pathways or factors. Furthermore, the factors involved in the antiviral response against VSV after 24 hours of poly I∶C pretreatment Human ethics approval was not required as neither human subjects nor primary human tissues were utilized. Production of primary mouse embryonic fibroblasts was completed with full approval from the McMaster Animal Review Ethics Board. All work with viruses was completed with full approval from the McMaster Presidential Biosafety Advisory Committee.−/−IRF9−/− mice −1 penicillin, 100 µg•ml−1 streptomycin and 2 mM L-glutamine. Experiments were performed with cells at passages four to eight. All cells were incubated at 37°C in a humidified 5% CO2 incubator as previously described Primary MEFs derived from WT C57Bl/6 and IRF3Vero cells (ATCC) were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% FBS. HSV-1 (KOS strain) expressing green fluorescent protein (HSV-1gfp) −/−9−/− MEFs Cells were mock-treated, or treated with 8.5 nM poly I∶C  in serum-free OptiMEM medium (Life Technologies) in the presence of 50 µg/ml DEAE-dextran (Pharmacia) for 1 hour. Full growth medium replaced OptiMEM for additional indicated amounts of time, unless otherwise noted. In all experiments, DEAE-dextran was used in controls not treated with poly I∶C to ensure that subsequent cellular responses were not influenced by the former. It was previously determined that 8.5 nM poly I∶C induces maximum protection in IRF3For IFN treatments, cells were pretreated with 100 U/ml IFNβ  for 24 hours prior to challenge with HSVgfp.−/−9−/− MEFs and left on for another 5 hours. For experiments utilizing the iNOS inhibitor L-NIL, please refer to Inhibition of iNOS section, below. Cells were challenged with HSV-1gfp to determine if a soluble factor confers resistance to infection in the absence of both IRF3 and IRF9. Viral replication was quantified 24 hours post-infection as assessed by GFP fluorescence. To ensure no residual poly I∶C in the transferred supernatants was conferring resistance to HSV-1 replication, absorbance of the supernatants was measured. In complete MEF media, poly I∶C was serially diluted 1∶5 with concentrations ranging from 2.2×10−5–8.5 nM. This concentration range corresponds with that used in antiviral assays described previously Supernatants from mock-treated cells and cells treated with 8.5 nM poly I∶C for 5 hours were transferred to naïve IRF32−, an oxidized metabolite of nitric oxide. For this, a Griess reaction was performed as previously described 2 (BDH) ranging from 0–20 µM prepared in alpha-MEM. Griess reagent  was prepared according to the manufacturer's instructions and subsequently added to standards and samples. The absorbance of the supernatants in the plates was read at 550 nm after a 10 minute incubation at room temperature with Griess reagent.MEFs were seeded in 96-well plates to approximately 70% confluency. After 24 hours, the cells were incubated with poly I∶C for 5 hours as described −/−9−/− MEFs were seeded at a confluency of ∼80% overnight prior to treatments in 12-well plates for antiviral assays and in 96-well plates to assess nitrite release via Griess assay. The cells were treated with 0–200 µM DETA-NO  or with 0–200 µM of the control NONOate, DETA  diluted in complete MEF media for 5 hours. Following treatment, cells were challenged with HSV-1 described earlier in an antiviral assay and viral replication was quantified as assessed by GFP fluorescence. Fresh DETA or DETA-NO was added after each medium change.WT and IRF3RNA was isolated from cells using TRIzol reagent (Life Technologies). A random 6-mer primer (0.2 ng) and 50 U of Superscript II (Life Technologies) were used to reverse transcribe 300 ng of DNase-treated RNA  in a total reaction volume of 20 µl. Subsequently, qRT-PCR was performed in triplicate using Universal PCR Master Mix and gene-specific TaqMan primers (Life Technologies) in a total volume of 25 µl.Data were analyzed via the ΔΔCt method. Gene expression was normalized to Gapdh, the housekeeping gene, and expressed as fold change over the mock-treated group . TaqMan specific primers used in this study include iNOS (Mm00440502_m1), IRF1 (Mm00515191_m1) and Gapdh (Mm99999915_g1).The iNOS inhibitors AMG and L-NIL , were diluted in complete medium to a final in-well concentration of 10 µM. To investigate the efficacy of the iNOS inhibitors, cells were pretreated for 2 hours with iNOS inhibitors prior to a 5 hour treatment with poly I∶C. RNA was then collected using TRIzol and prepared for qRT-PCR as described above. Expression of iNOS transcript levels both with and without inhibitor were compared using TaqMan specific primers to determine efficacy of each of the iNOS inhibitors used.To investigate the role of iNOS in the antiviral response, cells seeded the previous day in 12-well plates to 80% confluency were treated as described above. Cells were subsequently challenged with HSV-1gfp as indicated previously.The NF-κB inhibitor Bay 11-7082  was prepared in 1% DMSO, and used at concentrations ranging from 1–10 µM. Cells were incubated with this compound for 0.5 hours prior to mock and poly I∶C treatments. DMSO was included in mock- and poly I∶C-treatments.−/−9−/− MEFs, cells were seeded on glass coverslips overnight at 60% confluency. Cells were either mock- or poly I∶C-treated with and without Bay 11-7082. Following 5 hours of treatment, cells were fixed with 10% formalin and subsequently permeabilized with 0.1% Triton X-100 diluted in 1× PBS for 10 minutes each. An overnight blocking step at 4°C followed in 1×PBS with 2% goat serum. A 1∶200 dilution of NF-κB p65 antibody  was added to the coverslips for 1 hour at room temperature. A secondary anti-rabbit IgG Alexafluor 488 antibody diluted 1∶1000 (Life Technologies) was hybridized for 1 hour at room temperature. Nuclei were stained with Hoescht dye diluted 1∶10000 for 10 minutes. All antibody and Hoescht dilutions were in 1×PBS with 2% goat serum. Images were taken and analyzed using a Leica DM IRE2 inverted microscope with Openlab software (Improvision). Nuclear translocation of cells that received poly I∶C and the inhibitor was plotted as a percent of untreated cells (mock-treated) corresponding to each dilution of Bay 11-7082.To determine the efficacy of Bay 11-7082 in inhibition of NF-κB in IRF35′-CAGACATCGAGGAAGTGAAGGATCA-3′) and a scrambled sequence  were designed (Thermo Scientific). The scrambled sequence was used to account for nonspecific knockdown of IRF1. The selected target sequences were tested so as not to match any known murine gene (other than murine IRF1) sequences by using NCBI nucleotide BLAST at http://www.ncbi.nlm.nih.gov/BLAST/. Transfection was performed according to the manufacturer's protocol. IRF1 siRNA and scrambled siRNA were used at a concentration of 50 µM. IRF1 gene expression following knockdown was quantified by qRT-PCR.An oligonucleotide specific for IRF1, (Data are represented as the mean of three replicates ± standard error of the mean (SEM). A one-way ANOVA with a Tukey post-test was used to compare the means of 4 concentrations of Bay 11-7082 in inhibition of NF-κB nuclear translocation. An unpaired t-test was used to compare the means of two groups where indicated. All analyses were performed using GraphPad Prism 5.0 software.


Mu killer contains a partial inverted duplication of the mudrA transposase gene and two copies of the terminal inverted repeat A (TIRA) region of the master MuDR element of maize. Mu killer can effectively silence single copy MuDR/Mu lines, and it is proposed that a ∼4 kb hairpin RNA is generated by read through transcription from a flanking gene and that this transcript serves as a substrate for siRNA production. Mu killer was sequenced, except for a recalcitrant portion in the center of the locus, and shown to be co-linear with mudrA as originally proposed. The ability of the dominant Mu killer locus to silence a standard high copy number MuDR/Mu transposon line was evaluated. After two generations of exposure, about three quarters of individuals were silenced indicating reasonable effectiveness as measured by the absence of mudrA transposase transcripts. Mu killer individuals that effectively silenced MuDR expressed two short antisense transcripts. In contrast, Mu killer individuals that failed to silence MuDR expressed multiple sense transcripts, derived from read through transcription initiating in a flanking gene, but no antisense transcripts were detected. MuDR/Mu comprise the mutator family of maize Class II transposable elements , that could swiftly silence the minimal mutator system  primers. A 1.3 kb amplicon was obtained for the “right” half using the acm1-Mu killer chimeric primer acm1-muk_86r (5′ CCCCTACTTGTTGTTGAGATAATTG 3′) and mudrA-1298r primer. Genomic DNA targets were amplified in a 25 μl reaction using 20 ng genomic DNA as template, 0.5 μM of each primer, 2 mM MgSO4, 0.2 mM dNTPs, 1× HiFi Buffer, and 1 unit HiFi Platinum Taq  per reaction under the following conditions: 94°C for 2 min followed by 35 cycles of 94°C for 30 s, 60°C for 45 s, and 72° for 1.5 min; there was a final extension at 72°C for 10 min. The left and right Mu killer amplicons were purified using the Qiagen  PCR Purification Kit, TOPO subcloned, and sequenced using M13 forward and M13 reverse primers. The Mu killer sequence segments were assembled and validated by replicate sequencing and are available under GenBank numbers JX067398  and JX067399 .The s Figure . A 1.8 kMu killer, 0.7 kb amplicon with the muk-flank (5′ CCTTTGGTCAGTTCGGTTATCTCTG 3′) and mudr-219r (5′ AGGAGAGACGGTGACAAGAGGAGTA 3′) primers; bz2-mu2: 0.2 kb amplicon with the bz2-637f (5′ CAGAGAGGTGCCAACAGAAGT 3′) and Mu-TIR (5′ AGAGAAGCCAACGCCAWCGCCTCYATTTCGTC 3′) primers.Genomic DNA was extracted from adult leaf tissue using the CTAB method of Rogers and Bendich . GenotypMu killer, 0.5 and 0.7 kb amplicons with the muk-flank (5′ CCTTTGGTCAGTTCGGTTATCTCTG 3′) and mudr-219r (5′ AGGAGAGACGGTGACAAGAGGAGTA 3′) primers; 0.5 kb (spliced) and 0.6 kb  mudrA with the mudrA-2345f (5′ AGGAATGGCAACACACTGGGAAAC′ 3′) and mudrA-2938r (5′ TTCAGTGACTTCCTCTGCTACGTC 3′) primers; and tubulin6 (a DNase I control transcript), 0.5 kb (cDNA) with the tub6-475f (5′ AGTATGCCACTCCCTTGGTG 3′) and tub6-979r (5′ GGCACACATCATGTTCTTGG 3′) primers. Other primers used to amplify Mu killer were: amy-muk512f (5′AGTCCAGTCCGAGATAATTGC 3′) and mudrA-899r (5′ TTGTCCGTATCCAAACTTCCCT 3′). PCR amplification parameters were identical to those described above. PCR products were resolved on a 2% agarose/TBE gel stained with ethidium bromide.Total RNA was extracted from either whole spikelets containing 1 mm (pre-meiotic) to ∼2 mm (late prophase I meiotic stage) anthers or dissected 1–2 mm anthers using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. RNA quantity and quality were assessed with the Quant-iT RiboGreen RNA Assay Kit (Invitrogen) and agarose gel electrophoresis, respectively. Approximately 1 μg of total RNA was treated with DNase I and reverse transcribed into cDNA with an oligo(dT) primer using the SuperScript III Reverse Transcription system (Invitrogen) as per manufacturer’s instructions. Approximately 20 ng cDNA was used as template for RT-PCR. Target, expected amplicon size, and corresponding primer pairs are: Mu killer were used as generated from Cross 2  kernels were selected and grown in Stanford, California in summer 2008, and the presence or absence of Mu killer was confirmed via PCR on genomic DNA. Anthers were staged from the upper florets and then dissected onto dry ice and stored at −80°C. Total RNA was extracted from four biological replicates of mutator active and Mu killer anthers (15–20 anther sample size) and quantified and qualitatively assessed as described above. DNase I-treated total RNA (1 μg) was amplified and labeled with either Cy3 or Cy5 using a Low RNA Input Fluorescent Linear Amplification Kit . Eight hundred twenty-five nanograms of each labeled cRNA was hybridized for 17 h at 60°C on a 4 K × 44 K Agilent in situ synthesized oligonucleotide platform along with spike-in controls for calculating target RNA concentrations. Data acquisition, image processing, spot flagging and removal, and normalization were performed as described in Skibbe et al., Individuals from ears segregating for mutator active and siblings silenced by Mu killer acts as a dominant locus that effectively silences the minimal mutator system  to a high copy mutator bz2-mu2//bz2-ref spotted kernel line that has been demonstrated to have both a high forward mutation frequency and a large impact on the transcriptome of developing maize anthers  background; when mutator activity is silenced, kernels are uniformly bronze. After Cross 1 all progeny have purple seed, requiring genotyping to identify the Mu killer carriers. As expected, all these individuals were also carriers of the MuDR/Mu transposon system. Because Mu killer-mediated silencing occurs most effectively when Mu killer is inherited through the female parent, an additional cross was performed by a bz2 tester strain. If Mu killer is highly effective, then the expectation was that half of the progeny ears would segregate 1:1 purple:bronze and half of the progeny ears would be fully bronze. Among the fully bronze cohort, there are four classes. Two of the classes lack the bz2-mu2 reporter gene and will be bronze only, independent of Mu killer. Classes 2a and 2b .e Figure , it is hMu killer to eliminate mudrA transcripts. Plants were grown from bronze kernels of Cross 2a (Mu killer carrier parent), using genotyping to pick plants that inherited Mu killer, to compare to plants derived from spotted kernels of the parental bz2-mu2/bz2 active mutator line. Anthers were chosen for analysis because they are the final organs differentiated and thus represent the location most likely to experience silencing.In Crosses 2a and 2b, ears contained the expected 50% purple kernels, but the remaining kernels were a mixture of spotted (active mutator) and bronze (loss of mutator activity). The mutator system can silence spontaneously, and there is a very wide variation in this frequency and in the percentage of spotted kernels during progressive spontaneous silencing . These initial results are consistent with Mu killer acting as a reasonably efficient silencing agent of high copy mutator lines when present for two generations of plant growth.The selected primers span the third intron of d Figure , lane g;d Figure , generatMu killer transcript has been proposed to be a hairpin ∼4 kb in length, with an inversion point ∼1.3 kb into the mudrA region of the MuDR transposon. The structure of the Mu killer locus is based primarily on Southern blot evidence. To examine the proposed structure in more detail, the Mu killer locus was amplified using primers that are specific to mudrA, the TIR adjacent to mudrA, and the flanking amy4 and the acm1 genes to validate the gene order. The left and right halves of Mu killer were amplified in contiguous molecules of 1.8 and 1.3 kb, respectively, and fully sequenced and deposited into GenBank. The Mu killer locus begins with terminal inverted repeat A (TIRA) of MuDR and extends 1.298 kb into the mudrA gene on both the left and right halves. PCR amplification attempts using primers internal to 1.298 kb were unsuccessful indicating that the central region of MuDR is not present. Attempts to span the two halves using long-range PCR methods also failed; this result raises the possibility that an insertion/deletion polymorphism, a large rearrangement of unknown origin, or an exceptionally stable DNA hairpin structure between the left and right halves of the Mu killer locus prevents PCR amplification.The structure of the Mu killer locus, Slotkin et al.  or inactive (gDNA “−”) in silencing based on PCR confirmation of Mu killer but lacked Mu killer cDNA, along with two individuals that did accumulate mudrA cDNA were selected for further analysis along with positive and negative controls. The transcript accumulation in these four individuals was interrogated using primers positioned at four locations: (1) ∼500 bp upstream of Mukiller (amy4-flank) (2) at the junction of the amy4/Mu killer (amy4-muk_512f), (3) ∼900 bp into the Mu killer gene (mudrA-899r), and (4) ∼1300 bp into the Mu killer gene (mudrA-1298r). When the amy4-flank primer was paired with the two internal Mu killer primers, amplicons of the expected size  accumulated in the two Mu killer transcript “+” individuals but no transcripts were detected in either of the Mu killer transcript “−” individuals. When the amy4/Mu killer chimeric primer was paired with the two internal Mukiller primers, amplicons of the expected size (without any splice variants) accumulated in the two Mu killer transcript “+” individuals. In the Mu killer transcript “−” individuals, amplicons of an unexpected size were detected  shared by Mu killer and MuDR. Additional analysis using RT-PCR amplification employing oligonucleotides further into Mu killer (mudrA-899r and mudrA-1298r) demonstrated that longer transcripts  plus antisense mudrA  in the region shared with the 0.74 kb transcript type.Several unexpected molecular observations were made while assessing accumulation of s Figure . A 0.74 s Figure ; howevers Figure  can origamy4 flanking region. Similarly, the minor 1.8 and 1.4 kb transcripts diagrammed in Figure Interestingly, the major 0.65 kb transcript results from splicing of a short intron with canonical 5′ donor and 3′ acceptor sites in the MuDR: they contained Mu killer but generated mudrA cDNA. In these individuals, no antisense transcript types were identified that extended into the amy4 flanking primer region, that is, both the 0.74 and 0.65 kb transcript types were missing. We therefore propose that production of the antisense 0.74 and 0.65 kb transcripts is required for Mu killer-mediated silencing of mudrA. In individuals that failed to silence mutator, RT-PCR amplification products were only identified for the combination of the chimeric amy4/Mu killer primer paired with the two internal mudrA oligonucleotides  and 33,700 (Mu killer). There were many examples of stage-specific transcripts in both biological sample types. Two types of differences in transcript expression comparing Mu active to Mu killer silenced lines were analyzed: (1) “Off/On” differences represent expression that was detected in one sample but not in the other and (2) “Up/Down” differences are the differential expression class and include probes where expression was at least 1.5-fold higher in one sample than the other . Venn diagrams summarizing On/Off differences for Mu active vs. Mu killer backgrounds for both anther stages are shown in Figure Mu active background but expressed in Mu killer (green numbers).We found that the pre-meiotic 1 mm anther transcriptome contains 34,338 transcripts in an active mutator background and slightly more  transcript types in Mu killer has a major impact on the anther transcriptome compared to an active mutator line. Mu killer impact is dynamic, because only 381 transcript types were OFF in Mu active anthers at both developmental stages. In the reverse comparison (present in Mu active but absent in Mu killer) there are 1156 transcripts at 1 mm and 2425 at 2 mm with 301 transcripts absent in Mu killer for both anther lengths. Collectively, the differences between the two lines are more than 10% of the anther transcriptome. It is already established that high copy mutator lines express a diverse suite of stress response genes, compared to silenced sister lines  or red  and the same status by teal (both “off”) or orange (both “on”). Affected pathways within a sample of five pathway classes were then categorized by their color scheme. As seen in Table Mu killer but “on” in Mu inactive (blue). The combined “different” effects  is more than the combined “same” effects , again indicating that Mu killer has distinctive aspects to its silencing mechanism. The many pathways affected in the “Carbohydrate biosynthesis” pathway class are displayed in Figure Mu killer (blue) and four pathways have “on” reactions only in Mu killer (red).The transcripts that were “on” in http:/www.stanford.edu/~walbot/maizecyc/mukiller_onoff.html. These insights can guide future work in metabolomics and proteomics to discover whether the transcriptomic differences reported here result in distinctive cellular properties.Interestingly, three of the pathway classes in Table Mu killer: effectiveness in silencing standard high Mu copy number lines, molecular organization and expression of the locus, and impact on the anther transcriptome. After two generations of Mu killer exposure, 77% (10/13) high copy number MuDR/Mu individuals tested were silenced based on loss of transcripts characteristic of mudrA. Thus Mu killer-mediated silencing is considerably more frequent than the spontaneous ∼10–20% loss of mutator activity per generation generally observed  that generate sense mudrA and mudrB RNA while antisense transcripts result from termination failure in the region separating these two genes (Walbot and Rudenko, MuDR sense transcript accumulation (Kim and Walbot, Mu killer element producing 0.74 and 0.65 kb antisense mudrA transcripts was associated with effective silencing. Because these transcripts terminate in the flanking amy4 locus, it is possible that antisense RNA stability or effectiveness depends on the sequences contributed by the flanking gene. Both antisense transcripts are complementary to a short region of about 250 bases in authentic mudrA transcripts, including part of the second exon and the first exon including the 5′ untranslated region. Because the 0.65 and 0.74 kb RNAs cannot form large hairpin structures, it is presumed that small RNAs derived from them target either translation or transcription of mudrA. This is a new model to explain how Mu killer silences MuDR. It is possible that the minor longer transcript types (Figure Standard s Figure .Mu killer individuals that failed to silence MuDR, we found sense rather than antisense transcripts derived from Mu killer. These read through transcripts initiated in the amy4 flanking locus, and also included the non-transcribed portion of TIRA, and mudrA-homologous sequences. A complex splicing pattern was observed, utilizing canonical 5′ donor and 3′ acceptor sites that are not used in authentic mudrA transcripts (Figure MuDR is transcribed independent of its insertion sites, it is thought that transcription at MuDR loci is driven primarily by the enhancer elements located within the element TIRs; there is no evidence for read through transcripts when MuDR is inserted within the coding region of bz2-mu4 (Hershberger et al., Mu killer and the flanking sequence context unexpectedly highlight that both MuDR promoters and the insulator functions of the TIRs can be suppressed.In s Figure . BecauseMu killer. Endogenous hMuDR elements reside in every maize line tested; they are co-linear with MuDR but contain point mutations and other small defects that make them incompetent to program Mu element activities. They are also impotent in regulating MuDR (Rudenko and Walbot, MuDR persisted, or won the contest. The newly arisen Mu killer element can silence MuDR with reasonable frequency; however, we also found that when Mu killer fails to silence MuDR it switches from producing antisense to producing sense mudrA transcripts. In this case too, it appears that MuDR is victorious. The stability of the Mu killer transcription states is unknown, but it will be interesting in future studies to assess the persistence of Mu killer over a multiple generation time scale to determine if, like hMuDR elements, it ultimately fails to contain MuDR/Mu elements.If there is an “arms race” between transposon and host mechanisms to suppress them, aberrant transposon forms could be a potent weapon in host-mediated defenses as originally proposed for The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We sought to investigate whether there is a generalised hippocampal deficit in IL-1RI−/− animals. Therefore, in the current study we compared wildtype (WT) mice to IL-1RI−/− mice using a variety of hippocampal-dependent learning and memory tasks, as well as tests of anxiety and locomotor activity. We found no difference in performance of the IL-1RI−/− mice compared to WT mice in a T-maze working memory task. In addition, the IL-1RI−/− mice showed normal learning in various spatial reference memory tasks including the Y-maze and Morris mater maze, although there was a subtle deficit in choice behaviour in a spatial discrimination, beacon watermaze task. IL-1RI−/− mice also showed normal memory for visuospatial context in the contextual fear conditioning paradigm. In the open field, IL-1RI−/− mice showed a significant increase in distance travelled and rearing behaviour compared to the WT mice and in the elevated plus-maze spent more time in the open arms than did the WT animals. The data suggest that, contrary to prior studies, IL-1RI−/− mice are not robustly impaired on hippocampal-dependent memory and learning but do display open field hyperactivity and decreased anxiety compared to WT mice. The results argue for a careful evaluation of the roles of endogenous IL-1 in hippocampal and limbic system function.Interleukin-1 (IL-1) is a key pro-inflammatory cytokine, produced predominantly by peripheral immune cells but also by glia and some neuronal populations within the brain. Its signalling is mediated via the binding of IL-1α or IL-1β to the interleukin-1 type one receptor (IL-1RI). IL-1 plays a key role in inflammation-induced sickness behaviour, resulting in depressed locomotor activity, decreased exploration, reduced food and water intake and acute cognitive deficits. Conversely, IL-1 has also been suggested to facilitate hippocampal-dependent learning and memory: IL-1RI Interleukin 1 (IL-1) is a key pro inflammatory cytokine that is primarily produced in the periphery by immune cells but can also be synthesized by glia and neurons within the brain. Interleukin 1 signalling is mediated by the binding of IL-1 α or IL-1β to the interleukin type one receptor (IL-1R1). Interleukin 1 signalling can be blocked by IL-1 receptor antagonist (IL-1ra) which binds to the IL-1R and blocks signal transduction (see Interleukin 1 (IL-1) has been shown to play a key role in sickness behaviour resulting in depressed locomotor activity, decreased exploration, reduced food and water intake, However, there is also evidence that endogenous IL-1 is actually necessary for memory performance in normal healthy animals. Studies in rats by the Yirmiya group showed that when IL-1ra is administered intracerebroventricularly it impairs performance in the Morris water maze −/− have a generalised hippocampal deficit rather than a specific visuospatial learning deficit. Therefore, we have investigated the nature and degree of cognitive impairment in IL-1RI−/− mice. We also evaluated locomotor activity and anxiety in these mice using open field and elevated plus maze tests. In the light of the behavioural findings we also analysed aspects of hippocampal morphology in IL-1RI−/− and WT animals.Our laboratory has an interest in the role of IL-1 in cognitive function. Since many learning and memory tasks can be confounded by aspects of sickness behaviour, such as decreased locomotor activity, motor speed and consumption of food rewards, our laboratory has developed novel ‘escape from shallow water’ tasks to test working and reference memory in T-maze −/− mice were used. C57BL/6 female mice were obtained from Harlan, UK and IL-1R1−/− mice, B6.129S7-Il1r1<tm1Imx>/J (Stock # 003245), were obtained from the inbred colony of Prof. Kingston Mills in the Trinity College Bioresources Unit. In later experiments with male mice, both C57BL6 and IL-1RI−/− mice came from inbred colonies in the same Bioresources unit in TCD. The knockout mice, at the time of their initial arrival from JAX  had been backcrossed 7 times to a C57 background and thus C57BL/6 mice were an appropriate control strain. All mice were housed in cages of five at 21°C with a 12∶12 hour light dark cycle (lights on from 0800 to 2000) with food and water ad libitum. All animal experiments were performed in accordance with Republic of Ireland Department of Health & Children licenses, after ethical approval by the TCD (Trinity College Dublin) Animal Research Ethics Committee. All efforts were made to minimise both the suffering and number of animals used.In all of the behavioural tasks described below 5–8 month old female or male C57BL/6 and IL-1R1To assess hippocampal-dependent working memory we used the paddling T-maze task as previously described To investigate hippocampal dependent reference memory, we used the “paddling” Y-maze visuospatial task as we have previously described To investigate locomotor and rearing activity the open field task was used. Briefly, mice were placed in a box (58 cm×33×19 cm). The number of times the mouse reared and crossed the squares in the box (distance travelled) was recorded for three minutes.The elevated plus maze task was used to test for anxiety-like behaviour in mice The water maze consisted of a tank 120 cm in diameter, 60 cm in depth filled with water to a depth of 24 cm. The water was made opaque with the use of a white tempura paint powder . A camera was fixed to the ceiling above the water maze and connected to the computer-based tracking programme Ethovision 3.1 . Mice were trained using extra maze visual cues to find the location of a hidden platform (15 cm in diameter and 23 cm in height) submerged 1 cm below the water surface and 13 cm from the edge of the pool wall. The pool was divided into four quadrants namely, north, south, east and west; using virtual bisectors using Ethovision. The platform was placed in one of these four different quadrants which were 13 cm from the edge of the pool. The groups were counterbalanced so that there were mice going to each of the four quadrants. The training protocol used was as described by Goshen, consisting of three trials per day per mouse, for 3 days, with a 1 hour break between trials, followed by a probe trial the following day The spatial discrimination beacon water maze is an adapation of the water maze task where spatial memory and spatial-discrimination are assessed using two visually identical beacons. Contextual fear conditioning (CFC) was recorded using a box (40 cm×10 cm×16 cm) with a transparent wall that had a floor containing metal rods which were wired to a shock generator . The mice were placed into the box and allowed to explore for 2 minutes. Following exploration, a shock of 0.4 mA was administered for two seconds. An inter trial interval of 2 minutes was followed by a second shock of 0.4 mA for two seconds followed by a further 30 seconds of exploration before returning to the home cage. Fear conditioning was assessed 48 hours later for duration of 5 minutes. Freezing was regarded as the complete absence of movement, except those related to respiration as originally described −/− mice and WT mice were left in the Phenotyper home cage for a period of 60 hours  with free access to food and water. The recordings taken over the 60 hours were then binned into 4 hour periods and presented as 15 consecutive periods, showing night and day activities. Three Phenotyper home cages were used per strain, with five mice in each cage. The total activity for each strain was averaged across these three cages for night and day. Since the software did not allow us to view separate traces for individual animals, statistically the groups are therefore presented as n = 3, but this figure represents the activity of 15 animals of each strain.Circadian rhythm was recorded with the use of Phenotyper home cage (45 cm×45 cm) Noldus, Nottingham, U.K) and Ethovision 3.1 . IL-1R1−/− mice were indeed unresponsive to IL-1β, chemokine (C-X-C motif) ligand 1 (CXCL1) and IL-6 expression were analysed using the following primer pairs CXCL1: forward 5′-CACCCAAACCGAAGTCATAGC-3′ and reverse 5′-AATTTTCTGAACCAA and IL-6: forward 5′TCCAGAAACCGCTATGAAGTTC-3′ and reverse 5′-CACCAGCATCAGTCCCAAGA-3′. Plasma levels of CXCL1 and IL-6 levels were analysed 3 hours post injection of IL-1β (25 µg/kg i.p) or saline using R&D systems duo-set enzyme-linked immunosorbent assays (R&D systems UK) as previously described A subset of mice were terminally anaesthetised and transcardially perfused with heparinised saline 3 hours after peripheral injection of IL-1β (25 µg/kg) or saline. The hypothalmus and hippocampus of the brains were removed and stored at −80°C until use. As we have previously described −/− mice (n = 6). Sections were dewaxed in Xylene and Histoclear then rehydrated through a series of decreasing alcohols ranging from 100–70% concentration and treated with 0.2 M boric acid, pH 9, 65°C for 30 min and cooled to room temperature. Non-specific peroxidase activity was eliminated by incubating sections in 1% H2O2 in ethanol (1 ml H2O2/100 ml) for 10 minutes. Sections were washed in PBS and blocked using 10% normal horse serum. Sections were incubated with SY38 , at room temperature overnight. Sections were then washed in PBS before incubation with biotinylated horse anti-mouse secondary IgG . Sections were then washed in PBS and ABC was applied for 30 minutes followed by diaminobenzidine (DAB) reaction in the presence of ammonium nickel chloride (0.06% w/v) to intensify staining.Formalin-fixed, paraffin-embedded sections were cut from the brains of WT (n = 4) and IL-1R1−/− mice and WT mice was determined by mono-chrome pixel density analysis of digitally captured images using Cell A imaging software . Areas of uniform staining within each of the strata of the hippocampal formation (avoiding unstained areas such as blood vessels) were quantified using pixel density analysis. One group of quantifications  was performed on each section. Transmittance measurements were made at a light intensity within the linear transmittance range (0–255 Lux). In the dorsal hippocampus  the area of highest transmittance  was chosen as an internal standard and all other transmittances were subtracted from this layer. A similar approach was taken in more ventral hippocampus , using the CA3 neuronal layer as internal standard to calculating density ratios for radiatum and dentate gyrus polymorphic layer with respect to stratum lacunosum moleculare. The adjusted values for each stratum were then used in ratio calculations to derive a ratio measure of synaptic density as determined by the following equations. For the dorsal ratio: RatioDORSAL =  /Tcorpus callosum–TDG molecular), while for the ventral region RatioVENTRAL =  (TCA3–Tradiatum OR DG polymorphic)/TCA3–Tlacunosum moleculare).The density of synaptophysin staining in IL-1R1−/− brains, the ventricles on each section were outlined and an area measurement in pixels obtained. Using a stage micrometer of 1 mm in length the measurement in pixels was converted to mm2 (Image J area (pixels)/27.8 = area (mm2)). Areas were calculated for sections from each animal at 2.0 and 2.3 mm posterior to Bregma and summed and multiplied by the summed section thickness (2×10 µm: 0.02 mm).Ventricular volume was estimated Using Image J software , and 10 µm sections from WT and IL-R1All statistical analyses were performed using GraphPad Prism 5 for Windows. Data from CFC, elevated plus maze, open field, flag and probe trials and synaptic density were analysed using t-tests while Mann-Whitney tests were used to compare non-parametric ventricular volumes. One Way ANOVA was used to compare cytokine/PCR data while repeated measures two-way ANOVA with strain as between subjects factor and trial block as within subjects factor were used for repeated measures analyses of performance across multiple test sessions. Bonferroni post-hoc tests were used to test for differences at specific time points where main effects of strain or interactions of strain and trial were observed.−/− mice (n = 11) and WT (n = 20) mice in a visuo-spatial reference memory Y-maze task , nor an interaction , but there was a significant effect of trial block  where performance of both strains improved as training continued.Initially all experiments were carried out with female mice. We assessed the performance of IL-1R1aze task  across f−/−) were trained, for 12 days, to alternate their arm choices to escape from the shallow water maze. The performance of each mouse was assessed for 10 trials per day, for 12 days and these data were then divided into 3 blocks of 4 days (training block 1 (days 1–4) and block 2 (days 4–8) block 3 (days 9–12)). There was no impairment on this task in the IL-1R1−/− mice compared to their controls  and WT mice (n = 10). The data  as factors. There was no effect of strain  and no interaction of session and strain  but there was a significant effect of session  indicating that all animals had a memory for the context in which shock was received. Bonferroni post-hoc tests showed that both strains significantly increased freezing between pre-shock and post-shock sessions (p<0.001).In the contextual fear conditioning paradigm we recorded, for a period of 5 minutes, the time spent freezing 48 hours following exploration and foot shock at 0.4 mA for IL-1R1The data  reveal t−/− mice were more active in the open field compared to controls, showing a significantly greater total distance travelled (p = 0.0002). Their rearing activity in the open field was also significantly higher  indicating that exploratory activity is also increased in IL-1RI−/− mice . These data suggest a less anxious phenotype in the IL-1R1−/− mice. Latency to emerge from the initial closed arm was not significantly different between IL-1R1−/− and WT controls  and WT mice (n = 20) to locate the hidden platform in the Morris water maze spatial memory task during 3 days of 3 trials with an inter-trial interval of 1 hour . The protocol used to test this was that of Avital and colleagues, in order that we might replicate their original findings with the IL-1rKO mouse −/− mice displayed a similar rate of learning in the water maze compared to their controls. The IL-1R1−/− mice showed slightly, but statistically significantly, shorter latencies than the WT to get to the platform. Two-way ANOVA of the latency showed a significant effect of strain  and a significant effect of trial number  but no interaction  and there were no significant differences at any individual time point by Bonferroni post-hoc tests. There was also a slight, but non-significant, difference between the strains on distance swam . Both strains learned the task well . Furthermore, no obvious differences could be observed in the types of routes taken by the 2 strains as assessed by mouse tracking in Ethovision.We assessed the ability of IL-1R1−/− and wild-type animals with p = 0.9635 at 9 trials and p = 0.6970 at 27 trials (unpaired t test of time in target quadrant). In the non-spatial flag trial in which a flag was added to the platform in its existing location, both strains reached the platform more quickly than in the hidden platform version of the maze but there was no significant difference between strains on this parameter (p = 0.3593) as seen in −/− and WT mice perform with equivalent proficiency in both hidden and visible platform versions of the maze.In a probe trial conducted after 3 days of training, during which the platform was removed from the pool , both st−/− mice and their controls to locate the hidden platform in a spatial discrimination beacon water maze task. New mice, naïve to any other behavioural experiment, were used for this task. Briefly mice were trained to discriminate between two identical beacons, one of which had a hidden platform beneath the surface of the water, depending on their allocentric spatial locations. Animals were started from various positions in the maze, some close to the correct beacon, some close to the incorrect beacon and some equidistant from both beacons.We assessed the ability of IL-1R1−/− and WT mice learned about the spatial location of the platform to the same extent.Consistent with our previous watermaze experiment, a probe trial conducted after 72 trials  showed tHowever, given (i) that previous studies have implicated IL-1 in spatial memory performance and (ii) that other aspects of watermaze performance may be more sensitive to disruption of hippocampal function  or saline i.p at 3 hours were assessed by ELISA and RT-PCR. As shown in −/− mice treated with IL-1β but these molecules were robustly expressed in WT mice treated with IL-1β. Plasma cytokine responses to IL-1β were equally limited in both male and female IL-1RI−/− mice. Similarly we assessed the CNS transcription of mRNA for these IL-1β -sensitive genes. IL-1β induced robust transcription of both cytokine transcripts in WT animals but did not produce any change from basal levels in either male or female IL-1RI−/− mice. The same patterns were true for hippocampal transcription (data not shown). Thus, IL-1RI−/− mice, of both genders, are completely unresponsive to IL-1β stimulation. We also analysed these animals for IL-1β protein (plasma) and mRNA  and found no evidence of elevated basal IL-1β in these animals.Given the failure to find any substantial spatial learning deficit in the IL-1RI−/− mice, animals were housed under a reversed light cycle such that housing rooms are dark during the daytime and cognitive experiments were therefore performed during the animals’ ‘subjective night’ or more active period. We reasoned that it may be informative to assess the circadian rhythm of IL-1R1−/− mice compared to WT controls. We measured total activity of a cohort of 15 female IL-1R1−/− mice and 15 female WT in PhenoTyper home cage, 45 cm×45 cm,  captured by Ethovision . As expected, over a period of three nights and two days plotted in 4 hour blocks, both the IL-1R1−/− and WT mice were clearly more active in the night phase compared to the day  and male WT (n = 9) mice on a visuo-spatial reference memory Y maze task (−/− mice compared to their controls (WT). There was no effect of strain  or any interaction  but a significant effect of trial block  where performance of both strains improved as training continued.Since previous published studies on cognitive deficits in IL-1RIaze task  across f−/− mice show equivalent, unimpaired, contextual fear memory compared to their controls i.e they spend similar time freezing compared to the WT . Similarly, as shown in −/− mice (p = 0.0035). −/− mice spend a significantly longer time in the open arms of the elevated plus maze compared to the WT mice (p<0.05). This increased time in the open arms suggests that male IL-1R1−/− mice are less anxious, as was observed in the female mice (−/− mice are into open arms than those of WT mice (p = 0.0160 t-test), thus indicating a decrease in anxious behaviour in the IL-1RI−/− mice.Distance travelled and the number of rears in the open field for a period of 3 minutes was recorded. Data are expressed as mean±SEM and significant differences were analysed by unpaired t-tests. Based upon an activity  in the male mice . Since iale mice . These d−/−(n = 6) and WT mice (n = 4) was assessed with the use of ImageJ. −/− mice (p = 0.1714) as assessed by MannWhitney test following Kolmogorov–Smirnov test, although there were was a trend towards enlarged ventricular volume in the IL-1RI−/−.The ventricular volume in IL-1R1−/− mice and WT mice in either dorsal hippocampus  or in more ventral hippocampus . Similar results were observed for the DG polymorphic layer (data not shown). Unpaired t tests showed that there were no statistically significant differences (p≥0.1324), although there was generally more variability in values in the IL-1RI −/− animals.We also investigated whether there was a difference between the two strains in synaptic density in regions of the dorsal and ventral hippocampus. In −/− and WT mice on multiple hippocampal-dependent, spatial memory tests including T-maze working memory, Y and Morris water maze tests of reference memory, and contextual fear conditioning. There was, however, a subtle deficit in choice behaviour in IL-1R1−/− mice on a spatial discrimination beacon test in the watermaze. IL-1R1−/− mice also showed a significantly increased distance travelled and rears in a novel open field and showed decreased anxiety in the elevated plus maze. These effects were similar in male and female mice.In the current study we were unable to detect significant differences between IL-1R1−/− mice show increased locomotor activity with respect to wild-type controls in open field experiments. However the activity data in the continuous activity monitoring boxes suggest that their general activity is not significantly different to WT controls, indicating that this increased activity is likely to reflect a response to novelty rather than increased activity per se. Consistent with this, rearing activity, which is an exploratory behaviour, was also significantly increased in the open field. This type of increased activity in open field could potentially indicate a spatial learning impairment in these mice, although our data from the spatial memory tests performed do not support this possibility. Furthermore, our data from the elevated plus-maze test suggest that IL-1RI−/− are actually less anxious than wild type controls: knockout animals spent significantly more time in the open arms and less time in the closed arms than WT controls. These data are indicative of decreased anxiety in these mice. Although the time spent in open arms could potentially be confounded by the increased locomotor activity already described, we also found that when corrected for total activity  the arm entries into open arms as a proportion of total arm entries maintained the stronger tendency to visit the open arms in IL-IRI−/− mice  which could be important, not only in interpreting certain tests of cognition but also during anxiety, and which could also be a key target for modulation in sickness behaviour.Since general hyperactivity does not appear to be the explanation of the current data, decreased anxiety in IL-1RI−/− mice were not definitive, with no signficant effects on ventricular volume or hippocampal synaptic density in dorsal or more ventral regions, although changes in the lamination of the hippocampus in ventral regions make the analysis conducted here difficult in the most ventral regions. There is a recognised double dissociation between the dorsal hippocampus and ventral hippocampus with respect to spatial memory and anxiety −/− and littermate controls, the possibility that there are more pronounced structural or morphological differences in the anxiety-associated ventral hippocampus.Preliminary investigations into a neuropathological basis for these decreased anxiety effects in IL-1RIIt is also important to note that the described differences in open field activity and decreased anxiety also represent important potential confounding factors in studies of cognitive function. That is to say, in cognitive tasks that are aversively motivated, such as the MWM and contextual fear conditioning, an animal with decreased anxiety may show differences in these cognitive tasks because of its different response to the aversive stimulus rather than because of some cognitive impairment. Aspects of sickness behaviour can significantly confound cognitive testing It has been reported that endogenous IL-1 plays an important role in hippocampal-dependent learning and memory. Avital and colleagues showed that IL-1rKO mice are impaired on the Morris water maze task, displaying a slower rate of learning  compared to the WT −/− mice. Previous studies have suggested that spatial working memory and spatial reference memory reflect distinct psychological processes, subserved by different neural mechanisms. Therefore, the lack of effect on alternation behaviour in our mice compared with the deficits reported by Avital and colleagues could reflect the different nature of the memory processes involved. However, a simple visuospatial Y-maze spatial reference memory task, which like the MWM, relies on use of visuospatial cues to escape water, was also solved equally easily by WT and IL-1RI−/− mice. We next performed contextual fear conditioning experiments but we found robust and equivalent freezing responses in WT and IL-1RI−/− in male and female mice.The current experiments began by demonstrating normal, spatial working memory performance in a T-maze escape from water alternation task in the IL-1RI−/− mice: showing longer latencies to the hidden platform and travelling greater distances to find it. We did not observe such rapid learning of the task in WTs in our study and the probe test performed after 9 trials of spatial training revealed only a slight preference for the training/goal quadrant (33% in target quadrant). This rapid learning in controls, rather than ‘slow’ learning by the IL-1R1−/− may constitute a key difference between our studies and those of Avital and colleagues. Given this rather short training protocol of 3 trials per day for 3 days with a 1hr inter trial interval and the concomitant mild preference for the goal quadrant displayed by both groups, we continued training after the 9 trials for a further 18 trials. Even after this much longer training session we found that the IL-1R1−/− mice were not significantly different to WTs on this spatial reference memory task. Importantly, after this extended period of training, both groups now showed a much stronger preference for the training quadrant in the probe test (>40% time in training quadrant). Indeed, IL-1RI−/− and WT animals performed similarly on probe trials performed after 9 trials and 27 trials. They were also similar on the visible ‘flag’ trial. Therefore, it is clear that IL-1RI animals can learn this task and in the current study do so at the same rate as WT controls.In an attempt to replicate previous studies we also assessed the Morris watermaze (MWM), following the exact protocol of Avital et al −/− mice −/− mice again showed an equivalent preference for the training quadrant, suggesting that both groups had learned about the spatial location of the platform to the same extent. In contrast, the IL-1R1−/− mice displayed a subtle increase in the number of times they approached the wrong (decoy) beacon . Bannerman et al., −/− mice and their ability to learn about the spatial location of the platform during the beacon task, their deficit in choice behaviour may also reflect impaired behavioural inhibition rather than impaired associative spatial learning, as discussed above.In a recent study by Bannerman and colleagues −/− and wild types observed in open field and elevated plus maze experiments were the same in males as they were in females. These data indicate that gender is not an important factor in either the cognitive or anxiety tests used in the current study.The present results are apparently at odds with those studies showing that IL-1rKO mice are impaired in visuospatial memory in both the standard, open field MWM and during CFC −/− and wild type animals and these studies showed that overall distance travelled was equivalent in both strains and that both strains showed the expected dark phase peak in activity. However, the interaction of strain and time did indicate that IL-1RI−/− were perhaps slightly less active in the dark phase and perhaps slightly more active in the light phase. This leaves open the possibility that the strains may show other behavioural differences if tested during subjective night but does not offer an obvious explanation for the differences in cognitive function observed in our studies with respect to those of the Yirmiya group.It is also of note that Goshen and co-workers demonstrated the effect of IL-1β pathways on behaviour using mice raised in reverse cycle −/− animals are more prone to certain infections −/− mice. Undetected infection in the colony is an additional factor that should be considered to interrogate differences between divergent results in different animal units.Another possibility that might explain differences between findings at different sites is the health status of the animals. IL-1 is a key mediator in immune responses and IL-1RI−/−; B6.129S7-Il1r1tm1Imx/J; Stock number 003245) were obtained from an in-house colony, originally imported from Jackson Laboratories, USA. The initial generation of these mice Il1r1 was generated by homologous recombination in 129/SvEv AB1 ES cells and targeted mutant mice were subsequently backcrossed 7 times to C57BL/6 background (that was not C57BL6/J). The IL-1RI−/− animals supplied by JAX have remained at generation N5+N2F2 (since October 2009). As such they were initially a C57/129S7 hybrid and were backcrossed 5 times onto a C57 background before importation by JAX and a further 2 times onto a C57BL6/J background after importation by JAX. We have subsequently maintained them as an inbred colony and have used C57BL6 as our control strain. The studies of Koo and Duman have also been performed with these mice. A recent (March 2011) 32 single nucleotide polymorphism panel analysis of the JAX mice, with 27 markers covering all 19 chromosomes and the X chromosome, and a further 5 markers distinguishing between C57BL/6J and C57BL/6N sub-strains, was performed on the re-derived living colony at The Jackson Laboratory Repository. 26 of 27 markers throughout the genome indicate a C57BL/6 genetic background (http://jaxmice.jax.org/strain/003245.html). Furthermore, 3 of 5 markers that distinguish C57BL/6J from C57BL/6N indicated that the mice originally sent to JAX were on a C57BL/6N genetic background or a mixed C57BL/6J;C57BL/6N genetic background. As such, we would argue that C57BL/6 mice are an appropriate control strain. It is of interest that the studies of Koo and Duman were also performed with C57BL/6 controls, but not C57BL6/J originating from JAX. Our data demonstrating decreased anxiety, increased activity and a limited effect on cognitive performance are consistent with the Koo and Duman studies −/− animal: the B6;129S1-Il1r1tm1Roml/J (Stock Number:003018), which is maintained as a hybrid strain (129S1/Sv * C57BL/6 cross). Thus a 129/Sv X C57BL/6 cross was used as the control strain for their initial and subsequent studies −/− mice, the effects of deletion of IL-1RI−/− may be different depending on the background strain of mouse.Another possible reason for the divergent results observed between our study and those of the Yirmiya group is a difference in strains used in these studies. In our studies female or male Interleukin 1 receptor 1 knock-out mice  are confounding factors that could be misinterpreted as cognitive impairments.It is worth pointing out that there are also experiments with IL-1ra −/− mice are not robustly impaired on hippocampal-dependent learning and memory tasks but do display a lower level of anxiety compared to WT mice. The study suggests that IL-1 signalling is involved in normal anxiety responses and neuroanatomical and neurochemical investigations for the basis of these effects should continue in knockout animals and littermate controls.In conclusion, these results suggest that, contrary to prior published studies, IL-1R1