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The Relative Contribution of NMDARs to Excitatory Postsynaptic Currents is Controlled by Ca2+-Induced Inactivation

NMDA receptors (NMDARs) are important mediators of excitatory synaptic transmission and plasticity. A hallmark of these channels is their high permeability to Ca2+. At the same time, they are themselves inhibited by the elevation of intracellular Ca2+ concentration. It is unclear however, whether the Ca2+ entry associated with single NMDAR mediated synaptic events is sufficient to self-inhibit their activation. Such auto-regulation would have important effects on the dynamics of synaptic excitation in several central neuronal networks. Therefore, we studied NMDAR-mediated synaptic currents in mouse hippocampal CA1 pyramidal neurons. Postsynaptic responses to subthreshold Schaffer collateral stimulation depended strongly on the absence or presence of intracellular Ca2+ buffers. Loading of pyramidal cells with exogenous Ca2+ buffers increased the amplitude and decay time of NMDAR mediated EPSCs (EPSPs) and prolonged the time window for action potential (AP) generation. Our data indicate that the Ca2+ influx mediated by unitary synaptic events is sufficient to produce detectable self-inhibition of NMDARs even at a physiological Mg2+ concentration. Therefore, the contribution of NMDARs to synaptic excitation is strongly controlled by both previous synaptic activity as well as by the Ca2+ buffer capacity of postsynaptic neurons.

INTRODUCTION
In the mammalian central nervous system, excitatory synaptic transmission is mediated by glutamate which co-activates postsynaptic NMDA and AMPA receptors (AMPAR and NMDAR). Fast synaptic currents are mediated by AMPAR channels, whereas NMDAR channels generate slower and longer lasting currents (Forsythe and Westbrook, 1988;Bekkers and Stevens, 1989;Stern et al., 1992;Spruston et al., 1995). NMDARs are highly permeable to Ca 2+ (Ascher and Nowak, 1988) and act as gatekeepers for the Ca 2+ influx into dendritic spines during synaptic activity (Perkel et al., 1993;Malinow et al., 1994). NMDAR function can be modulated by a large number of extracellular agents, including Mg 2+ , glycine, Zn 2+ , polyamines and protons (Collingridge and Lester, 1989;Hollmann and Heinemann, 1994), as well as by the intracellular activities of protein kinases and protein phosphatases (Kotecha and MacDonald, 2003).
In addition, numerous studies have shown that an increase in intracellular calcium concentration ([Ca 2+ ] i ) causes a reversible reduction of NMDA-activated currents, irrespective of the source of calcium (Legendre et al., 1993;Vyklicky, 1993;Medina et al., 1994;Kyrozis et al., 1995;Umemiya et al., 2001). The mechanism of Ca 2+ induced inactivation of NMDARs (CIIN) involves calmodulin binding to the C-terminal of the GluN1 subunit and a subsequent reduction in the channel's open probability (Ehlers et al., 1996;Zhang et al., 1998). The functional consequences of CIIN, however, are not well understood. If calcium entry via NMDAR suffices to suppress further activation of the channels, it could mediate an important negative feedback regulation for synaptic excitation under physiological conditions (Rosenmund et al., 1995). This question has not been directly addressed in the past, because most NMDAR-mediated responses were recorded at low extracellular Mg 2+ concentration, with prolonged ( 1 ms) agonist application and without exact estimates of NMDAmediated Ca 2+ influx. However, even at resting membrane potentials ( −70 mV) and in the presence of physiological Mg 2+ concentrations NMDARs still act as the main synaptic source of Ca 2+ entry (Kovalchuk et al., 2000), suggesting the possibility that CIIN shapes the postsynaptic Ca 2+ dynamics and the kinetics of excitatory postsynaptic potentials (EPSPs). In order to explore the role of CIIN in synaptic transmission in neuronal networks, we addressed the following questions: (i) Is NMDAR-mediated Ca 2+ influx during unitary synaptic events sufficient to produce detectable CIIN? (ii) Does CIIN depend on membrane potential due to the voltage-dependent Mg 2+ block of NMDARs? (iii) Does CIIN affect unitary EPSP kinetics and temporal summation of postsynaptic potentials?
We report that manipulating the Ca 2+ buffer capacity of hippocampal CA1 pyramidal neurons strongly affects the amplitude of single, subthreshold NMDAR-mediated EPSPs. Moreover, upon high-frequency afferent stimulation, simultaneous relief from Mg 2+ block and CIIN increased the contribution of NMDARs to postsynaptic EPSPs and significantly prolonged their decay time. Our findings suggest that Ca 2+ flux induced during unitary synaptic events is sufficient to produce detectable inhibition of NMDARs. Repetitive activation of excitatory synapses results in a significant prolongation of the integration window for synaptically evoked action potentials (APs).

MATERIALS AND METHODS
All experimental protocols were performed in accordance with the Kazan Federal University regulations on the use of laboratory animals (ethical approval by the Institutional Animal Care and Use Committee of Kazan State Medical University N9-2013) or by the state government of Baden-Württemberg, Germany. All efforts were made to minimize animal suffering and to reduce the number of animals used.
Patch electrodes were pulled from hard borosilicate capillary glass (Sutter Instruments flaming/brown micropipette puller).
Hippocampal CA1 pyramidal cells were identified visually using IR-video microscopy. Whole-cell recordings from these neurons were taken at room temperature using a HEKA EPC-7 amplifier (List Elektronik). To evoke synaptic currents, glass electrodes filled with ACSF were placed in the stratum radiatum within 50-100 µm of the body of the recorded neuron. Inhibitory synaptic transmission was blocked during recordings by the addition of 10 µM gabazine to the perfusion ACSF. The intersweep interval was 6 s. In the voltage-clamp experiments the command voltage was corrected for the liquid junction potential. AMPAR and NMDAR mediated currents were pharmacologically dissected using the AMPAR and NMDAR antagonists, CNQX (10 µM) and APV (50 µM), respectively. After recording the total current responses (containing both AMPAR and NMDAR components, 100 sweeps), AMPAR channels were blocked by bath application of CNQX (10 µM) and another 100 sweeps containing only NMDAR responses were recorded. To confirm that the remaining current after CNQX application was NMDAR-mediated; APV (50 µM) was applied at the end of every experiment. AMPA currents were obtained by subtraction of the averaged NMDA response from the averaged total response. AMPA/NMDA ratios were calculated as the peak AMPAR-mediated current amplitudes divided by the peak NMDAR-mediated current amplitudes.
During recordings, membrane resistance was monitored, and data from cells in which the membrane resistance varied by >15% were discarded from the analysis. Throughout the article n refers to the number of the experiments in the group.
For statistical analysis, the Mann-Whitney test has been used and data are presented as mean ± SD, unless otherwise stated.

Ca 2+ Entry During Unitary Subthreshold Synaptic Responses is Sufficient to Trigger CIIN
The physiological significance of CIIN depends on two major questions: (i) Is the onset of CIIN fast enough to affect the amplitude of the NMDAR response during a unitary synaptic event? and (ii) Is the concentration of Ca 2+ entering the spine during subthreshold EPSPs/EPSCs sufficient to trigger CIIN?
Depending on these parameters, the effect of CIIN can be either nearly instant, modulating all postsynaptic responses, or can be mostly cumulative, being pronounced during repetitive activity at high frequencies. Manipulation of [Ca 2+ ] i by changing the intracellular Ca 2+ buffer capacity provides a powerful tool to discriminate between those two scenarios. Hippocampal CA1 pyramidal neurons have naturally low endogenous buffer expression (Scheuss et al., 2006). To evaluate the kinetics of CIIN, we examined the effect of intracellular buffer loading (10 mM EGTA) into CA1 pyramidal neurons, on the amplitude of Schaffer-collateral evoked NMDAR-mediated EPSCs (nEPSCs). Experiments were carried out in Mg 2+ -free ASCF. Responses were measured either at −70 mV or at +50 mV and were compared to the amplitude of AMPAR-mediated EPSCs (aEPSCs) recorded from the same cell at −70 mV. At −70 mV, the relative amplitudes of nEPSCs recorded in the presence of EGTA were substantially higher compared to those measured with buffer-free pipette solution ( Figure 1A). Accordingly, the AMPA/NMDA ratio recorded with EGTAcontaining intracellular solution was 0.7 ± 0.26 (n = 6) and under EGTA-free conditions it was 2.44 ± 0.55 (n = 7, p = 0.001). Ratios of aEPSCs recorded at −70 mV to nEPSCs acquired at +50 mV were still slightly lower in the presence of the buffer, however the apparent difference was not significant (EGTA-free 0.94 ± 0.28, n = 7); EGTA-containing 0.81 ± 0.24 (n = 6; p = 0.45; Figure 1A). These data show that at −70 mV EGTA loaded into the cell prevents CIIN by buffering incoming Ca 2+ , resulting in increased nEPSC amplitudes compared to those in buffer-free conditions. Whereas at +50 mV, when Ca 2+ entry is negligible, the nEPSC amplitude is practically insensitive to buffer loading due to lack of CIIN. The latter also indicates that buffer loading does not trigger any long lasting voltage-independent change in synaptic NMDAR conductance.
To test whether the degree of CIIN can be increased by prolonged subthreshold synaptic stimulation, we measured and compared the amplitude ratios of the second (NMDA2) and fifth (NMDA5) nEPSCs to the first nEPSC (NMDA1), using a five-pulse stimulation protocol (10 Hz) in neurons loaded with buffer-free or EGTA-containing intracellular solutions ( Figure 1B). The averaged NMDA2/NMDA1 ratio was increased slightly, but not significantly, by buffer loading (EGTA-free: 1.23 ± 0.11, n = 6; EGTA-containing: 1.36 ± 0.21, n = 5; p = 0.662). Later responses, however, were clearly enhanced by buffering Ca 2+ : the NMDA5/NMDA1 ratio was 0.9 ± 0.15 in neurons patched with EGTA-free solution (n = 6) and 1.28 ± 0.21 in EGTA-containing neurons (n = 5; p = 0.009). These data indicate that CIIN does alter the NMDAR contribution to unitary responses, with a stronger impact on NMDAR-mediated currents during prolonged repetitive activity.

In the Presence of Mg 2+ CIIN Affects NMDAR-Mediated Currents in a Voltage-Independent Manner
The next question was whether under physiological conditions, when NMDARs are heavily controlled by extracellular Mg 2+ , CIIN could still influence nEPSCs? The magnitude of the Mg 2+ block at resting membrane potentials is nearly maximal, resulting in a robust reduction of the NMDAR contribution to postsynaptic Ca 2+ influx. On the contrary, at less negative potentials, upon partial relief of the block, enhanced Ca 2+ influx through the channels might still have significant consequences on NMDAR function. In other words, the nonlinearity of NMDAR-mediated Ca 2+ entry due to Mg 2+ block could give rise to a voltage dependence of CIIN. To test this hypothesis, we investigated the effect of intracellular buffer loading on current voltage (IV) relationships of evoked synaptic NMDARmediated currents. Figure 2A shows averaged evoked nEPSCs measured at −70, −35, 0, 35 and 50 mV with buffer-free, EGTA-containing (10 mM) and BAPTA-containing (1 mM) intracellular solutions. All responses were normalized to the mean EPSC amplitude obtained at 50 mV, where reduced Ca 2+ entry through the channels should have a minor effect on the amplitudes of nEPSCs. Normalized nEPSCs measured at 35 mV were nearly identical, irrespective of the intracellular buffer content. However, responses recorded at −35 and −70 mV with EGTA (nEPSC −35 : −0.32 ± 0.07; nEPSC −70 : −0.11 ± 0.03; n = 6) or BAPTA (nEPSC −35 : −0.34 ± 0.09; nEPSC −70 : −0.12 ± 0.03; n = 5) in the patch pipettes were more than twofold larger than those collected with bufferfree intracellular solution (nEPSC −35 : −0.17 ± 0.04; nEPSC −70 : −0.05 ± 0.01; n = 7; Figure 2B). The enhancement of NMDAmediated EPSCs in the presence of intracellular Ca 2+ buffers was highly significant at both negative recording potentials (−35 mV, p 0.001; −70 mV, p < 0.001 one way ANOVA). However, after normalization to the values obtained at −70 mV, the normalized nEPSC −35 amplitudes in all three groups were not different (−3.71 ± 0.5, −2.72 ± 0.6 and −3.2 ± 0.9 for buffer-free, EGTA-and BAPTA-containing solutions respectively; p > 0.05 one way ANOVA; Figure 2C), indicating that the magnitude of CIIN did not depend on the strength of the Mg 2+ block. Thus, CIIN can drastically reduce nEPSC amplitudes even in the presence of Mg 2+ at physiological concentrations.

The Relative Contribution of NMDAR to the Postsynaptic Responses is Strongly Controlled by CIIN
To further substantiate the modulatory role of CIIN under physiological conditions and estimate its impact on the amplitude of compound EPSCs we compared AMPAR-and NMDAR-mediated responses measured at −70 and −35 mV in neurons patched with buffer-free and buffer-containing pipette solutions. At both holding potentials, the relative amplitudes of nEPSCs recorded from the cells dialyzed with buffer-free intracellular solution were significantly smaller compared to those measured with EGTA (10 mM) or BAPTA (1 mM), as indicated by much smaller AMPA/NMDA ratios (Figures 3A,B).
To evaluate the effect of CIIN on the relative NMDAR contribution to the compound response, we reconstructed weighted synaptic IV-curves of aEPSCs and nEPSCs recorded with buffer-free and buffer-containing solutions. Both AMPAR- and NMDAR-mediated responses were normalized to the averaged aEPSC amplitude measured at −70 mV ( Figure 3C). As expected aEPSC amplitudes did not depend on the intracellular buffer content and the IVs of the aEPCSs were nearly linear. However, the weight of the NMDAR contribution to the compound EPSCs strongly depended on the presence of Ca 2+ buffers. In the cells dialyzed with buffer-free solution at −70 mV the contribution of the nEPSC was 6 ± 1% of the compound response. The impact of NMDARs increased at −35 mV (24 ± 8%) but was still significantly lower than that of AMPARs (n = 8; p < 0.001; Figure 3D). In neurons loaded with buffers, the contribution of NMDAR channels was strongly enhanced at −70 mV (10 mM EGTA: 18 ± 6%, n = 6; 1 mM BAPTA 21 ± 4%, n = 5), moreover, at −35 mV weighted nEPSC amplitudes were significantly larger than aEPSCs (59 ± 10% and 56 ± 5%, p < 0.05 for EGTA-and BAPTA-containing solutions respectively). Thus relief from CIIN gave rise to a three to fourfold enhancement in NMDAR contribution to excitatory postsynaptic responses.

CIIN Moderates the EPSP Decay Time and Action Potential Firing Window
The increased NMDAR contribution in CIIN-free conditions may substantially prolong EPSP duration and as a result, increase the time window for AP generation. To test these possibilities, we examined the consequences of intracellular BAPTA (1 mM) loading on the EPSP decay time constant and neuronal firing properties. Experiments were carried out in current clamp mode, and postsynaptic CA1 pyramidal cells were kept at resting membrane potential (around −65 mV). To reach different levels of postsynaptic depolarization, Schaffer collateral inputs were stimulated with 50, 20 and 10 Hz trains of three stimuli. The stimulation intensity was the minimal and sufficient intensity to trigger, with 40-60% probability, an AP in response to the 3rd stimulus in 50 Hz trains. Reduction of the stimulation frequency to 20 and 10 Hz decreased both contribution of the EPSPs temporal summation and pairedpulse facilitation to the peak depolarization reached by the 3rd EPSP. After recording 50-75 sweeps at each stimulation frequency, NMDARs were blocked by bath application of the receptor antagonist APV (50 µM) and additional 50 responses were collected at 50, 20 and 10 Hz, respectively. To estimate the contribution of NMDARs to the EPSP duration we compared the decay time constants (tau) of the averaged 3rd EPSPs before and after APV application. Sweeps with APs (occurring at 50 Hz stimulation) were excluded from EPSP decay analysis.
In cells patched with buffer-free intracellular solution APV application caused a small, but non-significant acceleration of EPSP decay. The change in the EPSP time constant did not depend on stimulation frequency, the averaged tau values were (in milliseconds) 52 ± 12 vs. 38.9 ± 10 (50 Hz), 52.7 ± 17.5 vs. 37.6 ± 12.2 (20 Hz) and 51.6 ± 9.5 vs. 37.4 ± 12 (10 Hz) before and after drug application respectively (n = 5; p > 0.05; Figure 4A). However, in the BAPTA loaded neurons, activation of synaptic NMDARs had a drastic effect on EPSP decay. At 50 Hz tau values were, in control 74 ± 23, and decreased to 36 ± 14 ms in the presence of APV (n = 5; p = 0.008; Figure 4B). At 20 Hz the acceleration of EPSP decay constants by APV was still significant (58.4 ± 21 vs. 34 ± 12 ms; p = 0.029), while at 10 Hz, block of NMDARs did not cause a meaningful tau reduction (51 ± 9.2 vs. 36.1 ± 6.24 ms; p = 0.2). Note that the time constants measured in the presence of APV were very similar to those in the neurons patched with buffer-free and BAPTA-containing solutions.
In line with the prolongation of EPSP decay, AP delays (latency), measured as the interval between the 3rd stimulus artifact and the peak of the AP, were significantly longer in the BAPTA loaded neurons. Figure 4C shows superimposed traces recorded from neurons recorded with buffer-free (black) and BAPTA-containing (red) pipette solutions. Cumulative probability plots (right) show the shift towards longer AP delays in the presence of BAPTA (pooled data from five cells in each group; p < 0.001, Kolmogorov-Smirnov test). Thus, ablation of CIIN and consequent enhancement of the NMDAR contribution to the EPSP duration considerably prolonged the window for AP generation.

DISCUSSION
It is generally accepted that the main functional role of NMDARs is related to their high permeability to Ca 2+ , which confers on NMDARs a central role in both synaptic plasticity and neuronal survival under physiological conditions and neuronal death under excitotoxic pathological conditions (Paoletti et al., 2013).
Functional consequences of NMDARs modulation by various signaling molecules and biochemical cascades under physiological conditions were extensively studied over the last two decades. However the functional role of CIIN remains poorly understood.

CIIN as a Mechanism of NMDARs Self-Regulation Under Physiological Conditions
The phenomenon of Ca 2+ induced inhibition of NMDARs, has been well documented and explored at the level of intracellular molecular mechanisms (Legendre et al., 1993;Medina et al., 1994;Rosenmund et al., 1995;Ehlers et al., 1996;Wang and Wang, 2012;Paoletti et al., 2013;Bajaj et al., 2014;Yang et al., 2014). However, an important question remained open, namely whether the Ca 2+ entry associated with single NMDAR mediated synaptic events under physiological conditions is sufficient to self-inhibit NMDAR mediated responses. In other words, do the mechanisms governing CIIN operate on the EPSCs time scale (milliseconds)?
These aspects of CIIN have not been addressed in previous studies, where CIIN was triggered either by Ca 2+ entry through voltage gated calcium channels or by the prolonged activation of NMDARs (Medina et al., 1994(Medina et al., , 1995(Medina et al., , 1996. These studies also did not strictly quantify the magnitude of NMDAR ''selfinhibition'', especially under physiological conditions. However, Ehlers et al. (1996)  We have found that Ca 2+ entering trough NMDAR during a unitary synaptic event can strongly attenuate the amplitude of nEPSCs indicating that the CIIN operates on a rapid time scale of a few milliseconds. This data is in agreement with our previous findings on recombinant channels where in outside out patches, Ca 2+ influx triggered by brief (1 ms) activation of Ca 2+ permeable AMPARs was sufficient to reduce current amplitude of a co-expressed and co-activated Ca 2+ -impermeable NMDAR mutant (Rozov et al., 1997). Moreover, in physiological Mg 2+ concentrations, even around resting membrane potential, where the strength of the Mg 2+ block is nearly maximal, NMDARs can still conduct a sufficient amount of Ca 2+ , to produce a nearly fourfold reduction in the channel's function. Indeed, according to Kovalchuk et al. (2000) under these conditions, subthreshold afferent stimulation gives rise to detectable [Ca 2+ ] i in the spines of CA1 pyramidal cells which is almost exclusively mediated by NMDARs. Together with the fact that Ca 2+ influx through NMDARs is detectable up to at least +20 to +40 mV (Burnashev et al., 1995;Kovalchuk et al., 2000) this suggests that CIIN is operative under physiological conditions.
Our data strongly suggest that this elevation in [Ca 2+ ] i is sufficient to trigger CIIN and onset of the inhibition is fast enough to shape individual postsynaptic responses. This finding is in perfect agreement with data on the magnitude of CIIN on the single channel level (Ehlers et al., 1996). Thus, we provide the first evidence that under physiological conditions synaptic NMDARs in cells with low buffer capacity are drastically selfinhibited by NMDAR-mediated Ca 2+ influx.
However, in the vast majority of GABAergic interneurons the contribution of NMDARs to the Ca 2+ homeostasis is strongly moderated by an increased endogenous buffer capacity (Freund and Buzsáki, 1996). On the other hand, the presence of endogenous buffers like parvalbumin (PV), calretinin (CR) or calbindin (CB) can effectively reduce the magnitude of CIIN, increasing the contribution of NMDARs to the postsynaptic response. Indeed, synaptic expression of the GluN1 subunit in the hippocampal PV positive interneurons is several fold lower than that in CA1 pyramidal cells (Nyiri et al., 2003), nevertheless, the values of AMPA/NMDA ratios measured in these neurons are very similar (Fuchs et al., 2007). Thus, relief from CIIN by endogenous Ca 2+ buffers changes the main job of NMDARs from the synaptic Ca 2+ supplier to the active postsynaptic contributor to the amplitude and decay of EPSPs. Functionally this can minimize the role of NMDAR channels in the induction of long-term plasticity, but increase their impact on EPSP temporal summation and AP firing profile (Figure 4). It has been found recently that NMDA spikes occurred in multiple dendritic branches of layer 2/3 pyramidal neurons both spontaneously and as a result of sensory input have a major role in enhancing neuronal output in neocortex (Palmer et al., 2014). Contribution of CIIN to the local NMDA spikes, therefore, may also have influence on the number of output APs thus affecting sensory processing and network activity in the cortex.

Possible Role of CIIN During Aging and in Neuropsychiatric Disorders
Change of NMDARs function have also been implicated in the development of psychotic symptoms in the number of neuropsychiatric illnesses (Lakhan et al., 2013). Along with this the expression of endogenous Ca 2+ buffers changes during aging and some neurological disorders (Bu et al., 2003;Riascos et al., 2011). Thus, the selective vulnerability of the basal forebrain cholinergic neurons to degeneration in Alzheimer's disease has been attributed to the age-related loss of CB from these neurons and a consequent rise in intracellular Ca 2+ (Riascos et al., 2011). Under these conditions CIIN may play an intrinsic compensatory role counteracting intracellular Ca 2+ elevation by reducing NMDAR-mediated Ca 2+ entry.
Interestingly, alteration in expression of Ca 2+ buffers often aligns with the alteration of NMDAR function. For instance, agedependent reduction of CR expression in hippocampal granular cells coincides with down regulation of GluN1 immunoreactivity (Gazzaley et al., 1996). Finally, a number of neurological disorders are associated with dysregulation of both NMDAR function and endogenous Ca 2+ buffer synthesis (Heizmann and Braun, 1992;Paoletti et al., 2013;Kook et al., 2014). It has been suggested that PV alterations in schizophrenia can consequently lead to the hypofunction of NMDARs. Schizophrenia is often attributable to NMDAR hypofunction, this might reflect dysregulation of the receptor rather than a deficit in the number of NMDARs (Kantrowitz and Javitt, 2010;Gonzalez-Burgos and Lewis, 2012). In addition, the variation in extracellular Ca 2+ concentration under certain conditions may also attenuate the impact of the CIIN. For example, in vivo measurements of extracellular Ca 2+ concentration in primates during seizures has shown that the Ca 2+ level drops to within the 100 µM range (Pumain et al., 1985). In this case reduced CIIN may increase the window for synaptic integration due to the prolongation of the NMDAR mediated response and lead to neuronal overexcitability.
In conclusion, our findings suggest that Ca 2+ induced inactivation of NMDARs operating on the time scale of EPSCs may contribute to the cell-specific fine tuning of excitatory synaptic transmission under normal and pathological conditions.

AUTHOR CONTRIBUTIONS
Study conception and design: AD, NB and AR. Acquisition of the data: FV, YZ and AR. Analysis and interpretation of the data: FV and AR. Analysis of the data: MM. Drafting of manuscript: AD, NB and AR. Critical revision: NB and AR.

ACKNOWLEDGMENTS
This work was supported by the program of competitive growth of Kazan University, the subsidy allocated for the state assignment in the sphere of scientific activities, the RFBR grant (14-04-01457), the Bundesministerium für Bildung und Forschung (Bernstein Center for Computational Neurosciences 01GQ1003A) and by the support of the A * MIDEX project (n • A * M-AAP-TR-14-02-140522-13.02-BURNASHEV-HLS) funded by the ''Investissements d Avenir'' French Government program, managed by the French National Research Agency (ANR). We thank David Jappy for useful comments on the manuscript.


extfieldsofstudy: Biology Medicine

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Genetic Diversity of Crimean Congo Hemorrhagic Fever Virus Strains from Iran

Background: Crimean Congo hemorrhagic fever virus (CCHFV) is a member of the Bunyaviridae family and Nairovirus genus. It has a negative-sense, single stranded RNA genome approximately 19.2 kb, containing the Small, Medium, and Large segments. CCHFVs are relatively divergent in their genome sequence and grouped in seven distinct clades based on S-segment sequence analysis and six clades based on M-segment sequences. Our aim was to obtain new insights into the molecular epidemiology of CCHFV in Iran. Methods: We analyzed partial and complete nucleotide sequences of the S and M segments derived from 50 Iranian patients. The extracted RNA was amplified using one-step RT-PCR and then sequenced. The sequences were analyzed using Mega5 software. Results: Phylogenetic analysis of partial S segment sequences demonstrated that clade IV-(Asia 1), clade IV-(Asia 2) and clade V-(Europe) accounted for 80 %, 4 % and 14 % of the circulating genomic variants of CCHFV in Iran respectively. However, one of the Iranian strains (Iran-Kerman/22) was associated with none of other sequences and formed a new clade (VII). The phylogenetic analysis of complete S-segment nucleotide sequences from selected Iranian CCHFV strains complemented with representative strains from GenBank revealed similar topology as partial sequences with eight major clusters. A partial M segment phylogeny positioned the Iranian strains in either association with clade III (Asia-Africa) or clade V (Europe). Conclusion: The phylogenetic analysis revealed subtle links between distant geographic locations, which we propose might originate either from international livestock trade or from long-distance carriage of CCHFV by infected ticks via bird migration.

Introduction
Endemic regions of Crimean Congo Hemorrhagic Fever (CCHF) have been reported in Africa, the Middle East, Eastern Europe and Western Asia (Elevli et al. 2010). In the past 10 years, Turkey, Bosnia, and Iran have been reported the most frequent outbreaks of CCHF worldwide (Mohammadi and Razmi 2014).
The virus is transmitted to humans through the bite of infected Ixodid ticks (mostly of the Hyalomma genus) (Mehravaran et al. 2013, Champour et al. 2014, or direct contact with infected blood, by meat from infected animals or by nosocomial transmission (Chinikar et al. 2012b).
CCHFV is a single stranded RNA virus with segmented negative sense genome consisting of a small (S), a medium (M) and a large (L) segment. The S RNA segment encodes the nucleocapsid (N) protein, and the M RNA segment encodes the glycoprotein precursor, resulting in the two envelope glycoproteins Gn and Gc, while the L segment encodes the putative RNA-dependent "RNA" polymerase (Papa et al. 2002). It has been proved that segmented RNA viruses have the potential of segment reassortment and form new distinct genomic variants if the host cells are subject to dual infection by more than one genetically distinct virus (Hewson et al. 2004b, Lukashev 2005. CCHFVs are relatively divergent in their genome sequence and grouped geographically into seven distinct clades based on the S-segment sequence analysis (Han and Rayner, 2011). West-Africa in clade I, Central Africa in clade II, South-Africa and West-Africa in clade III, Middle-East and Asia in clade IV, Europe in clade V and Greece in clade VI (Deyde et al. 2006, Hewson et al. 2004a). The clade IV may be devided into two distinct clades, Asia-1 and Asia-2 (Hewson et al. 2004a).
Beside the genome analysis on S segment, CCHFVs are divided into six clades based on M-RNA sequences; clade IV (Asia or Middle East) comprising strains in China, Pakistan, Oman, and South Africa, clade III (S Africa or W Africa 2) comprising those in Uzbekistan, Tajikistan, China, Pakistan, Iran, Iraq, South Africa and Nigeria, clade I (W Africa1) comprising those in Congo, Senegal, China, clade V (Europe) comprising those in Russia, Kosovo and Turkey, clade VI (Greece) including isolated strain from Greece, and clade VII (Mauritania) including isolated strain from Mauritania (Papa et al. 2005, Morikawa et al. 2002, Morikawa et al. 2007).
In Iran, the first phylogenetic study on Ssegment of CCHFV in 2004 showed that the CCHFV strains were clustered within clade Asia 1 with the highest similarity to Matin strain from Pakistan (Chinikar et al. 2004), previously a pioneering genetic characterization study had showed that the Iranian strain (ArTeh 193-3) obtained from ticks in the North-Eastern region of Khorasan Province in 1978 clustered within clade Africa I and had similarity with CCHFV strains from West Africa, mainly from Senegal and Mauritania (Sureau and Klein 1980).
In 2008, further phylogenetic analysis in tick populations in Isfahan province in Central Iran, demonstrated that a variant strain clustered within clade IV (Asia 1) with the highest similarity to an Iraqi strain (Chinikar et al. 2012a).
Genetic analyses in 2012 of one strain from Northern Iran illustrated that the Russian CCHFV genome is circulating in this area, and accordingly the existence of European clade V in Iran was confirmed .
According to the previous phylogenetic studies, there are seven clades of CCHFV and Iranian strains of CCHFV distributed within three clade I (Africa I), clade IV (Asia 1) and clade V (Europe) .
The main objective of this study was to obtain new insights into the molecular epidemiology of CCHFV in Iran by analyzing the partial and complete nucleotide sequences of the S and M segments of CCHFV genome obtained from Iranian patients and several sequences available from GenBank. encompassed all geographical areas and were randomly selected and investigated in a span of 11 years between 2002 and 2013. A total of 50 human sera samples were selected among previously RT-PCR positive samples which were stored in the Arboviruses and Viral Hemorrhagic Fevers Laboratory (National Ref. Lab) sample bank; Pasteur Institute of Iran and also transferred samples to the this laboratory during the study. Geographical distribution and the year of sampling related to each strain are shown in Table 1.

Serological and molecular identification
To investigate each human serum sample for the presence of CCHFV-specific antibodies, an ELISA was initially used to detect IgM (Garcia et al. 2006). Molecular analysis was subsequently applied. For this purpose, viral RNA was extracted from 140 μl of serum using a QIAamp RNA Mini kit, according to of the manufacturer's instructions (QI Agen GmbH, Hilden, Germany) (Yashina et al. 2003). Specific primers for amplification of partial S, whole S and M segments were designed by CLC main workbench software version 5.0, based on available CCHFV sequences in GenBank.
To amplify the partial of the S-segment, a primer pair (PSF5'-GAATGTGCATGGGTT AGCTC-3') and (PSR 5'-GACATCACAA TTTCACCAGG-3') was designed and used to amplify a 536 bp section. For reverse transcription, 50 °C (30 min) used. An initial enzyme activation step at 95 °C for 5 min was succeeded by 40 reaction cycles carried out with 30 sec at 94 °C, 30 sec at 50 °C, and 45 sec at 72 °C followed by a final incubation at 72°C for 10 min.
For amplification of the partial of the Msegment, the primers of PMF 5'-TGCAC TTGAGCATCTGCAA-3' and PMR 5'-AG CTGATTCCTGTCCTTTC-3' was designed and used to amplify a 557 bp section of the M-segment. For reverse transcription, 50 °C (30 min) used An initial enzyme activation step at 95 °C for 15 min was succeeded by 40 reaction cycles carried out with 30 sec at 94 °C, 30 sec at 45 °C, and 1 min at 72 °C followed by a final incubation at 72 °C for 10 min (Rodriguez et al. 1997). PCR products were amplified using one-step RT-PCR, according to Rodriguez et al. The amplified products were visualized by ethidium bromide agarose gel staining (Yadav et al. 2012).

Sequencing
The PCR products were then sequenced using Big Dye Terminator V3.1 Cycle sequencing Kit with Modified Sanger Sequencing Method by ABI Genetic Analyzer 3130. For full-length S-segment sequencing, internal primers were designed as follow: Forward: 3'AATGCAAACACGGCAGCTTT 5' and Reverse: 3'GGAACTGTGAGACAG TCGGG5'. The obtained sequences were optimized (gaps and noises were deleted) (Chinikar et al. 2010).

Sequence alignments and phylogeny analysis
In addition to the 50 CCHFV sequences ob -130 http://jad.tums.ac.ir Published Online: January 05, 2016 tained, several sequences available from GenBank at www.ncbi.nih.gov were incorporated into the alignments for phylogenetic analyses ( Table 2). The sequence alignment was performed by ClustalW and a scaled phylogenetic tree generated by the Maximum Likelihood (ML) with Kimura 2-parameter distance using Mega5 software. Bootstrap confidence limits were based on 1000 replicates. This method evaluates the topologies of different trees and chooses the best tree based on a specified model. This model is based on the evolutionary process that can account for the conversion of one sequence into another (Tonbak et al. 2006, Aradaib et al. 2011.
Samples included in this study were correlated to the prevalence of the CCHFV from each region. Accordingly, 11 sera were selected from the Southeast of Iran, 10 sera from Central Iran, 9 sera from Northeast Iran, 8 sera from Southern Iran, 4 sera from West, 3 sera from Northwest, 3 serum samples from North, 1 serum sample from East and 1 serum sample from Southwest.

Partial sequence phylogenetic analysis of S segment
The phylogenetic analysis conducted by the Neighbor Joining (NJ) and Maximum-Likelihood (ML). Only ML are presented here, however phylogenetic tree that were created using the NJ algorithm had the same topology and strongly supported the same phylogenetic groups as the ML tree.
Phylogenetic analysis using the partial Ssegment (~500 bp) demonstrated, of 50 sequenced CCHFV obtained from Iranian patients, 40 sequences (80%), mainly in the Southeast and East of Iran, were located within clade IV (Asia 1). Two Iranian sequences (4%), both in the Southeast of Iran, formed a distinct cluster in the clade IV (Asia 2). These two Iranian sequences fell in the subgroup consisting of the India. Notably, two sequences had strong similarity to each other with 100 % bootstrap support. Seven sequences (14%) of Iranian strains, mainly in the Northwest of Iran, were located within clade V (Europe). One sequence (Iran-Kerman/22) showed itself as out-group and had the highest differences with other clades.
No Iranian sequences were positioned within clade III (S Africa and/ or W Africa 2), clade II (Central Africa), clade I (W Africa 1), and clade VI (Greece AP92).

Complete genome sequence phylogenetic analysis of S segment
The topology of complete genome sequence analysis of S-segment (~1500 bp) was in accordance with partial genome sequence analysis of the S-segment. The phylogeny of the complete S segment nucleotide sequences from strains obtained in Gene-Bank indicated that eight major groups could be recognized. The Iranian S segment from strain Iran-Kerman/22 was least similar to all other S segments and stands as an outgroup and considered as clade VII (Iran) (Fig. 2).

Partial sequence phylogenetic analysis of M segment
Based on phylogenetic analysis on the partial sequence of M segment (~520 bp), six clades were distinguishable. The tree showed that seven Iranian strains clustered in the clade III (S Africa and/ or W Africa) and V (Europe) with other strains of Middle East and Europe.
The Iranian CCHFV strains Iran-Kerman/ 43, Iran-Tehran/65 and Iran-KhRazavi/72 in accordance with the Iranian strains (DQ 446216 and DQ446215) formed a separate cluster within the clades III and showed maximum closeness with the Pakistan (AJ 538199). In addition, Iran-Gilan/69 and Iran-Isfahan/78 strains fell in clade V (Europe) and showed maximum association with the isolates from Russia, Kosovo and Turkey. Interestingly, Iran-Kerman/22 showed the highest similarity to clade V in the M segment analysis (Fig. 3).

Discussion
In 1992, the first complete nucleotide sequence of S-RNA segment of Chinese strain C68031 of CCHFV was determined (Marriott and Nuttall 1992). Afterwards, during an out-break of CCHF in the United Arab Emirates between 1994 and 1995, nested reverse transcriptase polymerase chain reaction (RT-PCR) amplifying of the partial S-RNA seg-136 http://jad.tums.ac.ir Published Online: January 05, 2016 ment of CCHF virus was developed and used for analysis of CCHFVs (Schwarz et al. 1996).
Phylogenetic studies relied on the sequence data of the S-RNA segment have demonstrated genetic diversity for many CCHFV strains from different regions of the world (Iashina et al. 2002). Despite the fact that recombination is relatively scarce in CCHFV genome (Chare et al. 2003) and a partial sequence of S-RNA segment can be used for phylogenetic analyses, but it is recommended to use the full sequence data of the S-RNA segment for obtaining more precise phylogenetic analysis, as the possibility of recombination is still expected (Lukashev 2005).
There have been several genetic analyses of CCHFVs obtained from Iran; however, all the reported Iranian strains were obtained mostly from the South-East of Iran and just four obtained from Central and recently one strain from the Northern regions of Iran (Chinikar et al. 2012a, Chinikar et al. 2004, Chinikar et al. 2010. What makes this study unique is in that a large number of CCHFV strains have been characterized genetically by partial and complete nucleotide sequencing of virus small (S) and medium (M) segments with a wide geographical distribution. Fifty partial sequences of S segment and six partial sequences of M segment and seven complete sequences of S segments from various locations have been studied.
In this study, we have used a bioinformatics approach to analyze an alignment by estimating the phylogenetic relationship between the obtained sequences from Iranian patients and GenBank available data. Phylogenetic analysis based on large collections of partial and complete sequences of the S segment has indicated the existence of eight distinct clades for CCHFV (Drosten et al. 2002). Accordingly, the most surprising finding in this study is discovery of a novel genomic variant of CCHFV. In other words, this study suggests the emergence of VII-IRAN clade for CCHFV in Iran as a novel clade based on S segment analysis.
In general, this phylogenetic analysis based on sequences of S-RNA of CCHFVs reveals that the majority of CCHFV sequences from Iran belongs to clade IV (Asia 1), and clade V (Europe), which is in consistent with previous reports from Iran (Chinikar et al. 2004, Chinikar et al. 2012b). In addition, for the first time, two sequences (Iran-Zahedan/19 and Iran-SistanBalochestan/ 85, which were obtained from the South-East Iran, were seen within clade IV (Asia 2) with the highest proximity to strains from India (JN572089).
The data of this study are in conformity with previous studies which showed similarity between Iranian S segment of CCHFV strains with Pakistan, Afghanistan (Chinikar et al. 2004) and Iraq (Chinikar et al. 2012a), Interestingly findings regarding all phylogeny studies during these years is that CCHFV strain Iran/ArTec 193-3 is likely fade away from Iran and it has been never reported since 35 years ago when it was reported as the first CCHFV strain from Iran by Sureau (Sureau and Klein 1980).
Our data based on the S segment shows that CCHFVs are grouped in eight different clades and have correlation with their geographical location (Morikawa et al. 2007). Previous phylogenetic studies based on L-RNA segment sequences have showed that the L tree topology is similar to the S tree topology (Hewson et al. 2004b). However, the phylogenetic topology based on M-RNA segment sequences of CCHFVs is different from that of S-RNA segments (Seregin et al. 2004, Ahmed et al. 2005. These analyses show that CCHFVs are likely to be grouped in six different phylogenetic clades based on M-RNA sequences (Deyde et al. 2006, Carroll et al. 2010. To obtain a phylogenetic tree based on M segment, a partial sequence data can be used (Morikawa et al. 2007), indicating that re-137 http://jad.tums.ac.ir Published Online: January 05, 2016 combination within the M segments is not common during evolution of the CCHFVs. However, genetic re-assortment occurs frequently in CCHFVs, when host ticks co-infected with different types of CCHFVs (Deyde et al. 2006). The reason why M-RNA segment re-assortment is more frequently observed is not clear, however, strong interrelation between N protein encoded in the S-RNA segment and RNA polymerase encoded in the L-RNA segment may be required to produce viable virus (Chamberlain et al. 2005).
Different examples of RNA segment reassortment can be seen in the M segment phylogenetic tree. Tree based on M segment nucleotide and deduced amino acid sequence differences were very similar to each other. Like Mauritanian strain ArD39554 that belongs to group III in S and stands as an outgroup VII in M segment trees, surprisingly in our obtained strains, the Iranian strain Iran-Kerman/22 forms a unique group and considered as clade VII in S tree (Fig. 2), whereas it grouped within group V (Europe) with 100 % bootstrap support for this topology in M tree (Fig. 3), suggesting that it representing M segment re-assortment.
Other potential M segment re-assortment events include Iranian strains Iran-Kerman/43 and Iran-KhRazavi/72 that were in group IV (Asia 1) in S tree, while cluster within group III in the M segment tree.

Conclusion
The CCHFVs in Iran have multiple origins with vivid geographical relationships between virus strains. Moreover, phylogenetic results reveal subtle links between distant geographical locations, which may originate either from livestock trade or from long-distance carriage of virus by infected ticks during bird migration.
According to all previous phylogeny studies, four genetic lineages of CCHF viruses, clade IV (Asian 1 and II), clade V (Europe) and new clade VII (Iran) exist in Iran. Based on mentioned above data, having a high genomic variation of CCHFV, have been made Iran as a miniature model of the world for genetic analysis of CCHFV with various strains for CCHFV.


s2fieldsofstudy: Biology Medicine

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What is CRISPR/Cas9?

Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 is a gene-editing technology causing a major upheaval in biomedical research. It makes it possible to correct errors in the genome and turn on or off genes in cells and organisms quickly, cheaply and with relative ease. It has a number of laboratory applications including rapid generation of cellular and animal models, functional genomic screens and live imaging of the cellular genome.1 It has already been demonstrated that it can be used to repair defective DNA in mice curing them of genetic disorders,2 and it has been reported that human embryos can be similarly modified.3 Other potential clinical applications include gene therapy, treating infectious diseases such as HIV and engineering autologous patient material to treat cancer and other diseases. In this review we will give an overview of CRISPR/Cas9 with an emphasis on how it may impact on the specialty of paediatrics. Although it is likely to have a significant effect on paediatrics through its impact in the laboratory, here we will concentrate on its potential clinical applications. We will also describe some of the difficulties and ethical controversies associated with this novel technology.

CRISPR/Cas9 is a gene-editing technology which involves two essential components: a guide RNA to match a desired target gene, and Cas9 (CRISPR-associated protein 9)—an endonuclease which causes a double-stranded DNA break, allowing modifications to the genome (see figure 1).

Figure 1
The CRISPR/Cas9 system.1 Clustered regularly interspaced palindromic repeats (CRISPR) refers to sequences in the bacterial genome. They afford protection against invading viruses, when combined with a series of CRISPR-associated (Cas) proteins. Cas9, one of the associated proteins, is an endonuclease that cuts both strands of DNA. Cas9 is directed to its target by a section of RNA. This can be synthesised as a single strand called …

INTRODUCTION
Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 is a gene-editing technology causing a major upheaval in biomedical research. It makes it possible to correct errors in the genome and turn on or off genes in cells and organisms quickly, cheaply and with relative ease. It has a number of laboratory applications including rapid generation of cellular and animal models, functional genomic screens and live imaging of the cellular genome. 1 It has already been demonstrated that it can be used to repair defective DNA in mice curing them of genetic disorders, 2 and it has been reported that human embryos can be similarly modified. 3 Other potential clinical applications include gene therapy, treating infectious diseases such as HIV and engineering autologous patient material to treat cancer and other diseases. In this review we will give an overview of CRISPR/Cas9 with an emphasis on how it may impact on the specialty of paediatrics. Although it is likely to have a significant effect on paediatrics through its impact in the laboratory, here we will concentrate on its potential clinical applications. We will also describe some of the difficulties and ethical controversies associated with this novel technology.

OVERVIEW OF CRISPR/CAS9
CRISPR/Cas9 is a gene-editing technology which involves two essential components: a guide RNA to match a desired target gene, and Cas9 (CRISPR-associated protein 9)-an endonuclease which causes a double-stranded DNA break, allowing modifications to the genome (see figure 1).

Correction of genetic disorders
One of the most exciting applications of CRISPR/Cas9 is its potential use to treat genetic disorders caused by single gene mutations. Examples of such diseases include cystic fibrosis (CF), Duchenne's muscular dystrophy (DMD) and haemoglobinopathies. The approach so far has currently only been validated in preclinical models, but there is hope it can soon be translated to clinical practice.
Schwank et al used CRISPR/Cas9 to investigate the treatment of CF. Using adult intestinal stem cells obtained from two patients with CF, they successfully corrected the most common mutation causing CF in intestinal organoids. They demonstrated that once the mutation had been corrected, the function of the CF transmembrane conductor receptor (CFTR) was restored. 4 Another disease in which CRISPR/Cas9 has been investigated is DMD. Tabebordbar et al recently used adeno-associated virus (AAV) delivery of CRISPR/Cas9 endonucleases to recover dystrophin expression in a mouse model of DMD, by deletion of the exon containing the original mutation. This produces a truncated, but still functional protein. Treated mice were shown to partially recover muscle functional deficiencies. 5 Significantly, it was demonstrated that the dystrophin gene was edited in muscle stem cells which replenish mature muscle tissue. This is important to ensure any therapeutic effects of CRISPR/Cas9 do not fade over time. Two similar studies have described using the CRISPR/Cas9 system in vivo to increase expression of the dystrophin gene and improve muscle function in mouse models of DMD. 6 7 Other studies have used CRISPR/Cas9 to target duplication of exons in the human dystrophin gene in vitro and have shown that this approach can lead to production of full-length dystrophin in the myotubules of an individual with DMD. 8 CRISPR/Cas9 could also be used to treat haemoglobinopathies. Canver et al 9 recently showed BCL11A enhancer disruption by CRISPR/Cas9 could induce fetal haemoglobin in both mice and primary human erythroblast cells. In the future such an approach could allow fetal haemoglobin to be expressed in patients with abnormal adult haemoglobin. This would represent a novel therapeutic strategy in patients with diseases such as sickle cell disease or thalassaemias.
Knock-in of a fully functional β-globin gene is much more challenging, which is the reason for this somewhat unusual approach.

Treatment of HIV
Another potential clinical application of CRISPR/Cas9 is to treat infectious diseases, such as HIV. Although antiretroviral therapy provides an effective treatment for HIV, no cure currently exists due to permanent integration of the virus into the host genome. Hu et al showed the CRISPR/Cas9 system could be used to target HIV-1 genome activity. This inactivated HIV gene expression and replication in a variety of cells which can be latently infected with HIV, without any toxic effects. Furthermore, cells could also be immunised against HIV-1 infection. This is a potential therapeutic advance in overcoming the current problem of how to eliminate HIV from infected individuals. After further refinement, the authors suggest their findings may enable gene therapies or transplantation of genetically altered bone marrow stem cells or inducible pluripotent stem cells to eradicate HIV infection. 10 Engineering somatic cells ex vivo to treat malignancy or other diseases There has been increasing interest in the possibility of using CRISPR/Cas9 to modify patient-derived T-cells and stem/progenitor cells which can then be reintroduced into patients to treat disease. This approach may overcome some of the issues associated with how to efficiently deliver gene editing to the right cells.
T-cell genome engineering has already shown success in treating haematological malignancies and has the potential to treat solid cancers, primary immune deficiencies and autoimmune diseases. Genetic manipulation of T-cells has previously been inefficient. However, Schumann et al recently reported a more effective approach in human CD4 + T-cells based on the CRISPR/Cas9 system. Their technique allowed experimental and therapeutic knockout and knock-in editing of the genome in primary human T-cells. They demonstrated T-cells could be manipulated to prevent expression of the protein PD-1, which other work has shown may allow T-cells to target solid cancers. 11 There is also interest in using CRISPR/ Cas9-mediated genome editing in pluripotent stem cells or primary somatic stem cells to treat disease. For example Xie et al 12 showed the mutation causing β-thalassaemia could be corrected in human induced pluripotent stem cells ex vivo. They suggest that in the future such an approach could provide a source of cells for bone marrow transplantation to treat β-thalassaemia and other similar monogenic diseases.

LIMITATIONS
A number of challenges remain before the potential of CRISPR/Cas9 can be translated to effective treatments at the bedside. A particular issue is how to deliver gene editing to the right cells, especially if the treatment is to be delivered in vivo. To safely deliver Cas9-nuclease encoding genes and guide RNAs in vivo without any associated toxicity, a suitable vector is needed. AAV has previously been a favoured option for gene delivery. 1 However, this delivery system may be too small to allow efficient transduction of the Cas9 gene. 1 A smaller Cas9 gene could be used, but this has additional implications on efficacy. 1 A number of other non-viral delivery systems are under investigation and this process requires further optimisation.
Another significant concern is the possibility of offtarget effects on parts of the genome separate from the region being targeted. Unintentional edits of the genome could have profound long-term complications for patients, including malignancy. The concentration of the Cas9 nuclease enzyme and the length of time Cas9 is expressed are both important when limiting off-target activity. 1 Although recent modifications in the nuclease have increased specificity, further work is required to minimise off-target effects and to establish the long-term safety of any treatment.
The therapeutic applications of CRISPR/Cas9 considered in this article have predominantly been directed at somatic cells. A particularly controversial issue surrounding CRISPR/Cas9 is that of gene editing in Figure 1 The CRISPR/Cas9 system. 1 Clustered regularly interspaced palindromic repeats (CRISPR) refers to sequences in the bacterial genome. They afford protection against invading viruses, when combined with a series of CRISPR-associated (Cas) proteins. Cas9, one of the associated proteins, is an endonuclease that cuts both strands of DNA. Cas9 is directed to its target by a section of RNA. This can be synthesised as a single strand called a synthetic single guide RNA (sgRNA); the section of RNA which binds to the genomic DNA is 18-20 nucleotides. In order to cut, a specific sequence of DNA of between 2 and 5 nucleotides (the exact sequence depends upon the bacteria which produces the Cas9) must lie at the 3' end of the guide RNA: this is called the protospacer adjacent motif (PAM). Repair after the DNA cut may occur via two pathways: non-homologous end joining, typically leading to a random insertion/deletion of DNA, or homology directed repair where a homologous piece of DNA is used as a repair template. It is the latter which allows precise genome editing: the homologous section of DNA with the required sequence change may be delivered with the Cas9 nuclease and sgRNA, theoretically allowing changes as precise as a single base-pair.
embryos. It has already been shown that CRISPR/ Cas9 technology can alter the genome of human embryos 3 which theoretically could prove useful in the preimplantation treatment of genetic diseases. However, any genetic modification of the germline would be permanent and the long-term consequences are unclear. Many oppose germline modification under any circumstances, reasoning that an eventual consequence could be non-therapeutic genetic enhancement. 13 It is clear that the ethical boundaries, within which CRISPR/Cas9 can be used, remain to be fully determined.

Clinical bottom line
▸ CRISPR/Cas9 technology has the potential to revolutionise the treatment of many paediatric conditions. ▸ A number of practical and ethical challenges must be overcome before this potential can be realised at the bedside.


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Cannibalism Affects Core Metabolic Processes in Helicoverpa armigera Larvae—A 2D NMR Metabolomics Study

Cannibalism is known in many insect species, yet its impact on insect metabolism has not been investigated in detail. This study assessed the effects of cannibalism on the metabolism of fourth-instar larvae of the non-predatory insect Helicoverpa armigera (Lepidotera: Noctuidea). Two groups of larvae were analyzed: one group fed with fourth-instar larvae of H. armigera (cannibal), the other group fed with an artificial plant diet. Water-soluble small organic compounds present in the larvae were analyzed using two-dimensional nuclear magnetic resonance (NMR) and principal component analysis (PCA). Cannibalism negatively affected larval growth. PCA of NMR spectra showed that the metabolic profiles of cannibal and herbivore larvae were statistically different with monomeric sugars, fatty acid- and amino acid-related metabolites as the most variable compounds. Quantitation of 1H-13C HSQC (Heteronuclear Single Quantum Coherence) signals revealed that the concentrations of glucose, glucono-1,5-lactone, glycerol phosphate, glutamine, glycine, leucine, isoleucine, lysine, ornithine, proline, threonine and valine were higher in the herbivore larvae.

Introduction
Cannibalism is the process of feeding on parts of or whole individuals of the same species [1]. This phenomenon is taxonomically widespread but uncommon in most animal taxa [2]. Cannibalism may be advantageous since feeding on individuals of the same species, i.e., conspecifics, may represent a source of high quality nutrients. It could also eliminate competitors and predators, thereby making available valuable ecological resources [3,4]. If cannibalism produces such positive effects it could be expected to occur more commonly than it actually does. Perhaps negative effects associated with cannibalism outweigh the benefits under some circumstances, or for some species cannibalism is intrinsically not as beneficial as it is assumed. When ingesting conspecifics, cannibals expose themselves to injuries from prey that fight back and to the pathogens and parasites present in the victim [2,5]. Cannibalism may also reduce the chances of finding a mate. However, it could also be possible that conspecifics simply do not represent the best quality diet for some animal species.
Cannibalism has been reported in many insect species, both carnivores and herbivores. One example is the larval stages of Helicoverpa armigera (H. armigera, Hübner, Lepidotera: Noctuidea), the cotton bollworm, when kept under laboratory conditions [6]. H. armigera is one of the worst insect pests of agriculture worldwide, attacking a wide range of food, fiber, oil, and fodder crops as 2 of 9 well as many horticultural and ornamental crops. It thereby causes economic damage amounting annually to millions of dollars in crop losses and environmental damage due to the extensive use of insecticides required to control its populations [7,8]. Cannibalism in Lepidoptera is commonly associated with environmental factors such as high population densities but it may also have a genetic component. In Helicoverpa virescens, a close relative of H. armigera, inheritance has been shown to determine different levels of cannibalism over a geographic range [3]. Due to its economic and ecologic relevance, H. armigera has become a model system for entomology, yet the impact of cannibalism on its metabolism has not been evaluated. Therefore, in the present study it was investigated how cannibalism shapes the H. armigera metabolism and influences its development. H. armigera larvae were fed with conspecifics of the same size (henceforth "cannibals") or with an artificial plant diet ("herbivores"). It was expected that if conspecifics represent a good quality diet, the growth rate and survival of cannibals and herbivores should be at least similar. It was also examined whether cannibals and herbivores had similar metabolic fingerprints, which were determined by two-dimensional nuclear magnetic resonance.

Results and Discussion
Helicoverpa armigera cannibals were smaller than the herbivores (Figure 1), a result in agreement with previous reports [6]. The present study, however, pioneers in finely dissecting the consequences of cannibalism on the physiology of this insect. It is not known how often H. armigera larvae cannibalize each other in nature nor if there are larvae that are entirely cannibalistic or if larvae actually switch back and forth between herbivory and cannibalism. However, this study indicates that pure cannibalism has a negative impact on the development of H. armigera. Previous reports also showed that, under laboratory conditions, cannibalism negatively affected pupation with cannibals showing a six-fold decrease in successful pupation [6]. If this negative effect on pupation also exists in nature, it is not known. In this study no anatomical differences associated with size differences were observed between cannibals and herbivores. Strong differences were detected, however, in the 1 H-13 C HSQC (Heteronuclear Single Quantum Coherence) spectra of larval D 2 O extracts. NMR metabolomics studies are vastly dominated by 1 H spectroscopy for the profiling of samples ( Figure S1), with 1 H-13 C HSQC being the preferred technique for compound identification. This is mainly due to the higher sensitivity of 1 H spectroscopy; however, 1 H-13 C HSQC can also be used for the profiling itself. The inherent disadvantage in low sensitivity is compensated by the increment in the number of signals detected per sample as a result of the spreading of the 1 H signals on the 13 C dimension [9,10]. Signals in the 1 H-13 C HSQC spectra were manually defined as 135 regions of interest (ROIs) using rNMR ( Figure S2).
The chemical shifts of the 135 ROIs (the centroid of the 1 H-13 C cross-peaks) were analyzed with SpinAssign to generate a list of candidate metabolites present in the extracts (Table S1). The chemical shifts of the candidate metabolites were confirmed using two additional public databases (HMBD and BMRB).
In parallel, the 135 ROIs were batch integrated (all the ROIs for all the spectra at once) in rNMR to generate a matrix of signal integrals (Table S2). This ROI data matrix was subjected to principal component analysis (PCA). PCA scores showed a distinct signature for the chemical profiles of cannibal larvae, with the formation of two separated clusters along PC1 (Figure 2), a pattern also displayed when 1 H NMR data obtained from the same samples were used for PCA ( Figure S3, Table S3). Interestingly, within-group variation along PC2 was larger in the cannibals. Since all larvae used for the assay were randomly chosen from the main population, it is unexpected that this phenotypic difference has a genetic component; rather, it must be caused by an environmental factor. The difference in variation along PC2 might be the result of the higher homogeneity of the plant diet ingested by the herbivores. All larvae, cannibals and herbivores, were provided with an amount of food larger than they could eat to prevent metabolic differences caused by starvation. It is possible that some cannibals ingested more heads while other cannibals ingested proportionally more thoraxes or abdomens, generating more variation in the metabolic profiles of cannibals. The combination of the PCA loadings with the identification of the metabolites producing the different ROIs showed that several of the metabolites with the largest variability were grouped into three classes: monomeric sugars, fatty acid-related compounds and amino acids and amino acid-related compounds ( Figure 3). metabolomics studies are vastly dominated by 1 H spectroscopy for the profiling of samples ( Figure S1), with 1 H-13 C HSQC being the preferred technique for compound identification. This is mainly due to the higher sensitivity of 1 H spectroscopy; however, 1 H-13 C HSQC can also be used for the profiling itself. The inherent disadvantage in low sensitivity is compensated by the increment in the number of signals detected per sample as a result of the spreading of the 1 H signals on the 13 C dimension [9,10]. Signals in the 1 H-13 C HSQC spectra were manually defined as 135 regions of interest (ROIs) using rNMR ( Figure S2). The chemical shifts of the 135 ROIs (the centroid of the 1 H-13 C cross-peaks) were analyzed with SpinAssign to generate a list of candidate metabolites present in the extracts (Table S1). The chemical shifts of the candidate metabolites were confirmed using two additional public databases (HMBD and BMRB). Figure 1. Growth rate of herbivore (blue) and cannibal (red) Helicoverpa armigera larvae. Weight of individual larvae was recorded from the moment a group of larvae was switched from herbivore diet to cannibalism. Larvae were in third instar at the beginning of the experiment. Bars represent mean and standard error of the mean; t-tests were computed to identify differences between diets at specific days (* p > 0.05).

Figure 1.
Growth rate of herbivore (blue) and cannibal (red) Helicoverpa armigera larvae. Weight of individual larvae was recorded from the moment a group of larvae was switched from herbivore diet to cannibalism. Larvae were in third instar at the beginning of the experiment. Bars represent mean and standard error of the mean; t-tests were computed to identify differences between diets at specific days (* p > 0.05). In parallel, the 135 ROIs were batch integrated (all the ROIs for all the spectra at once) in rNMR to generate a matrix of signal integrals (Table S2). This ROI data matrix was subjected to principal component analysis (PCA). PCA scores showed a distinct signature for the chemical profiles of cannibal larvae, with the formation of two separated clusters along PC1 (Figure 2), a pattern also displayed when 1 H NMR data obtained from the same samples were used for PCA ( Figure S3, Table S3). Interestingly, within-group variation along PC2 was larger in the cannibals. Since all larvae used for the assay were randomly chosen from the main population, it is unexpected that this phenotypic difference has a genetic component; rather, it must be caused by an environmental factor. The difference in variation along PC2 might be the result of the higher homogeneity of the plant diet ingested by the herbivores. All larvae, cannibals and herbivores, were provided with an amount of food larger than they could eat to prevent metabolic differences caused by starvation. It is possible that some cannibals ingested more heads while other cannibals ingested proportionally more thoraxes or abdomens, generating more variation in the metabolic profiles of cannibals. The combination of the PCA loadings with the identification of the metabolites producing the different ROIs showed that several of the metabolites with the largest variability were grouped into three classes: monomeric sugars, fatty acid-related compounds and amino acids and amino acid-related compounds ( Figure 3). HSQC data are primarily used for qualitative purposes, yet direct relative quantitation of HSQC signals is possible due to the intrinsic highly linear response of this NMR experiment [11]. Thus, in order to have a clearer view on the variation of individual metabolites among diets, the integrals of one ROI per metabolite were compared in cannibal and herbivore larvae. If a given metabolite produced more than one ROI, then ROIs that did not overlap with other metabolites were used for intensity comparison. Using this approach it was observed that the largest difference in concentration for all metabolites was detected for D-glucose which was much more abundant in the herbivore larvae ( Figure 4). The other identified monomeric sugar, D-glucono-1,5-lactone, was also more abundant in herbivores ( Figure 4). D-glucono-1,5-lactone is an oxidation product of D-glucose. The reaction is carried out by glucose oxidase, an enzyme secreted in the labial salivary glands of H. armigera [12]. HSQC data are primarily used for qualitative purposes, yet direct relative quantitation of HSQC signals is possible due to the intrinsic highly linear response of this NMR experiment [11]. Thus, in order to have a clearer view on the variation of individual metabolites among diets, the integrals of one ROI per metabolite were compared in cannibal and herbivore larvae. If a given metabolite produced more than one ROI, then ROIs that did not overlap with other metabolites were used for intensity comparison. Using this approach it was observed that the largest difference in concentration for all metabolites was detected for D-glucose which was much more abundant in the herbivore larvae ( Figure 4). The other identified monomeric sugar, D-glucono-1,5-lactone, was also more abundant in herbivores (Figure 4). D-glucono-1,5-lactone is an oxidation product of D-glucose. The reaction is carried out by glucose oxidase, an enzyme secreted in the labial salivary glands of H. armigera [12].  In the case of the fatty acid-related metabolites, glycerol phosphate was much more abundant in the herbivores ( Figure 5). Also more abundant in the herbivores were glycerol and choline; however, choline phosphate was more abundant in cannibals ( Figure 5). Choline belongs to a special class of nutrients termed "quasi-vitamins" or "conditionally essential" nutrients and its absence from artificial diets results in a somewhat reduced insect growth rate [13]. Choline is absorbed by the midgut cells of Pieris brassicae, a lepidopteran like H. armigera. It is then transported into the hemolymph and later converted, probably in the fat body, into phospholipids [14]. The phosphate ester of choline, choline phosphate, is the most abundant choline metabolite in the hemolymph of the silkworm (Bombyx mori L., also a lepidopter) and its concentration increases as the larvae approaches pupation [15]. Choline phosphate is involved in the biosynthesis of phospholypids for the formation of cell membranes.  In the case of the fatty acid-related metabolites, glycerol phosphate was much more abundant in the herbivores ( Figure 5). Also more abundant in the herbivores were glycerol and choline; however, choline phosphate was more abundant in cannibals ( Figure 5). Choline belongs to a special class of nutrients termed "quasi-vitamins" or "conditionally essential" nutrients and its absence from artificial diets results in a somewhat reduced insect growth rate [13]. Choline is absorbed by the midgut cells of Pieris brassicae, a lepidopteran like H. armigera. It is then transported into the hemolymph and later converted, probably in the fat body, into phospholipids [14]. The phosphate ester of choline, choline phosphate, is the most abundant choline metabolite in the hemolymph of the silkworm (Bombyx mori L., also a lepidopter) and its concentration increases as the larvae approaches pupation [15]. Choline phosphate is involved in the biosynthesis of phospholypids for the formation of cell membranes. In the case of the fatty acid-related metabolites, glycerol phosphate was much more abundant in the herbivores ( Figure 5). Also more abundant in the herbivores were glycerol and choline; however, choline phosphate was more abundant in cannibals ( Figure 5). Choline belongs to a special class of nutrients termed "quasi-vitamins" or "conditionally essential" nutrients and its absence from artificial diets results in a somewhat reduced insect growth rate [13]. Choline is absorbed by the midgut cells of Pieris brassicae, a lepidopteran like H. armigera. It is then transported into the hemolymph and later converted, probably in the fat body, into phospholipids [14]. The phosphate ester of choline, choline phosphate, is the most abundant choline metabolite in the hemolymph of the silkworm (Bombyx mori L., also a lepidopter) and its concentration increases as the larvae approaches pupation [15]. Choline phosphate is involved in the biosynthesis of phospholypids for the formation of cell membranes. As for the amino acids and amino acid-related compounds, they were more abundant in herbivores, with the exception of glutamate, which was slightly more abundant in cannibals, and putrescine, which showed comparable concentrations in cannibals and herbivores ( Figure 6). Putrescine has been shown to have a high incidence during diapause in H. armigera [16]. Diapause is a programmed developmental arrest coupled with the depression of metabolic activity, and in H. armigera diapause induction and preparation occur in the larval stage [17]. Thus, the fact that cannibals and herbivores contained the same amount of putrescine implies that both types of caterpillars in this study were found in a comparable developmental stage. This reinforces the fact that the observed differences in the metabolic profiles of cannibals and herbivores are the result of the diet and not the larval ontology. Some evidence considers cannibalism as adaptive [1,[18][19][20], and in the case of herbivore insects feeding on conspecifics, it may provide nutrient compositions that are more optimal than plant diets. For herbivores, conspecific predation may provide a meal with a higher carbon-to-nitrogen ratio (C:N ratio) than the normal plant diet and it has been suggested that cannibalism in such animals may result from a specific need for protein [21]. On the other hand, there is evidence that cannibalism has a negative impact on insect fitness. For instance, in larvae of the lepidopteran Spodoptera frugiperda, cannibalism reduces survival and pupal weight [22], and larval cannibalism in the lepidopteran Ascia As for the amino acids and amino acid-related compounds, they were more abundant in herbivores, with the exception of glutamate, which was slightly more abundant in cannibals, and putrescine, which showed comparable concentrations in cannibals and herbivores ( Figure 6). Putrescine has been shown to have a high incidence during diapause in H. armigera [16]. Diapause is a programmed developmental arrest coupled with the depression of metabolic activity, and in H. armigera diapause induction and preparation occur in the larval stage [17]. Thus, the fact that cannibals and herbivores contained the same amount of putrescine implies that both types of caterpillars in this study were found in a comparable developmental stage. This reinforces the fact that the observed differences in the metabolic profiles of cannibals and herbivores are the result of the diet and not the larval ontology. As for the amino acids and amino acid-related compounds, they were more abundant in herbivores, with the exception of glutamate, which was slightly more abundant in cannibals, and putrescine, which showed comparable concentrations in cannibals and herbivores ( Figure 6). Putrescine has been shown to have a high incidence during diapause in H. armigera [16]. Diapause is a programmed developmental arrest coupled with the depression of metabolic activity, and in H. armigera diapause induction and preparation occur in the larval stage [17]. Thus, the fact that cannibals and herbivores contained the same amount of putrescine implies that both types of caterpillars in this study were found in a comparable developmental stage. This reinforces the fact that the observed differences in the metabolic profiles of cannibals and herbivores are the result of the diet and not the larval ontology. Some evidence considers cannibalism as adaptive [1,[18][19][20], and in the case of herbivore insects feeding on conspecifics, it may provide nutrient compositions that are more optimal than plant diets. For herbivores, conspecific predation may provide a meal with a higher carbon-to-nitrogen ratio (C:N ratio) than the normal plant diet and it has been suggested that cannibalism in such animals may result from a specific need for protein [21]. On the other hand, there is evidence that cannibalism has a negative impact on insect fitness. For instance, in larvae of the lepidopteran Spodoptera frugiperda, cannibalism reduces survival and pupal weight [22], and larval cannibalism in the lepidopteran Ascia Some evidence considers cannibalism as adaptive [1,[18][19][20], and in the case of herbivore insects feeding on conspecifics, it may provide nutrient compositions that are more optimal than plant diets. For herbivores, conspecific predation may provide a meal with a higher carbon-to-nitrogen ratio (C:N ratio) than the normal plant diet and it has been suggested that cannibalism in such animals may result from a specific need for protein [21]. On the other hand, there is evidence that cannibalism has a negative impact on insect fitness. For instance, in larvae of the lepidopteran Spodoptera frugiperda, cannibalism reduces survival and pupal weight [22], and larval cannibalism in the lepidopteran Ascia monuste orseis produces smaller adults [23]. Considering the contrasting evidence, it is possible that cannibalism may have positive or negative effects, depending on the species involved and the environmental conditions. The present study with its pioneering nature provides a strong case for depletion in nutrient availability as a possible cause for the negative consequences of cannibalism observed in some insect species. Additionally, not only the total amount of but the ratios between different classes of nutrients can be critical in explaining the performance of cannibals. Particularly, the carbohydrate-to-protein ratio has been shown to be critical for the performance (growth rate × survival) of the orthopteran Chortoicetes terminifera [24]. In this species a 2:1 carbohydrate-to-protein ratio has been found as an optimal diet. If the amount of free amino acids that we detect in the bodies of H. armigera larvae at least partially reflects the amount of protein ingested, then the carbohydrate-to-protein ratio would be larger in the herbivores and this might be connected to their larger size. Furthermore, the carbohydrate-to-protein ratio in the insect diet influences the absorption efficiency of carbohydrates and proteins in the insect gut and this has been correlated with specific gut microbiotas and the functionality of the insect immune system [24] and this, in turn, could also explain the differences in the performance between herbivore and cannibal H. armigera larvae.

Insect Bioassay
Helicoverpa armigera eggs were purchased from Sumika Technoservice Corporation (Terazuka-shi, Hyogo, Japan). Larvae were fed from hatching with the artificial diet Silk Mate 2S based on mulberry leaves and manufactured by Nosan Corporation (Yokohama-shi, Kanagawa, Japan). Upon arrival to the laboratory larvae were kept in 2 mL Eppendorf tubes, one larva per tube with plenty of artificial diet. Once the larvae reached an average weight of 40 mg thirty larvae were transferred to Petri dishes, one larva per dish. Third-to fourth-instar larvae (aprox. 40 mg) were used because cannibalism is less common in earlier [6] and latter [25,26] larval stages of H. armigera in captivity and also to maximize the amount of sample available per larva for NMR analyses. Larvae used for the bioassay were randomly chosen from the main stock. Fifteen larvae were fed with the Silk Mate 2S diet (herbivore group) and the other 15 larvae (cannibal group) were fed with prey larvae from the main stock (larvae always fed with artificial diet). Larvae used as prey were selected to have a similar size as the cannibals they were going to feed. The jaws of the prey larvae were disabled by longitudinally splitting the larva's head using a scalpel, thereby preventing wounds caused by prey larvae fighting back. Every cannibal larva was offered one live, disabled prey larva in the morning and one more in the afternoon to ensure that cannibals were never devoid of food. The remains of the previous prey larva were always removed when placing the new one in the Petri dish to minimize the risk of infection for the cannibals. Insects were kept at 28 • C under natural light. After two days in the case of the herbivores and three days for the cannibals, larvae were killed by placing them at −20 • C. Larvae were sacrificed when they started showing signs of preparation for pupation, which happened at different times between diets. Whole larvae were freeze-dried and the water-free material was weighed and used for NMR analyses. The Ministry of Education, Culture, Sports, Science and Technology of Japan does not require ethical approval for working with insects. All efforts were made to minimize suffering of the larvae.

Sample Preparation for NMR
To minimize chemical shift variations due to pH differences across samples a 1 mM phosphate buffer (KPi) was used as solvent. It consisted of 10.7 g K 2 HPO 4 + 5.24 g KH 2 PO 4 in 1 L D 2 O (D 99.9% Cambridge Isotope) containing 1 mM DSS-d 6 (D 98% Cambridge Isotope) as internal standard. The buffer was directly measured with a pH-meter, which showed a value (pD) of 7.0. Experimental and theoretical approaches showed that pH = pD + 0.41 [27], thus the pH of the buffer was 7.41. Freeze-dried larvae were individually placed in 1.5 mL Eppendorf tubes and covered with 700 µL buffer. Larvae were ground-up with a plastic pestle. Samples were centrifuged at 17,500× g at 20 • C for 10 min. After recovering the supernatant dilutions were made against the larva with the smallest dry weight in the whole experiment in order to normalize the amount of insect material per sample. The final volume for all samples was 600 µL of deuterated solvent.

NMR Spectra Acquisition and Processing Parameters
Spectra were recorded using an Avance II 700 Bruker spectrometer equipped with a 5 mm inverse CryoProbe operating at 700.153 MHz for 1 H and 176.061 MHz for 13 C. Acquisition temperature was 298 K. The 1 H spectra were acquired using a water suppression pulse program with the following conditions: 0.38 Hz × point −1 , acquisition time = 1.31 s, relaxation delay = 2.0 s and 90 • pulse width = 8 µs. Sixty-four transients with 0 dummies were recorded per spectrum. FIDs (free induction decays) were Fourier-transformed using line broadening (LB = 1.0 Hz). The resulting spectra were manually phased and baseline-corrected and calibrated to the internal standard (DSS-d 6 ). 1 H-13 C HSQC spectra were acquired using the Bruker pulse program hsqcetgp for echo/antiecho gradient with the following conditions: relaxation delay = 2.0 s; 7043 Hz spectral width in f1 and 9804 Hz in f2; 90 • pulse widths = 10 µs for hydrogen, 15 µs for carbon. The partial spectral width (aliasing) in the indirect dimension (40 ppm in 13 C) allowed to reduce the acquisition time per spectrum without losing spectral resolution [28,29]. Qsine (SSB = 2.0) was used for the window function. Sixteen dummies and 64 transients were collected. FIDs were Fourier-transformed and the resulting spectra were manually phased, baseline corrected and calibrated using glucose (C1: δ 1 H = 5.22376, δ 13 C = 54.764) and allantoate (δ 1 H = 5.23749, δ 13 C = 62.8906) signals.

Multivariate Statistical Analyses of 1 H and 1 H-13 C HSQC NMR Spectra
1 H NMR spectra. Phased and baseline-corrected 1 H NMR spectra were binned from 10.0 to 0.5 ppm with a bin width of 0.02 ppm using the software automics [30]. Binning was performed applying the total scaling function of automics and the region from 5.0 to 4.6 ppm (HDO signal) was not binned. The matrix with the data of all the binned spectra (455 bins per spectrum) was exported to the software R (r-project.org) and processed with the package muma for NMR metabolomics [31]. Principal component analysis (PCA) was performed according to muma's manual and applying the following parameters: scaling = "pareto", imput = "mean", normalize = TRUE. Pareto scaling was chosen as the data stays closer to the original measurement. 1 H-13 C HSQC NMR spectra. Phased, baseline-corrected and calibrated 1 H-13 C HSQC NMR spectra were processed with rNMR (http://rnmr.nmrfam.wisc.edu/) to manually generate a list of regions of interest (ROI) with signals present across all samples. One hundred thirty-five ROIs were identified and the signals intensities in every ROI were obtained in batch mode with the following parameters: type = area, normalization = signal to noise. The matrix of ROIs intensities was exported to the software R and processed with the package muma for NMR metabolomics [30]. Principal component analysis (PCA) was performed according to muma's manual and applying the following parameters: scaling = "pareto", imput = "mean", normalize = TRUE.

Candidate Compounds
The 1 H-13 C cross-peak chemical shifts were recorded at the center of each ROI to generate a list of chemical shifts that was exported to the databases SpinAssign (http://prime.psc.riken.jp), HMBD (http://www.hmdb.ca/) and BMRB (http://www.bmrb.wisc.edu/metabolomics/) for compound identification by signal matching.

Conclusions
We present the first evidence of the dramatic changes in body chemical composition experienced by Helicoverpa armigera larvae when they become completely cannibalistic and the negative effect that this has on the growth of cannibals, and we elaborate on how these changes might shape the fitness of this insect.