Semantic memory is a crucial higher cortical function that codes the meaning of objects and words, and when impaired after neurological damage, patients are left with significant disability. Investigations of semantic dementia have implicated the anterior temporal lobe (ATL) region, in general, as crucial for multimodal semantic memory. The potentially crucial role of the ventral ATL subregion has been emphasized by recent functional neuroimaging studies, but the necessity of this precise area has not been selectively tested. The implantation of subdural electrode grids over this subregion, for the presurgical assessment of patients with partial epilepsy or brain tumor, offers the dual yet rare opportunities to record cortical local field potentials while participants complete semantic tasks and to stimulate the functionally identified regions in the same participants to evaluate the necessity of these areas in semantic processing. Across 6 patients, and utilizing a variety of semantic assessments, we evaluated and confirmed that the anterior fusiform/inferior temporal gyrus is crucial in multimodal, receptive, and expressive, semantic processing. Semantic memory or conceptual knowledge is a crucial higher cognitive function. It allows us to code and retrieve the meaning of words, objects, sounds, faces, etc., which has been acquired across a lifetime of verbal and nonverbal experience. Additionally and perhaps reflecting its core essence, semantic memory allows us to generate coherent concepts which license generalizations based on meaning rather than surface similarities . The forThe principal evidence for the importance of the ATL in semantic representation arose from the study of semantic dementia (SD), in which a selective pan-modal, pan-category semantic deficit arises from atrophy focused on the anterior temporal region, bilaterally . The affThe current investigation was concerned with the role that ventral aspects of the ATL might play semantic memory. This question was motivated not only on theoretical, but also clinical, grounds given that this region is often included in resection for temporal lobe epilepsy, unless a subtemporal procedure for hippocampectomy is used . In compThere are some strong hints in the literature that the ventral ATL might play an important role in multimodal semantic representation. Although this region is primarily considered to be the apex of the ventral visual stream , it is bThe current study directly tested the contribution of the vATL region to semantic processing through the implantation of subdural electrode grids, for the presurgical evaluation of patients with partial epilepsy and brain tumor. Unlike functional neuroimaging or TMS alone, this method offers dual unique opportunities: by placing a grid over a region of interest (ROI), it is possible not only to evaluate the functionally related cortical activity directly , but also to induce temporary disruption of the function at that site, within the same participant. This is not, of course, the first study ever to have used this method to probe the ventral ATL. In clinical neurosurgery, the same region has been regarded as an important language area—the basal temporal language area (BTLA)—ever since electrical stimulation of this cortical region was found to impair reading and naming , 1991, aAcross a series of investigations, therefore, we explored semantic processing of the vATL through the implantation of grid electrodes. The study adopted 3 key methodological approaches. First, we collected both cortical stimulation data and cortical-evoked responses in the same participants, so that we could directly compare functionally related LFPs and test if these same functions were disrupted by transient stimulation . Secondly, to license direct comparability with the previous neuropsychological and neuroimaging studies reviewed above, we utilized the same assessment materials and experimental methods leading not only to novel insights about the neural basis of semantic memory, but also to new clinical stimulation methods. Finally, we should note that, given the rarity of the clinical procedure and the limited assessment time with each patient, it was impossible to conduct the large experimental test battery with all patients. Instead, we conducted a core stimulation and evoked potential protocol (Investigations 1 and 2) with all patients and supplemented these with more in-depth investigations across pairs of cases to build up a more complete picture of the nature and extent of semantic processing in the ventral ATL.We recruited 6 patients with either intractable partial epilepsy (5 patients) or brain tumor 1 patient) who underwent subdural electrode implantation in the left (Patients 1–5) or right (Patient 6) hemisphere for presurgical evaluation. The subdural electrodes were constructed of platinum with an interelectrode distance of 1 cm and a recording diameter of 2.3 mm . The patients' demographics are summarized in Table  patient This study was approved by the ethics committee of the Kyoto University Graduate School of Medicine (no. C533), and the patients gave written informed consent.One hundred  line drawings were collected . All couElectrocorticogram (ECoG) was recorded during the picture naming task with a band-pass filter of 0.016–600 Hz and a sampling rate of 2000 Hz in Patients 1 and 3, and with a band-pass filter of 0.016–300 Hz and a sampling rate of 1000 Hz in Patients 2, 4, 5, and 6. Pictures were presented one at a time, every 5 s, on a PC screen.Supplementary Fig. 1). After confirming the reproducibility, grand average waveforms (for the whole test session) were computed . A time window of 1800 ms was set with activity in the 300 ms preceding the picture presentation serving as the baseline for measurement. Average waveforms were computed for the first and second half of the experiment to assess the consistency of the LFPs . To generate Figure 1C, we undertook the following steps. Consistent with the critical methodological factors for maximizing ATL-related semantic activations established by Previous studies have obtained important evidence about the vATL region by directly comparing results from contrastive neuroscience techniques . Details of the methodology for cortical stimulation and the subsequent cortical mapping have been described elsewhere .For mapping the vATL, after confirming the absence of positive  and negative  tongue motor responses, a series of semantic and language assessments was tested concurrent with the 4- to 5-s period of electrical stimulation at an intensity of 10–15 mA. For the assessment battery shown below , responsThe patients were asked to read aloud of a part of children's story (80–120 kana and kanji mixed words).The patients were asked to name of 6 familiar animate and 6 nonanimate objects. The 6 items in each category were printed in black on a white card and were shown simultaneously.Written names of the 6 nonanimate items used for picture naming were presented for reading aloud either in kanji or kana scripts . All 6 words were written in black on a white card and were presented to the patients to read. Kanji is a semi-opaque orthography, whereas kana has a direct one-to-one correspondence to its phonemic form (except for the unmarked pitch accent). Consequently, both types of reading require visual decoding and speech production, but kana reading requires much less lexical-semantic support to generate the correct pronunciation .The patients were asked to make gestures following a simple spoken sentence .From its spoken name, patients were asked to touch the target picture from 6 line drawings, all selected from the same animate/nonliving category.The target was probed with a written name in the center of the panel. Ten colored pictures (1 target and 9 foils) from the same category were surrounding the written probe. This test was conducted just for Patients 1 and 2 while stimulating the core ROI (see below for definition).The core semantic/language ROI within the vATL was defined as the cortical region, which exhibited both robust LFPs for naming (Investigation 1) and marked language impairment by stimulation. Regarding the definition of a robust LFP, N200 and N400/P400 have been reported to be language-specific . In addiRather than probing semantic and language performance on many tasks across a large number of grid locations, by using a limited number of trials per stimulation site, with clinically maximal stimulation , Investigation 3 focused on the semantic–language ROI (see above) and utilized a much greater number of trials to establish the relationship between graded levels of stimulation intensity and performance on an expressive (picture naming) and a receptive (written word-picture matching) semantic task. In a new approach, by using graded stimulation over a single target location, we were able to vary the intensity from 0 (sham) to 9 mA to investigate the finer, graded relationship between stimulation and impaired function. Both reaction time (RT) and accuracy were measured, given that RT can be more sensitive to the presence of mild impairments . The motA), and psycholinguistic variables , we confirmed the patients' normal semantic ability on the 2 assessments and used the data to form the baseline for the later stimulation study. The patients were tested on naming of 80 colored pictures (≥90% accurate) and on a 10-alternative (within category) written word-picture matching task comprising the same 80 target items (≥95% accurate). Both assessments were Japanese versions modeled on the Cambridge 64-item semantic battery test . Based o3The electrode pair in the core semantic–language ROI (see above) was selected. It was A12/A17 in Patient 1 and B14/B19 in Patient 2. In Patient 2, another core ROI was identified at a more medial position , 3, 6, and 9 mA .In both tasks, each trial started with a fixation period of 2 s, followed by display of the material  for 8 s, and thus an intertrial interval of 10 s. In the naming task, the patient was asked to name the picture aloud as soon as possible. In the matching task, the patient was asked to say the number shown next to the target picture. A 50-Hz electrical stimulation was given for 3 s from 1 s before the display of the material  to 2 s after the display of the material. Time-locked stimulation was given for intervention in each trial, that is, every 10 s to give intervention to all trials. First, we performed graded stimulation to the picture naming task at 4 levels of intensity in the ascending order . After 30 min break, we conducted the word-picture matching task in the same ascending stimulation order. Following the standard instructions for the Cambridge Semantic Battery, we conducted naming before word-picture matching so as to minimize any cueing/priming effects on naming . In all sessions, ECoG was carefully examined to monitor possible afterdischarges induced by the 3-s stimulation. All sessions were video-recorded and the verbal responses were recorded with a microphone that was placed in front of each patient. The RT for correct trials (without afterdischarges) was submitted to the analysis. Task performance was assessed by accuracy and RT for each stimulation intensity  and a difficulty(RT)-matched number judgment task . The aiNaming LFPs were collected using the same methods and materials as summarized in Investigation 1. Patient 4 and 5's naming LFPs are reported under Investigation 1 Fig. . The samStimuli were presented on the PC screen every 5 s. The participants were asked to respond to the stimuli by pressing 1 of 3 designated buttons. One session consisted of 51 trials and 6 sessions (3 synonym and 3 number judgment tasks) were performed alternately. The recording method and parameters for ECoG were the same as in Investigation 1 (naming) with a band-pass filter of 0.016–300 Hz and a sampling rate of 1000 Hz. The method of offline averaging of ECoG was the same as naming LFP.A similar methodology and approach to Investigation 3 was used in this study. Prior to surgery, Patient 4 completed the synonym and number judgment tasks as well as picture naming (the same items as Investigation 3). From these baseline assessments, we selected 60 correct trials for synonym judgment, 20 trials for number judgment, and 20 trials for naming during concurrent stimulation. Synonym judgment was split into 3 conditions (20 items for each cell) varying the type of word  but matching decision times. Trials for the number judgment task were selected so that the baseline decision times were equated to the synonym task. Baseline naming times were much faster than the synonym and number judgment tasks, and thus naming trials could not be matched by RT.For Patient 4, electrodes D08/D13 were identified as the core semantic–language ROI (see above). For Patient 5, electrodes E13 were identified as the core semantic–language ROI (stimulation was delivered to the pair of electrodes E13/E18). Stimulation was delivered time-locked to the onset of the task presentation for 3 s thereafter. A fixed intensity  was used. Afterdischarge was not induced. RTs, accuracy, and errors were recorded for later evaluation and analysis. The results are summarized in Figure T1-weighted volume data acquisition. MPRAGE volumetric scan was performed before and after implantation of subdural electrodes as a part of presurgical evaluations. In the volumetric scan taken after implantation, the location of each electrode was identified on the 2D slices using its signal void due to the property of platinum alloy  taken after grid implantation were non-linearly coregistered to the T1 volume acquisition taken before implantation , then to the MNI standard space (ICBM-152) using FNIRT of the FSL software (www.fmrib.ox.ac.uk/fsl/fnirt/). This method has been reported elsewhere for standardization of the electrode locations (A magnetization-prepared rapid gradient-echo (MPRAGE) sequence was applied for anatomical um alloy . To compocations .Supplementary Fig. 1 for the full LFP maps) were observed at and around the left (Patients 1–5) and right (Patient 6) anterior fusiform gyri . A negative activity with a peak latency of 230–300 ms was observed in 2 patients (1 and 3), followed by a positive/negative activity peaking at 310–450 ms in 5 cases (1–5). Patient 6 showed a negative activity with peak latency around 300 ms, similar to left cases. Figure 1A shows the LFP for each patient at this site. It is striking that this location is almost exactly the same as the site of peak activation observed for a range of different verbal and nonverbal semantic tasks using distortion-corrected fMRI or PET in neurologically intact participants —indicating a strong convergence of evidence across LFP, fMRI, and PET. In the subsequent investigations, therefore, the necessity of this region  was tested and confirmed in a series of direct stimulation experiments.In all 6 patients, LFPs were recorded during the semantic task of picture naming. Robust responses . At the strongest intensity level, RT was significantly slower than that before stimulation (P < 0.05). Accuracy also declined at this stimulation intensity. Additionally, this milder stimulation method evoked errors of commission as well as omission in Patient 1. All errors were semantically closely related , suggesting that the nature of the stimulation-induced impairment was semantic, like that found in SD patients. Patient 2's errors were dominated by omissions (the most common naming error type in SD).For 2 patients (1 and 2), the precise function of the same anterior fusiform–ITG region was more thoroughly investigated by using a novel graded stimulation protocol with expressive (picture naming) and receptive semantic tasks . As can be seen in Figure 3B, for both patients, the effect on naming was significantly greater than that on word-picture matching . This outcome directly mirrors SD patients' rapid decline in naming accuracy compared with word-picture matching as the disease progresses . In comparison, the nonsemantic number judgment task did not generate any reliable LFPs in this area, indicating that processing in the region is semantically selective. The results of direct stimulation mirrored the LFP findings. The analysis of Patient 4’s RT data revealed a significant stimulation × task interaction . This pattern  reflected the fact that 7 mA/3 s concurrent stimulation of the anterior fusiform–ITG significantly and substantially slowed synonym judgments for all word types  and naming responses , but had no effect on number judgments . On the more demanding synonym judgment task, concurrent stimulation also reduced Patient 4's accuracy from 100% at baseline to 65% (McNemar P < 0.001). As found in Investigation 3, the lower level of stimulation allowed generation of a semantically related naming error (penguin→ “pelican”), again underlining the notion that this region is crucial for semantic processing. There was no change in Patient 4's accuracy on the number judgment tasks. A very similar result was obtained for Patient 5. There was a significant stimulation × task interaction in response times . Again, this interaction reflected a substantial and significant slowing of RTs for synonym judgments  and naming , but a much smaller effect on number judgments . In comparison with Patient 4, Patient 5's accuracy on the naming and synonym judgment tasks was much more strongly suppressed by stimulation . All of the Patient 5's stimulation-induced naming errors were omissions. In comparison with the 2 semantic tasks, a small drop in accuracy in the number judgment task was not significant (McNemar P = 0.13). In summary, the stimulation and LFP methods produced highly convergent findings, namely that the anterior fusiform/ITG is a semantically selective region.Like Patients 1–3, robust LFPs were observed during naming in the anterior fusiform–ITG for Patients 4 and 5  The apparent disparity between stimulation of the vATL versus resection of it; (2) the localization of cortical stimulation; and (3) the relationship of the vATL to other regions implicated in multimodal semantic processing.From a general clinical perspective, there appears to be a disparity between the apparently minimal neuropsychological deficits of patients with ATL resection in the chronic phase versus the semantic impairment that follows from electrical stimulation of the vATL or is found in SD. Post-surgical language/semantic impairment is much more obvious in the acute stage, but diminishes over time. Using sensitive measures, however, various studies have found evidence for remaining semantic impairment underpinning the patients' anomia even in the chronic phase . The parand also the areas to which it is connected. Indeed, a combination of local and remote stimulation could amplify the observed impairment. To test this hypothesis, future studies are required, in which grid electrodes are implanted over the vATL-connected component of Wernicke's area to test if stimulation leads to a selective semantic impairment. While we cannot rule out this possibility at this stage, we think it may be unlikely for 3 reasons. First, previous neurophysiological studies have found that current density is maximal only beneath the stimulated electrode , we performed graded-intensity stimulation at a single location (the anterior fusiform–ITG) to investigate the relative effects on expressive (naming) and receptive (word-picture matching) semantic tasks. This new methodology was inspired by previous studies of the disease progression in SD patients, which have used this neurological model to establish the relationship between the degree of damage to the underlying semantic system and the resultant performance level across different tasks . Thus, lIn conclusion, the present systematic, combined stimulation and LFP study located semantic processing in and around the anterior fusiform–ITG. This neuroanatomical location is in good agreement with the activation foci for semantic processing in various functional neuroimaging studies. The measured semantic function and induced transient impairment at the anterior fusiform–ITG was found to be both multimodal  and pan-category , and graded stimulation of the region elicited a similar form of semantic impairment as observed across the disease progression of SD. Taking these findings together, the anterior fusiform–ITG appears to be critically important for semantic processing. A future prospective neuropsychological study of patients who need additional resection of the vATL during subtemporal hippocampectomy, which utilizes advanced neurosurgical techniques to avoid resection of the anterior ventral temporal surface and the temporal stem , is needSupplementary material can be found at http://www.cercor.oxfordjournals.org/.26282218 (R.M.) and 26293209 (A.I.), grant-in-aid for Exploratory Research 26560465 (R.M.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), grants-in-aid for Research on Measures for Intractable Diseases (H26-Nanjii-Ippan-051) (A.I.) from the Japanese Ministry of Health, Labor and Welfare, and SPIRITS (Supporting Program for Interaction-based Initiative Team Studies) (R.M.) from Kyoto University. Funding to pay the Open Access publication charges for this article was provided by an RCUK block grant to the University of Manchester.This research was supported by an MRC-UK (MR/J004146/1) program grant (MALR), grants-in-aid for Scientific Research (B)


Recent efforts to evaluate the contribution of neonicotinoid insecticides to worldwide pollinator declines have focused on honey bees and the chronic levels of exposure experienced when foraging on crops grown from neonicotinoid-treated seeds. However, few studies address non-crop plants as a potential route of pollinator exposure to neonicotinoid and other insecticides. Here we show that pollen collected by honey bee foragers in maize- and soybean-dominated landscapes is contaminated throughout the growing season with multiple agricultural pesticides, including the neonicotinoids used as seed treatments. Notably, however, the highest levels of contamination in pollen are pyrethroid insecticides targeting mosquitoes and other nuisance pests. Furthermore, pollen from crop plants represents only a tiny fraction of the total diversity of pollen resources used by honey bees in these landscapes, with the principle sources of pollen originating from non-cultivated plants. These findings provide fundamental information about the foraging habits of honey bees in these landscapes. The extent to which non-crop plants may be contaminated by insecticides is not known. Here, the authors show that pollen collected by honey bees living in areas of intensive maize production is contaminated by a wide range of pesticides throughout the growing season, with the principle pollen source being non-crop plants. The declines of honey bees and other pollinators have been at the forefront of recent scientific publications and popular press. Accumulating evidence suggests that no single stressor alone is responsible for declines. Rather, it is probably a combination of abiotic and biotic factors acting in synchrony, to have a negative impact on pollinator populations12345789101112131516171816192223242572829303132Pollen collected by honey bees was collected, identified and screened for agricultural pesticides over a period of 16 weeks in 2011. We initiated this study after all planting of treated seeds in our study was completed, to minimize dust from pneumatic planters as a direct source of pesticide residues. Two Langstroth hives were placed at each of three sites: (1) an open meadow with wildflowers, woody shrubs and trees present , (2) the border of a maize field planted with seeds treated with the neonicotinoid clothianidin and three fungicides, and (3) the border of a maize field planted with seeds that received no pesticide treatment. To characterize the landscape surrounding honey bee hives, proportions of different land covers within a 2-km radius around each of the three sites were extracted from the 2012 Cropland Data Layer produced by the US National Agricultural Statistics Service using QGIS (QGIS Development Team 2015). Honey bee colonies remained at each site for the length of the growing season and foraging bees had free access to pollen from all crop and non-crop species in the vicinity. Analysis of forager-collected pollen revealed that in all cases honey bees foraged primarily on non-cultivated plants and residues of multiple pesticides were found throughout the season. Although a variety of agricultural pesticides were found at all sites, the contaminants likely to provide the greatest hazard to honey bees in our study were non-agricultural pyrethroid insecticides targeting nuisance pests such as mosquitoes.Pollen collected by honey bees was consistently contaminated with pesticides throughout the 16-week period and the overwhelming majority of pollen was collected from non-cultivated plants. Pesticide residue analyses of bee-collected pollen revealed contamination by up to 32 different pesticides spanning 9 chemical classes . The mosSinapsis arvensis, an insect-pollinated annual species that was particularly common at that site. Although pollen from non-cultivated plants dominated honey bee loads over the course of our experiment, foragers did occasionally visit soybean (Glycine max) and less frequently maize (Zea mays) to collect pollen, despite the distance from the meadow to surrounding crop fields , the herbicide metolachlor (83.3%) and the pyrethroid insecticides prallethrin and phenothrin (46.7 and 30% of samples) . Carbamap fields . Honey bAmbrosia artemisiifolia and two other pollen species, one unknown and another unidentified member of the Asteraceae. Although maize and soybean pollen was readily available in the vicinity during mid-summer, the majority of pollen collected by honey bees throughout the season came from non-cultivated plants , as well as the herbicides metolachlor and atrazine  . Of the d plants . ForagerPollen collected by honey bees at the maize field grown from pesticide-treated seed was contaminated with 28 pesticide residues, a number similar to the other sites. However, overall mean concentrations of active ingredients were significantly higher, up to 1,453 p.p.b. . FungiciFinally, we gathered honey bee toxicity data from the International Union of Pure and Applied Chemistry Pesticide Properties Database (http://sitem.herts.ac.uk/aeru/iupac), the US Environmental Protection Agency, the National Pesticide Information Center (http://npic.orst.edu/ingred/aifact.html#ecotox) and the open literature35This study demonstrates that the primary pollen resources used by honey bees living near field crop landscapes are non-cultivated plants, and that these pollen sources are frequently contaminated with a range of pesticides37Our study area is representative of a large portion of the United States that is characterized by intensive production of the top two crops, by land area, grown in the country—maize and soybeans377832333950) as an indicator of risk posed by a particular pesticide, a more comprehensive evaluation of pesticide risk to honey bees has been proposed. This approach incorporates the frequency of detection of pesticides in relevant matrices, namely pollen and nectar, to gain a more realistic picture of the risk of mortality posed to bees that are exposed to varying concentrations of pesticides with a range of toxicities50 values. Oral risk was calculated based on the amount of pollen a honey bee consumes in its lifetime during its role as a nurse bee (65 mg)50) and the frequency of encountering pesticide active ingredients in pollen. Nine active ingredients of the 32 detected in pollen samples were relevant in terms of posing a low , moderate  or high risk of mortality  to 50% of honey bees exposed to the chemicals in collected pollen43ca. 60 p.p.b. of neonicotinoid residues and may contribute to our results, as these dusts land on flowers that honey bees frequentThe pesticide concentrations we documented in pollen samples spanned levels that may have a range of effects from sub-lethal to acutely toxicλ-cyhalothrinλ-cyhalothrin, at all three sites . Conversely, highly toxic pesticides may pose relevant risk levels even if they are present only sporadically in pollen resources. These scenarios are especially important to consider in cases when two or more pesticides that exhibit synergy are detected simultaneously in pollen resources. We therefore calculated the potential risks posed by the frequency of synergistic mixtures between the fungicide propiconazole and certain neonicotinoidsee sites , 4, 5. Aλ-cyhalothrinλ-cyhalothrin for bumble beesNosema ceranaeGiven the nature of our study, whereby we allowed honey bees to visit flowering resources freely as they bloomed throughout the season, it is impossible to definitively determine the main source of pesticide contamination at any period. The frequent detection of the fungicides azoxystrobin and trifloxystrobin is probably the result of residual amounts of these active ingredients remaining in the soil of surrounding crop fields, given that these fungicides are common ingredients in the seed treatment formulations applied to maize seeds before planting. Coumaphos is a common miticide that is frequently used in bee-keeping, which probably accounts for its presence in honey bee pollen at all sites. Given that we initiated our study late in the spring when the planting of seed-treated crops in the region was complete, the detection of thiamethoxam and clothianidin in pollen samples at all sites was unlikely to be due solely to drift from planter dust (during the planting of treated seeds) landing directly on blooming flowers. Rather, it is more likely due to residual seed treatment compounds present in soils2643161752These findings provide guidance in filling crucial gaps in our current knowledge of the plants that honey bees preferentially visit  in landscapes that are dominated by monocultures of maize and soybean. For example, bees from all colonies in our study collected the vast majority of their pollen from non-crop plants even when pollen-producing field crops were abundant in the landscape, suggesting that pollen from these crops is not a preferred food source. Our work demonstrates that the pest-management practices employed both within crop fields and beyond have implications for honey bee and other pollinator populations in the area, as both urban and agricultural pesticides were relatively common in all types of pollen throughout the season. Finally, it is critical to point out that although we used honey bees as pollen collectors in this experiment, it is likely to be that a wider range of pollen-feeding animals, including many wild bees and other non-target invertebrates are also exposed to the same suite of pesticides810We selected six standard, or Langstroth, hives from the Purdue Honey Bee Lab  for inclusion in this study. Each consisted of two standard depth boxes with ten frames in each. Before the study, in early May of 2011, colonies were examined by pulling frames, ensuring that all colonies were queenright and of approximately equal strength. Sister queens from the same breeding line were used to ensure uniform genetic makeup of bees among treatments. Hives were then randomly assigned to each treatment. A bottom pollen trap  was placed in each hive and hives were placed on top of two 10 × 10 cm wooden blocks, ∼60 cm in length, to minimize contact with the ground. Bottom pollen traps are placed at the base (entrance) of hives and contain a metal screen with circular openings just large enough to allow honey bees to enter. Foragers with full pollen loads are unable to fit through these openings and as they attempt to move through pollen falls off and accumulates in a tray below. Vegetation was trimmed around the base of hives so the entrance was not blocked. All hives were placed on lands owned by Purdue University. At the agricultural sites, hives were placed 3 m from the field margin with the opening facing the maize field. The maize fields were ∼3 km apart. The non-agricultural site was located ∼12 km from the treated and untreated maize fields, but because of the intensive production of maize and soybeans in our study area Mephitis mephitis) on the following sample dates: Hive 1A (26 July), Hive 1B (24 May), Hive 2A  and Hive 2B (24 May). These ten sampling points were excluded from calculations of seasonal pesticide prevalence in pollen samples. Therefore, over the 16-week sampling period a total of 30 pollen samples were collected from colonies at the non-agricultural area, 24 samples from the colonies adjacent to the untreated maize field and 32 samples from the colonies adjacent to the neonicotinoid-treated maize field, for a grand total of 86 pollen samples across all sites. We added 30 ml of extraction solution (15 ml of 1% dH2O/acetic acid solution + 15 ml acetonitrile) to each tube and mixed thoroughly, followed by the addition of 6 g of anhydrous magnesium sulfate (MgSO4) and 1.5 g sodium acetate (NaAc). Tubes were mixed gently on an agitator for 10 min and then centrifuged. Fifteen millilitres of supernatant was removed and dispensed into a 15-ml Agilent dispersive Solid Phase Extraction tube containing 400 mg primary secondary amine, 400 mg C18 and 1,200 mg MgSO4. Two millilitres of toluene was added to each 15 ml Agilent tube, to aid in the extraction of planar pesticides from the pollen matrixPesticide residues were extracted from honey bee pollen using a modified QuEChERS methodThe data that support the findings of this study are available from the corresponding author on request.How to cite this article: Long, E. Y. & Krupke, C. H. Non-cultivated plants present a season-long route of pesticide exposure for honey bees. Nat. Commun. 7:11629 doi: 10.1038/ncomms11629 (2016).


The effects of prenatal nutrition on adult cognitive function have been reported for one generation. However, human evidence for multigenerational effects is lacking. We examined whether prenatal exposure to the Chinese famine of 1959–61 affects adult cognitive function in two consecutive generations. In this retrospective family cohort study, we investigated 1062 families consisting of 2124 parents and 1215 offspring. We assessed parental and offspring cognitive performance by means of a comprehensive test battery. Generalized linear regression model analysis in the parental generation showed that prenatal exposure to famine was associated with a 8.1 (95% CI 5.8 to 10.4) second increase in trail making test part A, a 7.0 (1.5 to 12.5) second increase in trail making test part B, and a 5.5 (−7.3 to −3.7) score decrease in the Stroop color-word test in adulthood, after adjustment for potential confounders. In the offspring generation, linear mixed model analysis found no significant association between parental prenatal exposure to famine and offspring cognitive function in adulthood after adjustment for potential confounders. In conclusion, prenatal exposure to severe malnutrition is negatively associated with visual- motor skill, mental flexibility, and selective attention in adulthood. However, these associations are limited to only one generation. Gestation is a crucial period for rapid brain development. Adequate nutrition during intra-uterine period is especially important for the formation of the brain, which lays the foundation for future cognitive development throughout childhood and adulthood. Evidence from animal studies shows that under-nutrition in early life leads to impaired learning performance in adult rats357et al. found that prenatal exposure to the Dutch famine did not affect the ability for abstract reasoning rates or rates of mental retardation among 19-year old menet al. showed that 59-year-old men and women who were exposed to the Dutch famine in utero had lower performance on a Stroop-like taskEpisodes of famine that occur in human history provide a natural experiment for testing the hypothesis in human beings. Stein The 1959-61 Chinese famine, which was caused mainly by a sudden and sharp drop in grain, was one of the most serious famines in human historyThis study aims to determine the multigenerational effects of parental prenatal exposure to the 1959–61 Chinese famine on adult offspring cognitive function in a large retrospective family cohort study.This study was conducted in the Suihua Beilin rural area of Heilongjiang province, China. A total of 2124 parents and 1215 offspring from 1062 families were involved in this study . Of the In parental generation, participants who have been exposed to famine during intra-uterine period performed worse on Trail Making Test (TMT) and Stroop Color-Word Test (SCWT) compared with the unexposed participants. Generalized linear regression model analysis showed that prenatal exposure to famine was associated with a 8.7 (95% CI 6.5 to 10.9) second increase in part A of TMT (TMT-A); a 7.7 (2.3 to 13.1) second increase in part B of TMT (TMT-B); and a 6.1 (−7.7 to −4.5) score decrease in SCWT in adulthood. A total of 157 subjects (70 exposed and 87 nonexposed) reacted faster than average and also had less than 40% correct responses, which indicates the possibility that they were inattentive to SCWT. The associations were minimally changed  after excluding the 157 participants and further adjustment for potential confounders . The powThe linear mixed model analysis in the offspring generation indicated that the offspring of one or both exposed parents have a significantly lower score on the SCWT test, without adjustment for confounders. Parental exposure to famine during intra-uterine period was associated with a 1.5  or a 1.3  score decrease in SCWT test in the offspring generation. However, the associations between parental famine exposure and offspring SCWT score [β = −0.5 (−1.3 to 0.3) for maternal exposure and β = −0.4 (−1.0 to 0.3) for paternal exposure] became non-significant after excluding 95 participants who were likely inattentive to SCWT and the adjustment for potential confounders .This large retrospective family cohort study from a unique famine cohort demonstrates that prenatal exposure to famine was negatively associated with performance on TMT and SCWT in adulthood. TMT is generally believed to be a test for cognitive domains of visual- motor skills and mental flexibility2022et al. reported that exposure to famine during the early stage of gestation induced worse performance on a selective attention task at age 56 to 59The effects of prenatal exposure to famine on adult cognitive function have been investigated in the Dutch famine cohort study. However, the results of these studies were not consistent. Stein and Groot found no overall association between prenatal famine exposure and cognitive performance at the age of 19Pregnancy and infancy are the key periods for brain formation, which is the foundation for the development of cognition in childhood and adulthood. It seems reasonable that prenatal exposure to famine is closely associated with adult cognitive function in this study. The biological mechanisms through which malnutrition in pregnancy and infancy influence brain development and cognitive function may be involved in each crucial period of brain development. Five key neurodevelopmental processes are involved during early development, namely, neuron proliferation, axon and dendrite growth, synapse formation, myelination, and neuron apoptosisSome studies report that a damaged brain caused by early nutrient deficiency may be recovered after nutrient repletion during a time period, especially during pregnancy and infancy, when the affected neurodevelopmental process is ongoing31Several recent studies suggest that adult cognitive function can be affected by early-life nutritional exposure through epigenetic modifications in the brain, such as DNA and histone methylation57This negative finding can be interpreted from the following three aspects. First, the absence of significant associations between parental prenatal exposure to famine and offspring cognitive function in adulthood may be attributed to the age of the offspring generation; for example cognitive development is still a dynamic ongoing process at around 27 years because brain development is now known to proceed until the third decade of lifeThis study should be integrated in the light of its limitations. First, the Chinese famine lasted for three years, which means that the exposed participants in this study actually experienced the famine during the intra-uterine and infancy periods. Therefore, it was difficult to distinguish the effects of exposure to famine on adult cognitive function during the intra-uterine and infancy periods. However, the previous study suggested that the intra-uterine period can be considered as the primary critical period36In conclusion, the current study indicates that prenatally severe malnutrition have negative effects on visual- motor skill, mental flexibility, and selective attention in adulthood. However, these associations are limited to only one generation.The participants were selected from the Suihua Beilin rural region of Heilongjiang province, which is located northeast of China. Suihua region is located in south central Heilongjiang province, and has a population of about 900 thousand, 550 thousand of which lived in Beilin rural area. This area suffered from severe famine from 1959 to 1961. The average grain production during 1956–58 was 2.1 million ton per year, which decreased to 1.3 million ton per year during 1959–61 and recovered to 2.0 million tons per year during 1962–64 .st 1959 and September 30th 1964, who were selected from the 111,536 households in Suihua Beilin rural area. To minimize misclassification of the famine exposure periods, 277 households who have a parent born between October 1st 1961 and September 30th 1962 were excluded because the exact dates of the start and end of the Chinese famine were difficult to confirm.In this study, participants were recruited based on the household unit that includes two generations . By means of the Suihua household registration record, 1856 households have both F1 parents born between October 1The 1579 families were further identified from a door-to-door census and invited to participate in this study. The additional family inclusion criteria were as follows: 1) both parents and their children can participate in this study; 2) they lived in the Suihua region for at least three generations; and 3) they provided a written informed consent. A total of 1148 households were selected to participate in the cognitive assessment. As some participants did not complete the assessments, 86 (7.5%) households were further excluded from the 1148 households. The total sample size in this study was 1062 households including 2124 F1 parents and 1215 F2 offspring.st 1959 and September 30th 1961 were classified as prenatal famine exposure, and parents born between October 1st 1962 and September 30th 1964 were classified as non-exposedBased on previously published criteria, F1 parents born between October 1All procedures comply with the Declaration of Helsinki and were approved by the Ethical Committee of Harbin Medical University.2.A detailed interview was conducted by a research staff to collect each participant’s basic information, which included offspring characteristics at birth  and parental and offspring characteristics in adulthood . Educational level was classified as > high school, high school, and < high school. Economic status was evaluated based on the mean annual income. According to the criteria of the 2002 China National Nutrition and Health Survey, 2000 Chinese yuan per person per year was used as a cutoff point for economic statusA comprehensive neuropsychological battery test was administered to all subjects (parents and offspring) in Chinese, including the following tests.The general intelligence of the participants was assessed using Part 1 of AH4 testAVLT assesses verbal learning capacity, as well as recall and retrieval from short-term and long-term memory. In this test, the examiner will read a semantically unrelated word list to the examinee. The subject is asked to recall these words after presenting the entire list. The learning phase and recalling phase are repeated three times. A five-minute nonverbal task is then conducted, after which the subject will recall the words for the fourth time. The subject will take a 20-minute nonverbal test, and then recall the words again for the fifth time. The indicators are recorded as follows: 1) AVLT short-term memory (AVLT-I): the sum score of recall accuracy of the first three repetitions with a full mark of 36; and 2) AVLT delayed recall (AVLT-II): the recall score of the fifth time with a full mark of 12This test assesses language and retrieval from long-term semantic memory. The subject is asked to name as many animal items as possible within one minute. The correct number of animals is then recordedIn this test, both visuo-spatial constructional ability and visuospatial memory are measured. The subject is asked to copy a figure (CFT-I), and then draw the figure from memory after about 25 minutes (CFT-II)41In part A of TMT (TMT-A), the subject is timed to connect the Arabic numbers 1 to 25 in sequence as fast as possible. In part B of this test (TMT-B), the Arabic numbers are surrounded by either squares or circles, and the subject is asked to switch between number and shapes. In this test, visual conceptual (TMT-A) and visuo-motor tracking (TMT-B) are assessed20This test measures executive function, specifically selective attention. The test used in this study was a short version of a single trail Stroop-like test. A name of a color was printed in one of the four different ink colors. Subjects had five seconds to identify the color of the ink rather than the color of the words spelled and to choose the right option out of four names of colors printed in different ink colors. Total test time was five minutes. The outcome of this test was the time in seconds (response time) of responding to each item, as well as the percentage of correct answers (score)22All statistical analyses were performed using SPSS Statistics v20.0 (IBM Corp. USA), with α = 0.05. We used chi-squared tests and analysis of variance to compare the differences in categorical and continuous variables among the groups, respectively, in parental and offspring generations.In the F1 generation, generalized linear regression models were used to investigate the associations between prenatal famine exposure and cognitive functions in adulthood. Sex, ageP value of the fixed effect was corrected by FDR. When the FDR corrected-P value of the fixed effect was significant, post-hoc Tukey test was used to perform the multiple (six times) pairwise tests among neither, maternal, paternal, and bilineal.In the F2 generation, to consider the correlation of characteristics between multiple offspring of the same parent, we applied mixed linear model to assess the associations between parental prenatal exposure to famine and cognitive functioning in adult offspring, with the family number as a random effect and parental famine exposure  as the fixed effect. In addition to offspring characteristics in adulthood , offspring characteristics at birth , which had been shown to be associated with cognitive performance505152To rule out the influence of inattention or indifference in Stroop task, we conducted a secondary analysis after excluding the subjects with faster than mean reactive time, but less than 40% correct responses.P value of the association between prenatal exposure to famine and adult cognitive function was statistically significant, the statistical power, which is the probability of correctly rejecting the null hypothesis given the specified sample size, was calculated using PS Power and Sample Size Calculations version 3.1.2When the How to cite this article: Li, J. et al. Multigenerational effects of parental prenatal exposure to famine on adult offspring cognitive function. Sci. Rep.5, 13792; doi: 10.1038/srep13792 (2015).


Autism spectrum disorders (ASD) comprise an etiologically heterogeneous set of neurodevelopmental disorders. Neuroligin-3 (NL-3) is a cell adhesion protein that mediates synapse development and has been implicated in ASD. We performed ex-vivo high resolution magnetic resonance imaging (MRI), including diffusion tensor imaging (DTI) and behavioral  tests at 3 different time points  on NL-3 and wild-type littermates to assess developmental brain abnormalities in NL-3 mice. MRI data were segmented in 39 different gray and white matter regions. Volumetric measurements, along with DTI indices from these segmented regions were also performed. After controlling for age and gender, the NL-3 knock-in animals demonstrated significantly reduced sociability and lower anxiety-related behavior in comparison to their wild type littermates. Significantly reduced volume of several white and gray matter regions in the NL-3 knock-in mice were also observed after considering age, gender and time point as covariates. These findings suggest that structural changes in the brain of NL-3 mice are induced by the mutation in the NL-3 gene. No significant differences in DTI indices were observed, which suggests that the NL-3 mutation may not have a profound effect on water diffusion as detected by DTI. The volumetric and DTI studies aid in understanding the biology of disrupting function on an ASD risk model and may assist in the development of imaging biomarkers for ASD. Autism spectrum disorder (ASD) comprises a complex and etiologically heterogeneous group of neurodevelopmental disorders with an unknown unifying pathogenesis. While ASD is defined by the presence of deficits in social interaction as well as restricted and repetitive patterns of behavior, ASD is nevertheless highly heterogeneous in terms of behavior and genetic abnormalities Magnetic Resonance Imaging (MRI) has been employed extensively to examine morphological changes in human ASD High resolution MRI studies of the adult NL-3 knock-in mouse brain (108 day-old) indicated reduced volume in several regions of the brain, compared to wild-type (WT) littermates. Despite, volumetric differences, no differences were noted in any of the DTI parameters in this study ad lib. With the exception of a weekly cage change, mice were not handled until after the completion of all behavioral tests. To avoid any confounding effects, similar conditions for housing, feeding and handling were used throughout the study. This study was started during the spring of 2011 (4/2011) and completed by in the fall of 2011 (10/2011). A few animals were randomly selected after each time point  for imaging. Details of the animal distributions including age, gender and strains for behavioral  and imaging studies are summarized in A breeding pair of NL-3 knock-in mice (JAX strain number 008475) was procured from The Jackson Laboratory , bred in-house at the University of Pennsylvania, and offsprings were genotyped using protocols from Jackson Labs. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC). At 2–4 days postnatally, litters were culled to two males and two females to ensure adequate nutrition for all pups and a more uniform social environment during development and to also ensure that litters were balanced in the numbers of males and females. Mice were group housed in a light and temperature controlled animal facility that is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Males were removed from breeding cages prior to the birth of pups. All efforts were made to minimize animal pain and discomfort. Water and standard rodent chow were available Social Approach Test  was performed on NL-3 knock-in mice and WT mice at 28 (juvenile), 48 (peri-pubescent) and 68 (early adult) days-of-age using a 3-chambered Plexiglas apparatus under dim lighting (<2 lux) during testing in order to minimize the general stress level of the mice, as described previously Anxiety-related behavior was also assessed in these animals using the elevated zero maze test, which was performed a day after the social approach test  at approximately the same time of the day. A circular variant of the elevated plus maze, the zero maze apparatus consists of a raised circular track divided into two open and two closed quadrants. The track had an internal diameter of 40.5 cm and a width of 5.1 cm and was elevated off the floor at a height of 40 cm. The closed quadrants had walls that were 11 cm high. The animal was placed into the center of a closed quadrant and observed for 5 min. Digitized video of each 5 min trial was scored manually to determine the total time spent in open and closed quadrants and the number of transitions between each open and closed quadrant After completion of the zero maze tests, a subset of NL-3 knock-in mice [n = 25 ] and WT littermates [n = 24 ] were sacrificed at each time point to perform high resolution MRI on extracted brain samples. Because we randomly selected a subset of animals after the zero maze test for imaging, not all animals had behavioral data at all three time points. The distribution of age, gender and strain of all animals used in this study is summarized in Prior to imaging, brain samples were removed from the fixative and switched back to PBS solution for 48 hours at 4°C to rehydrate the tissue. After 48 hours, the sample was removed and rinsed twice with fresh PBS solution and blotted using a soft tissue paper to remove any remaining PBS. The brain was then placed in a plastic tube filled with proton-free susceptibility-matching fluid .2/s, FOV (field of view)  = 17 mm×8.5 mm×10 mm; acquisition matrix size = 136×68×80, resulting in a 125 µm isotropic resolution. The diffusion-weighted images were acquired with diffusion weighting in 6 non-collinear directions with both positive and negative diffusion gradient amplitudes in addition to 2 reference scans with minimal diffusion weighting for a total of 14 data sets resulting in a total acquisition time of 13 hours and 19 minutes per sample.High resolution DTI was performed on a 9.4T, 8.9 cm vertical bore magnet equipped with a 55 mm inner-diameter 100 gauss/cm gradient tube and interfaced to a Direct Drive console  running the vnmrj 2.3.C software version. All MRI studies employed a horizontally mounted 2 turn, 11×20 mm (ID x length) solenoid RF coil for transmit and receive. The plastic tube containing the brain sample was placed inside the coil and the probe was mounted inside the magnet. Scout images were acquired in 3 orthogonal planes to localize the position and orientation of the brain sample. DTI data was then acquired using a 3D multi-echo pulsed-gradient spin echo sequence Image reconstruction was performed offline using in-house custom software developed in the IDL programming environment . Following application of a Gaussian filter and 3D Fourier transformation, the magnitude images from each echo train were summed in order to improve SNR. Data sets acquired with opposite polarity diffusion weighting gradients were then combined in order to minimize contributions of background gradients to the diffusion data −3 mm2/s), and radial diffusivity (RD). The regional brain volumes were computed and divided by the total brain volume from each individual sample to account for any differences in volumetric differences associated with extraction or fixation of the tissue.The b = 0 images for each mouse were registered to a general brain template in preparation for automated region based segmentation such that the same ROI was assessed from each specimen. The alignment and segmentation routines were performed using Advanced Normalization Tools (ANTs). The Camino Toolkit Descriptive statistics were computed for all study variables. Because animals were subjected to social approach and elevated zero maze tests repeatedly over time, a linear mixed effect model was used to analyze the behavioral data which accounts for possible correlation in outcomes collected from the same animal over time. Three factors were considered in this model: group (WT vs NL-3), gender  and age/time points  as covariates. The three-way interaction effect was first tested using a Wald test followed by testing the two-way interaction effects using likelihood ratio tests. As there was no evidence for interaction in the social sniffing time, the results from the final model that included only the main effects terms are reported. For the anxiety data, the results from a model that contains the main effects terms and an interaction term between group and age are reported.A linear regression model was used to analyze the cross-sectional volumetric and DTI data that included: group (NL-3 vs WT) as design factor and gender , and the actual age (in days), as covariates. The interaction effect was first tested and each region was analyzed separately. Because multiple tests or regression analyses were done for each of the 39 regions for the outcomes of DTI indices and volumes, a false-discovery rate (FDR) was computed to control the type I error and a cut off value of 0.05 was used to determine statistical significance. None of the interaction terms reached statistical significance. Therefore, we reported the results from the model that only included main effects for the three factors. The adjusted mean volume in the significant region was computed from the final model using the predictive average volume of a 50-day male mouse for NL-3 and wild type animal respectively.Using data from 28 animals that had both behavioral and imaging studies, the correlation between social, anxiety measures and segmented brain volumes were examined by calculating partial correlation coefficient through a linear regression model that have been adjusted for group, gender, and age. A p-value of <0.05 was considered significant. The correlation analysis was performed only on the selected brain regions which showed significant differences in volume between wild type and NL-3 mice. All statistical computations were performed using STATA 12  and statistical package for social sciences .We found no evidence of 3-way and 2-way interaction effects in social approach behavior . After controlling for gender and age, the NL-3 knock-in animals exhibited lower social preference than the WT animals. On an average, the NL-3 animals spent 8.7 seconds (95% CI: 1 to 15 seconds) less time sniffing the stimulus mouse in comparison to their wild type littermates (p = 0.019) . On the Raw data on volume and DTI indices at each time points are summarized in Correlation between the brain regions which demonstrated significant differences in volume (8 regions) and behavioral data  was also performed. No significant correlation between these brain regions and social sniffing time  was observed. However, a significant correlation between the volume of internal capsule  and medulla  and elevated zero maze test score was observed after accounting for strain, gender and age as covariates.We observed significantly lower volume from 6 brain regions and higher volume from the pons and medulla in NL-3 knock-in mice compared to the wild type littermates after considering age and gender as covariates. These volumetric differences were found at all the three stages of development  indicating that the NL-3 knock-in mutation causes alterations in these brain areas that are developmentally stable. While volumetric differences were observed, no significant differences in DTI parameters were observed between NL-3 knock-in and WT mice. These results are in agreement with a previously published MRI study on a smaller sample of 108-day-old male NL-3 knock-in mice The neuroligin and neurexin genes have been recognized in human autism association studies MRI has been used for examining both volume changes and white matter structural integrity in patients with ASD ASD comprises a set of neurodevelopmental disorders affecting socio-communicative behavior along with abnormalities in the sensorimotor skill learning, oculomotor control, and executive functioning. Some of these impairments may be related to abnormalities in the caudate nuclei, substantia nigra and somatosensory cortex as these regions have been reported to be abnormal in ASD. One of the core behavioral symptoms in autism is restricted and repetitive behaviors, and these behaviors have often been thought to be associated with the caudate putamen or striatum The link between neuroanatomical findings and behavioral symptoms is vital for understanding the role of structural changes in the etiology of ASD. Investigations of structural measures of the corpus callosum and other white matter regions have reported decreased size along with under connectivity associated with behavioral abnormalities in ASDs While our results of reduced volume in both gray and white matter of NL-3 knock-in agree with previous published reports in human ASD as well as in mouse models relevant to ASD, it is worth noting that there is significant heterogeneity in the literature with respect to volumetric differences in cortical thickness and morphology in ASD, with at times seemingly contradictory results, depending on the age, IQ, and clinical severity of the study population DTI is sensitive to the integrity of white matter and provides a better understanding of the neuroanatomical abnormalities in ASD. Several DTI studies have shed light on microstructural integrity of the white matter and developmental abnormalities in various brain regions in ASD in human We did not observe any significant correlation between brain volumes and social behavior of the mice. However a significant correlation between volume from internal capsule and medulla with anxiety score was observed in these animals. These preliminary studies in a small set of behavior assays used for assessing rodent behaviors indicate that imaging and behavior tests may act as independent parameters for the diagnosis of ASD in mouse models. Although more studies are needed, we believe that developmental studies involving behavioral and imaging assays in a genetically defined model of ASD, as performed in the current study, will aid in further understanding the biology of ASD in relevant mouse models.Our results should be interpreted with caution in light of limitations of the study, which include the following: 1) we have used only two behavioral assays - the social approach test and the elevated zero maze test, however other behavioral assays including studies of repetitive behaviors, ultrasonic vocalizations, social memory, open field locomotion, Morris water maze spatial learning and acquisition could provide a more detailed interpretation of behavioral phenotypes relevant to ASD as reported in previous studies In summary, reduced volume of several white and gray matter areas in the brains of NL-3 knock-in mice suggests that these structural changes are induced by the mutation in the NL-3 gene. Future imaging and behavior studies on NL-4 knock-in and knockout models, which have been suggested to be more relevant to ASD Table S1Distribution of animals used in the study.(DOCX)Click here for additional data file.


Rhabdoviridae family, was able to evade the protective immune response induced by the DNA vaccination of rainbow trout. The experiments comprised repeated passages of a highly pathogenic VHSV isolate in a fish cell line in the presence of neutralizing fish serum (in vitro approach), and in rainbow trout immunized with the VHS DNA vaccine (in vivo approach). For the in vitro approach, the virus collected from the last passage (passaged virus) was as sensitive as the parental virus to serum neutralization, suggesting that the passaging did not promote the selection of virus populations able to bypass the neutralization by serum antibodies. Also, in the in vivo approach, where virus was passaged several times in vaccinated fish, no increased virulence nor increased persistence in vaccinated fish was observed in comparison with the parental virus. However, some of the vaccinated fish did get infected and could transmit the infection to naïve cohabitant fish. The results demonstrated that the DNA vaccine induced a robust protection, but also that the immunity was non-sterile. It is consequently important not to consider vaccinated fish as virus free in veterinary terms.DNA vaccines encoding viral glycoproteins have been very successful for induction of protective immunity against diseases caused by rhabdoviruses in cultured fish species. However, the vaccine concept is based on a single viral gene and since RNA viruses are known to possess high variability and adaptation capacity, this work aimed at evaluating whether viral haemorrhagic septicaemia virus (VHSV), an RNA virus and member of  Novirhabdovirus genus within the Rhabdoviridae family [Oncorhynchus mykiss) in Europe, causing high mortalities in all fish stages [Viral haemorrhagic septicaemia virus (VHSV) is a negative-sense, single-stranded RNA virus, which belongs to the e family . VHSV ish stages .Currently, no commercial vaccine against VHS is available. Several vaccination strategies have been tested to control this disease, among them live attenuated vaccines, inactivated vaccines, and recombinant protein vaccines, but with limited efficacy or compromised safety aspects , 4. By cThe traditional DNA vaccine against VHS consists of a plasmid designed for expression of the viral surface glycoprotein (G) in eukaryotic cells. The immune protection induced by this vaccine start as early as 4 days after intramuscular injection, when an early, non-specific, and short-lasting antiviral protection is triggered. This early protection has been related to interferon-associated mechanisms, which are characterized by the overexpression of multiples interferon-stimulated genes with antiviral functions such as Mx, Vig-1, Vig-2, and Vig-8 . After 4Although the high efficacy of the DNA vaccine against VHSV has been consistent under experimental conditions, its protective effect might be threatened following repeated use under field conditions, due to the high variability of RNA viruses. Whether this variability might allow generation and selection of VHSV mutants, capable of evading the immunological protection induced by the DNA vaccine remains to be addressed.Evidence of how the genetic variability of VHSV allows adaptation to selective conditions was shown when rainbow trout were injected with a plasmid encoding a neutralizing single chain antibody (scAb) against the G protein of VHSV. In this case, a neutralization escape mutant was isolated from the survivors of the infection . SimilarWhile mutations in the G protein can affect the efficacy of the adaptive protection, mutations in e.g. the non-structural-protein (NV) may potentially affect the ability of the virus to bypass the innate protection induced by the DNA vaccine. This viral protein has been suggested to inhibit the apoptotic signal in virus infected cells at an early stage of virus infection, thus affecting the virulence of the VHSV .The aim of this work was to determine whether VHSV within repetitive passages under the selective pressure of DNA vaccine-induced immunity, would be able to develop mutants that could escape from the early and late protective mechanisms induced by the vaccine, and from neutralizing serum antibodies induced in DNA-vaccinated rainbow trout. Knowledge of these aspects is essential for the design of robust and safe DNA vaccination strategies for protection of rainbow trout against VHS under field conditions.The fish cell lines used in this study were EPC (epithelioma papulosum cyprini)  and BF2 50) per ml, in BF2 cells [To propagate VHSV, BF2 cell cultures were inoculated with low multiplicity of infection of the virus and maintained at 15°C until a complete cytopathic effect (CPE) was observed. The supernatant was collected and centrifuged at 4500 x g for 15 min at 4°C to eliminate cellular debris. The virus was stored at -80°C. The titer was determined using the method of 50% tissue culture infective doses  in the left epaxial muscle below the dorsal fin with 25 μl of purified DNA plasmid in saline solution , as described earlier [For  earlier . This st earlier . Non-vacRepeated passaging of VHSV was performed in fish vaccinated 1 week before inoculation with virus as well as in fish vaccinated 6 weeks before inoculation with virus. The fish were vaccinated with either 0,1 or 1,0 μg of the vaccine. Non-vaccinated fish were included as controls to confirm virulence the passaged virus.5 TCID50 ml-1 of the VHSV isolate DK3592b, hereafter called parental virus. After this, water flow was restored. Moribund fish, with clinical signs of VHS, were euthanized with an overdose of benzocaine and stored at −20°C until further analysis. At 21 days post infection, the surviving fish were euthanized with an overdose of benzocaine. The sampled moribund fish were dissected, and spleen, liver, heart, head kidney, and brain were collected and pooled per fish in MEM. Organs were homogenized in a TissueLyser (Qiagen) for 2 min at 20 Hz. The homogenate was centrifuged at 4500 x g for 15 min, and the supernatant was collected to be treated with gentamicin overnight at 4°C. After the antibiotic treatment, the virus content was titrated on BF2 cells and the samples stored at -80°C. These first-passage homogenates were used to infect new batches of fish vaccinated 1 or 6 weeks earlier (the second passage).In the first passage, each treatment group included two aquaria with 25 fish in each. The infection was carried out by immersion in 8 l water for 3 h in static water with a virus concentration of 1 x 10Due to the low amount of virus recovered from the first passage, the second passage was performed by I.P injection of 50 μl tissue homogenate supernatant into 2x10 fish. Subsequent passages were performed as the second passage with the exception that the tissue homogenates included organs from both survivors and moribund fish. The procedure for monitoring and sampling was performed as explained in the first passage .A challenge trial with vaccinated fish was performed to compare the performance of the parental virus with the virus from the last passage in vaccinated fish, hereafter called passaged virus, under the selective pressure induced by DNA vaccination. To obtain sufficient virus for immersion challenge, the passaged virus, was passaged once in BF2 cells and titrated as outlined above.4 TCID50 ml-1. After this, the water flow was restored. Two weeks post-infection, 10 non-vaccinated (naïve) fish were transferred into each aquarium to cohabitate with the vaccinated fish. The naïve fish were tagged by cutting a part of the tail fin. After another 2 weeks, all surviving fish were euthanized with an overdose of anesthetics. The spleen, heart, liver, kidney and brain were collected and pooled from each fish individually. The tissues were homogenated and used for virological examination. The persistence in vaccinated fish and the spread of the virus to naïve fish were calculated as the percentages of virus-positive fish.The comparative analyses included accumulated mortality, the virus carrier state of the vaccinated fish, as well as the ability of the virus to spread to co-habitant naïve fish. The fish were vaccinated with 1,0 μg of the DNA vaccine and challenged at 1 or 6 weeks post-vaccination, using virus passaged at similar times post-vaccination. Fish challenged with the parental virus served as controls. As an example, when the fish were challenged at 1 week post-vaccination the passaged virus used was VHSV-1W0,1 and VHSV-1W1,0. With each virus, the infection was performed in duplicates with 20 fish in each aquarium. The infection was carried out by immersion in static water in the 8 l aquaria for 3 h with a final concentration of the virus at 1x10Supernatants from tissue homogenates treated as described above were used to prepare 10-fold serial dilutions, which were inoculated onto BF2 monolayers with 60–80% confluency in 24-well tissue culture plates. The cultures were maintained at 15°C for 7 days when each well was examined. The wells with CPE were considered positive. The identity of the CPE-causing virus was confirmed by PCR as outlined below.An RT-PCR assay with primers specific for the VHSV nucleoprotein (N)-gene was used to confirm the identity of the passaged virus as well the virus re-isolated from sampled fish in the comparative challenge trial. Total RNA was isolated from 100 μl of each supernatant using the RNeasy mini kit (Qiagen). Subsequently, the cDNA was synthesized using the iScript kit (BioRad) following the manufacturer's recommendation. The qPCR reaction contained 1 μl of the cDNA, 7,5 μl of SuperMix, 0,03 μl of Rox, 0.3 μl of each primer VHSV-N-For 5’-AGG TCT CAG ATG TCA TCA AGG AG-3’ and VHSV-N-Rev 5’-CGG TGG AGC TCC TGA AGT T-3’, and 5.87 μl of free-nuclease water. PCR amplification program involved an initial step at 50°C for 2 min, and then a denaturation cycle 95°C for 2 min. These steps were followed by 40 cycles 95°C for 15 sec and 60°C for 1 min. Amplification was performed in MX Pro-Mx3005P thermocycler (Stratagene). Samples giving a signal within 25 cycles and an amplicon product with a melting temperature corresponding that obtained with the positive VHSV isolate DK3592b control, were considered as positive.For the first passage, serial 10-fold dilutions of VHSV isolate DK3592b 30 μl) were mixed in wells of 96-well plates with 15 μl of a 1/40 dilution of the heat treated (30 min at 45°C) neutralizing trout serum obtained after repeated immunization with the DNA vaccine (pcDNA3-vhsG) . Followi μl were in vitro passaged virus and the parental virus to the neutralizing effect of the immune serum was compared using the plaque neutralization assay (PNT), in which the titer of the serum is defined as the reciprocal value of the highest serum dilution reducing the number of plaques by 50% compared to a normal trout serum control [The susceptibility of the  control . To evalEach replicate in both the virulence and the persistence analyses was considered as an individual sample to evaluate intergroup variability. The statistical analysis for difference between treatments was performed by the Kruskal-Wallis test . Differences were considered significant at p<0.05.nd passage. This might suggest that the protection induced by 1,0 μg of the DNA vaccine was able to clear the virus in the immunized fish more efficiently than in fish vaccinated with 0,1 μg DNA, although the latter dose was still highly protective (data not shown).To test the ability of the virus to bypass the early protection induced by the DNA vaccine, we passaged VHSV in rainbow trout vaccinated 1 week before inoculation with virus. When the fish used for passaging had been vaccinated with a dose of 0,1 μg of the DNA vaccine, we were able to isolate virus from dead and vaccinated surviving fish in each of the successive 5 passages. When fish given 1,0 μg of the DNA vaccine were used, we were able to isolate virus only until the 2To test the ability of the virus to bypass the late protection induced by the DNA vaccine, the passaging was performed in rainbow trout vaccinated 6 weeks before inoculation. In this setup, VHSV was re-isolated from dead and vaccinated survivor fish in all 4 passages using both vaccine doses .After successive passages, the virus obtained from the last passage under selective conditions (passaged virus), was amplified by 1 passage on BF2 cells and later compared with the parental VHSV isolate.in vivo approach included three factors; mortalities induced in vaccinated and non-vaccinated fish, the capacity of the virus to infect and persist in vaccinated fish, and the ability of the virus to spread from vaccinated carriers to cohabitant naïve fish.The comparison for the The mortality rates caused by the passaged virus and the parental virus in vaccinated fish were both low, ranging between 0–12%. Only one of the duplicate aquaria with fish inoculated with the VHSV-1W1,0, and one of the duplicates inoculated with the VHSV-6W0,1, showed higher mortalities (about 20%) than the aquaria inoculated with the parental virus . Since tThe evaluation of the ability of the virus to infect vaccinated fish showed that both the parental and passaged virus were able to infect and persist in some of the vaccinated fish for at least 4 weeks post-challenge . The higThe analysis of whether vaccinated carriers infected with either the passaged or the parental virus were capable of transmitting the infection to naïve fish showed that even though only a few fish could be detected as carriers, transmission did take place for the passaged as well as for the parental virus, in at least in one of the replicates . The setAfter 11 successive passage in cell culture in the presence of neutralizing trout serum, we compared the ability of the passaged virus and the parental virus to escape from the neutralization by a trout immune serum. As shown in The glycoprotein gene of the 3 preparations of passaged virus and the parental virus was sequenced and compared to determine the presence of mutations. The 4 sequences had 100% nucleotide identity (not shown).This work focused on analyzing whether the fish rhabdovirus VHSV was able to mutate and escape from the protective mechanisms induced by the VHS DNA vaccine. Such mutants would represent a potential risk of reemergence of the disease in vaccinated fish populations, reducing the applied potential of the DNA vaccine. To our knowledge, this important aspect remains to be addressed for the otherwise extensively analyzed and highly protective fish DNA vaccines. Our results suggest that the immune response triggered by the vaccine is rather robust and not easily bypassed by the virus.The high genetic variability of RNA viruses is due to the high replication rates, large population size, and high mutation rates, which together generate a diverse spectrum of virus variants in every replication cycle and result in a population of one dominant virus variant along with multiple low-frequency virus variants. This phenomenon is known as the quasispecies theory and allows the virus to adapt rapidly to new environments, and to new hosts . When RNin vivo approach was used to evaluate the ability of VHSV to evade the early and late protection induced by the DNA vaccine in rainbow trout fingerlings, associated with innate and adaptive immunity, respectively [in vitro approach was applied, to evaluate the ability of the virus to evade the neutralizing effect of serum from rainbow trout immunized with the DNA vaccine.The experimental design included two approaches. First, an ectively , 33, 34.in vivo passaging, the initial inoculation of the fish was done by immersion. i.e. the natural route of infection. But from the second passage, the inoculation was performed by I.P injection due to the low amounts of virus recovered from each passage, and to avoid propagation of the virus in cell culture, which could compromise the selection procedure. In order to obtain enough virus to perform the infection in the final evaluation trial by immersion, one cell culture passage was required. Although this was a compromise, the risk of this significantly changing the virus population after several passages under selective conditions in vivo was considered to be small. Sequence analysis by next generation sequencing would be required to confirm this, but was beyond the scope of the present study.In the in vivo approach took into account that an escape mutant could have diverse strategies to bypass the immune protection induced by the DNA vaccine. Among these strategies, escape mutants could have increased virulence in vaccinated fish, causing higher mortality rates as a result of a more efficient viral replication. Alternatively, decreased virulence might reduce clearance, thereby allowing the virus to persist in the host and spread to cohabitant fish. However, the in vivo approach evaluation showed no advantage of any of the batches of passaged virus in comparison with the parental virus, neither considering virulence, persistence in vaccinated fish, nor the capacity of vaccinated fish carriers to transmit the infection to naïve cohabitant fish.The analysis of the According to Read et al. 2015, infected vaccinated chickens were associated with a higher risk of spread of highly virulent virus than the infected non-vaccinated chickens. The rationale behind this theory was that non-vaccinated chickens would die out rapidly and thereby allow the infection to be kept under control, while the infected vaccinated chicken would survive the infection and allow the virus to persist, replicate and spread in the host population for an extended period. Furthermore, the persistence in vaccinated chickens could promote the selection of hyperpathogenic virus strains that could cause a more severe disease .While this scenario might be true for some highly lethal viruses, our results suggest that it does not count for VHSV infections in rainbow trout. Although the virus causes high mortality, some individuals often survive and clearance from such fish is slower compared to DNA vaccinated individuals . Therefoin vitro aim was look at the adaptive humoral immune response to DNA vaccination alone and evaluate whether serial passages of VHSV in the presence of serum from rainbow trout immunized with the DNA vaccine, would favour propagation of neutralization escape mutants. The trout immune serum used here was obtained from a hyperimmunized fish [The zed fish .Previous works showed that viruses like VHSV and IHNV were able to generate escape mutants resistant to neutralization by monoclonal antibodies after a few passages in cell culture , 25. Anoin vitro analysis suggested that VHSV cannot easily escape even from the neutralizing serum antibodies, which is only one mechanism of the adaptive responses induced by DNA vaccination. However, while a single vaccination did not induce an antibody response in all individuals, particularly at lower temperatures [in vivo suggested that the broad nature of the immune response triggered by the vaccine, involving a range of both innate and adaptive mechanisms, made escape mutation incompatible with maintaining the viability and the infectious capacity of the virus.However, after 11 passages, there was no evidence of mutants escaping the neutralizing effect of the trout immune serum. This was confirmed by the fact that the passaged and the parental virus had a 100% identical glycoprotein gene sequence. In contrast to this, mutants escaping neutralization by mouse monoclonal antibodies carried one or few amino acid substitutions in the translated G gene . The experatures , our faiOur setup only included the most stringent selective condition by challenging vaccinated fish with virus carrying a G gene identical to the vaccine gene. It can therefore not be excluded that viral escape from DNA vaccine induced immunity might arise under less selective conditions, such as when the vaccine G gene is heterologous to that of the infecting virus, resulting in a less complete protection . The safIn conclusion, our results suggest a low probability of occurrence of escape mutants under optimal DNA vaccination conditions. In applied terms, this represents an additional advantage of this rather efficient vaccine against VHSV, making it a safe prophylactic tool. However, we also observed that some of the vaccinated fish can get subclinically infected and that the infection can be transmitted to naïve cohabitants if these are stocked with the vaccinated fish shortly after their exposure to the virus. Vaccinated fish from endemically infected zones should therefore be considered to be potential carriers in terms of trade regulations.