The female reproductive lifespan is largely determined by the size of primordial follicle pool, which is established following germ cell cyst breakdown around birth. Almost two-third of oocytes are lost during germ cell cysts breakdown, following autophagic and apoptosis mechanisms. To investigate a possible relationship between germ cell cyst breakdown and nutrition supply, we established a starvation model in mouse pups at birth and evaluated the dynamics of cyst breakdown during nutrient deprivation. Our results showed that after 36 h of starvation between 1.5 and 3 d.p.p., indicators of metabolism both at systemic and ovarian level were significantly altered and the germ cell cyst breakdown markedly decreased. We also found that markers of oxidative stress, autophagy and apoptosis were increased and higher number of oocytes in cyst showing autophagic markers and of TUNEL-positive oocytes and somatic cells were present in the ovaries of starved pups. Moreover, the proliferation of pre-granulosa cells and the expression of the oocyte-specific transcription factor Nobox were decreased in such ovaries. Finally, we observed that the ovaries of the starved pups could recover a normal number of follicles after about 3 weeks from re-feeding. In conclusion, these data indicate that nutrient deficiency at birth can generate a number of adaptive metabolic and oxidative responses in the ovaries causing increased apoptosis both in the somatic cells and oocyte and autophagy mainly in these latter and leading to a delay of germ cell cyst breakdown and follicle assembly. At first, one germline stem cell undergoes four times mitosis to form a 16-cell cyst, only one of these develops into an oocyte while the other 15 cells act as nurse cells. Such cells provide nutrients, proteins, mRNAs and organelles for the developing oocyte, transport their contents into the oocyte and undergo programmed cell death (PCD).8Germ cell cysts refer to a cluster of interconnected germ cells resulting from incomplete mitotic cytokinesis.10 It is not clear, however, whether oocytes in cysts die during or after cyst breakdown. In one model, one oocyte of a cyst dies following apoptosis and the large cyst breaks into two smaller cysts; this is repeated until a few individual oocytes remain.11 However, another study showed that cyst breakdown and apoptosis do not precisely coincide indicating that apoptosis is unlikely to be the major cause of cyst breakdown and that most of the oocytes actually die after cyst breakdown.6 Oocyte death during the fetal and early postnatal period has been described to occur by multiple causes and processes including apoptosis and autophagy.15 Several lines of evidence demonstrate that inducers of oxidative stress may act as signaling molecules in the pathway of apoptosis in several, if not all, cells and tissues.17 Oxidative stress occurs when the normal cellular redox balance is disturbed, resulting in dysregulation of redox-regulated processes and/or oxidative damage to cellular structures.19 As a matter of fact, oxidative stress can impair the ovary's function and normal development of follicles and has been associated with polycystic ovary syndrome (PCOS).21 Actually, in rodent models, increased ROS levels induce rapid primordial follicle loss and apoptosis in antral follicles.22 Whatever the exact mechanisms of oocyte apoptosis, in the mouse, more than two-third of oocytes die before and a few days after birth. At this time, cyst disappearance is concomitant with the formation of the primordial follicles (PFs). Processes extending from somatic cells have been observed between oocytes suggesting that somatic cells may physically separate oocytes.11 Physiologically, the size of the PF pool will largely determine the reproductive lifespan of female mammals.Likewise in the mouse, some oocytes within a cyst appear to receive nutrients and organelles from neighboring oocytes fated to undergo death.In vivo and in vitro exposure of neonatal mouse ovaries to E2, progesterone or phytoestrogens , inhibits cyst breakdown and PF assembly.24 Synthetic forms of estrogens, such as diethylstilbestrol (DES), bisphenol-A (BPA) and ethinyl estradiol (EE), also block cyst breakdown and alter PF formation.27 Disruption of Notch signaling by pharmacological inhibition, global deletion of Notch2 or Hes1  or conditional knockout of Lfng (a negative modulator of Notch) or Hes1 in somatic cells, result in abnormal oocyte maturation and multi-oocyte follicles (MOFs).31 Other signaling molecules and several growth factors appear to have a role in follicle assembly. Among these, the stem cell factor 35 and neurotrophins38 favor such process. Mutants for either bone morphogenetic protein 15 (BMP15) or growth differentiation factor 9 (GDF9), have ovaries with a significant proportion of MOFs.39 Both BMP15 and GDF9 are secreted by the oocyte early in ovarian differentiation, supporting the notion that signals from the oocyte to the granulosa cells are important for cyst breakdown and follicle formation. Mouse ovaries exposed to another TGFβ family member, activin A, showed an increased number of PFs.40 Sustaining a positive role of activin in PF formation, more MOFs were observed in mice overexpressing the activin antagonist, inhibin B.41 In addition, activin subunit expression is reduced in neonatal mice treated with E2, which blocks cyst breakdown.42 Mutants of another activin antagonist, follistatin, also have defects in oocyte development including a delay in cyst breakdown and follicle formation,43 while FST288, the strongest activin-neutralizing isoform, impaired germ cell nest breakdown and primordial follicle assembly by inhibiting pre-granulosa cell proliferation.44 The last TGFβ family member that has been implicated in follicle assembly is anti-Mullerian hormone (AMH). PF assembly is reduced in newborn rat ovaries treated with AMH while oocyte number is greater than controls.45 A study using mouse ovary organ culture implicates FSH in promoting cyst breakdown and PF formation.46 Finally, a study in the mouse ovary showed that cyst breakdown requires the activity of c-Jun N-terminal kinases (JNKs) that are likely necessary to downregulate E-cadherin expression in oocytes.47The processes of cyst breakdown and follicle assembly likely involve communication between oocytes and pre-granulosa cells that is mediated and regulated by several factors. As a matter of fact, a number of compounds can influence cyst breakdown and consequently the pool of PFs. 49 Mice lacking the factor in germline alpha , also encoding a bHLH protein, begin to lose oocytes at birth and oocytes still present are not enclosed in PFs.50 Disruption of Nobox (an oocyte-specific homeobox gene), results in increased mouse oocyte loss and a delay in cyst breakdown.52 Mice mutant for Foxl2, (a winged-helix forkhead transcription factor), are sterile with germ cell cysts that do not breakdown.53 Finally, siRNA knockdown of heterogeneous nuclear ribonucleoprotein K in rat ovary organ culture caused a block in cyst breakdown and follicle formation.54 Recent data showed that PCNA (proliferating cell nuclear antigen) can regulate primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries.55A number of transcription factors appear also to be important for PF formation. Mutant mice for the gene encoding the aryl hydrocarbon receptor (AHR), a basic helix loop helix (bHLH) transcription factor, form follicles at a faster rate than normal.Drosophila and Caenorhabditis elegans, starvation activates apoptotic checkpoints and autophagy in oogenesis and reduces the production of mature oocytes.57 In the perilous hours immediately after birth, a newborn mammal must survive the sudden loss of food supply from its mother. Under normal circumstances, newborns increase a metabolic response to ward off starvation until feeding occurs. Under these conditions, autophagy may become essential for viability of various tissues.58 The observation that in mouse the most part of the cyst breakdown occurring from 2 to 5 d.p.p., parallels the time when fully nutrient supply is restored by milk feeding, prompted us to investigate the influence of starvation on this process and PF assembly.Many animals alter their reproductive strategies in response to environmental stress. For example, in female In a first series of experiments, we characterize the dynamics of germ cell cyst breakdown in mouse ovaries from 0 to 4 d.p.p. Immunolocalization of the oocytes with the germ cell specific marker MVH (mouse Vasa homolog) in tissue sections, revealed that while almost all oocytes were located in germ cell cysts (indicated by arrowheads) at 0 d.p.p., more than half of them were individually enclosed into follicles (indicated by arrows) at 4 d.p.p. . BetweenTEM observations showed a frequent presence of autophagosomes, recognized for their characteristic spherical vesicle with double-layer membranes, suggesting macroautophagy, in the oocyte cytoplasm and of large number of mitochondria in some oocytes within cysts at 3 d.p.p. . On the P<0.01; P<0.01), while the amino acid metabolism appeared altered  increased transcripts of Sod1, Clrx1 and Txnrd1 and of the Bax/Bcl2 mRNA ratio in starved ovaries , no significant difference remained between the control and starved animals . At this10 These materials, derived by transfer from interconnected oocytes, could be used as material for autophagy. Under starvation condition, autophagy could be exacerbated and ultimately lead to apoptosis. Actually, several evidences are accumulating that autophagy and apoptosis may be triggered by common upstream signals, sometimes converging in combined autophagy and apoptosis. The alteration of metabolic parameters and the increased expression of a number of enzymes involved in oxidative stress alongside with enhance of both autophagic and apoptosis markers in the ovaries from starved pups, testify a general cellular stress induced by the nutrient deprivation as the trigger of the cell death processes. Under such conditions, both pre-granulosa cell proliferation and expression of some oocyte transcription factors , crucial for follicle assembly, appeared also to be impaired. This might explain the reduced rate of cyst breakdown. As Nobox is expressed mostly in primary (growing) oocytes and no change in transcripts level of other oocyte-specific genes such as Lhx8, Sohlh2, Figlα occur, oocyte growth could be also impaired in starved ovaries. Interestingly, the adverse effects of starvation on the ovaries appeared reversible. Actually, after about 3 weeks from pup re-feeding, the ovaries regained a normal number of follicles. This could indicate that cyst breakdown was only delayed and markers of oxidative stress, autophagy and apoptosis, increased in the starved ovaries. Despite these, the numbers of the remaining pre-granulosa cells and oocytes in such ovaries were apparently still sufficient to sustain the assembly of a normal number of follicles. Alternatively, this finding can be explained by the presence of ovarian or extra-ovarian stem cells able to sustain the ovary recovery.60The results of our first analyses concerning the dynamics of germ cell cyst breakdown, showed that a peak in this process occurred between 1 and 3 d.p.p. and was associated to a marked decrease of the oocyte number. Moreover, morphological and molecular markers of autophagy were found in some oocytes inside cysts. All together, these data support the existence of a relationship between cyst breakdown and oocyte degeneration but do not clarify whether oocyte death precedes, is concomitant or occurs after such process. At the same time, the presence of autophagic markers in oocytes within cysts, primordial follicles and the granulosa cells of primary follicles, but not inside primary follicles, suggests that, under normal condition, activation in one or a few cystic oocytes of autophagy perhaps related to a large number of mitochondria observed at TEM, can be a prerequisite for their survival after cyst breakdown. In line with our observations, some of the mouse oocytes in cysts have been reported to acquire many organelles, including mitochondria, and other cytoplasmic components before primordial follicle formation.61Dehydration might be a non-negligible factor in ovary molecular changes even though the starved pups rejected to suck water and the starvation time was not so long. The main objective of this investigation was to explore the effects of starvation and the influence of dehydration on mouse primordial follicles formation would be the next phase study.In conclusion, the present data indicate that nutrient deficiency at birth can generate a number of adaptive metabolic and oxidative responses in the ovaries causing increased apoptosis both in the somatic cells and oocyte and autophagy mainly in these latter and leading to impaired formation of the PF pool. Under the relatively short starvation time used by us (36 h), possibly thanks to such adaptive response, the ovary maintains the capability of regaining a normal follicle pool.adlibitum. Females were mated with males and the presence of the vaginal plug the morning after breeding, was considered as 0.5 day of pregnancy. For starvation protocols, pups were separated from mother at 1.5 d.p.p. for 36 h. To make sure to use pups that have received the same nutrition level before starvation, only animals with the same body weight (2.2±0.1 g) were selected. All procedures involving animals were approved by the Institutional Animal Care and Use Committees of Qingdao Agricultural University.CD1 mice were obtained from Vital River Laboratory Animal Technology Co. LTD  and maintained in the animal room of Qingdao Agriculture University, μm. The sections were heated at 60 °C in an air oven for 2 h, then immediately washed in xylene and rehydrated through a graded series of ethanol and soaked in PBS. Finally, they were transferred in 0.01 M sodium citrate buffer at high temperature (95 °C) for 10 min. After 1 h blocking with BDT , the sections were incubated with primary antibodies .TUNEL-positive cells were detected in paraffin sections of ovaries using the Bright Red Apoptosis Detect Kit . Briefly, the sections were heated at the 60 °C in an air oven for 2 h, then immediately washed in xylene and rehydration through a graded series of ethanol, and soaked in PBS. The samples were then treated with proteinase K for 15 min at room temperature, rinsed twice with PBS and incubated for 60 min at 37 °C in the dark in 100 The ovaries were dissected and immediately fixed in 2.5% glutaraldehyde in 0.2 M PBS (pH=7.2) overnight at 4 °C. The samples were processed and included in epoxypropane resin following standard transmission electron microscopy (TEM) procedures. Serial sections were cut at 50 nm using the EM UC7 ultramicrotome , stained with lead citrate and uranium and observed under HT7700 transmission electron microscope.β-Actin was used as housekeeping protein control.Protein extracts were obtained from six ovaries and using the Cell Lysis Buffer for WB . The proteins were separated on 10% SDS-PAGE gel and transferred onto Immobilon-PSQ Transfer Membrane . After blocking, the membranes were incubated with the appropriate primary antibody  overnigh−△△Ct method and normalized to β-actinThe mRNA was retrieved from two ovaries using the RNA Prep Pure Micro Kit , according to the manufacturer's descriptions and then reverse-transcribed into cDNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix . Thermal cycler program was set as 50 min at 42 °C, 65 °C for 15 min, and finally a cooling step at 4 °C. Quantitative PCR  was carrSamples of blood were collected from the mouse tail vein. For amino acid dosage, serum was separated from blood and de-proteinized with sulfosalicylic acid. Free amino acids in the supernatants were measured using an automated amino acid analyser . Glucose concentrations were determined by dropping blood samples onto an Accu-Chek Active test strips  and measuring by Accu-Chek Active Blood Glucose Meter .T-test was used to assess the difference between two groups 65 and one-way analysis of variance (ANOVA) for multiple comparison tests to analyze the effects of starvation on BECLIN1 protein level in


Oryza sativa) LNC. Spectral reflectance and biochemical composition were determined in rice leaves of different cultivars (Yongyou 4949 and Yangliangyou 6) throughout two growing seasons (2014–2015). Results demonstrated that HSL provided the best indicator for predicting rice LNC, yielding a coefficient of determination (R2) of 0.74 and a root mean square error of 2.80 mg/g with a support vector machine, similar to the performance of ASD (R2 = 0.73). Estimation of rice LNC could be significantly improved with the finer spectral resolution of HSL compared with MSL (R2 = 0.56).Fast and nondestructive assessment of leaf nitrogen concentration (LNC) is critical for crop growth diagnosis and nitrogen management guidance. In the last decade, multispectral LiDAR (MSL) systems have promoted developments in the earth and ecological sciences with the additional spectral information. With more wavelengths than MSL, the hyperspectral LiDAR (HSL) system provides greater possibilities for remote sensing crop physiological conditions. This study compared the performance of ASD FieldSpec Pro FR, MSL, and HSL for estimating rice ( Oryza sativa) is a daily necessity among people. The expansion of cities has resulted in a decrease in available land for paddies. In addition, immoderate application of fertilizers has given rise to serious environmental consequences, such as water eutrophication and soil hardeningRice  information, light detection and ranging (LiDAR) has undergone advanced developments in quantifying the 3D surface properties and processes in earth and ecological sciences9Recently, MSL/HSL systems have been applied to estimate leaf moisture contents under laboratory conditions1819With regard to pattern recognition algorithms, multiple linear regression methods such as partial least-squares regression2224The development of HSL offers a way to address the limitations of traditional optical remote sensing and LiDAR. However, the performance of HSL in quantifying LNC has yet to be directly compared with that of other remote sensing technologies. The current study compared the performance of an HSL system in discriminating rice LNC with that of the passive sensor ASD FieldSpec Pro FR  and an MSL system using SVM. The experiments were based on rice samples of different cultivars and growth states, grown in different places for two consecutive years (2014–2015). The objectives of this study were to (1) estimate rice LNC in different situations  with data collected by three sensors  and (2) compare their capability to predict rice LNC with SVM. This study focused mainly on the novel use of the LiDAR intensities of HSL and MSL to reflect foliar biochemistry.Located on the Jianghan Plain, Hubei Province is one of the largest provinces for rice plantations in China. The yield of Hubei Province ranked first in the nation’s yields in 2014Yongyou 4949 was grown in Junchuan County, Suizhou during the growing season of 2014. The crops were seeded on April 27, and transplanted on June 1. Six levels of urea fertilizer  were implemented. For every urea fertilizer level, three fields with identical cultivation conditions were used as replicates. The fields were designed as randomized blocks. Each plot was separated completely from the others by setting plastic films on the ridges of adjacent fields to avoid water leakage, thereby ensuring the precise application of N. The paddy rice samples were collected on July 15 and August 1, 2014.Yangliangyou 6 was grown in the experimental station of the Huazhong Agricultural University in Wuhan in the growing season of 2015. The crops were seeded on April 30, and transplanted on May 27. Four levels of urea fertilizer  were implemented. Replications and separation between fields were similar to the procedures used in 2014. The paddy rice samples were gathered on July 20, 22, 24, and 26, 2015.In each experimental field, at least six fully expanded second leaves from the top were selected randomly. The fresh leaf samples were sealed in plastic bags, kept in ice chests, and then transported to the laboratory for spectral measurementset al.The passive sensor ASD FieldSpec Pro FR  was used for spectrum measurements. The measurement was performed as described by Song, R2 for LNC is concentrated in the red-edge bands (700–760 nm) paired with the red-edge to near-infrared (NIR) bands (700–1100 nm), followed by blue to green bands (450–520 nm) paired with red-edge to NIR bands (740–1000 nm)32Previous rice LNC studies showed that the highest et al.One of the investigated active sensors was an MSL system developed by Wuhan University, operating at four wavelengths  covering VIS and NIR wavelengths. A detailed description of the system is provided in the study by Wei, et al.The HSL system employed in this study was developed by Wuhan University, and a detailed description can be found in the study by Du, All of the rice leaves were measured perpendicular at a distance of 4.2 m. Thus, the effects of incidence angle and distance were eliminated. The influence of certain factors, such as the dark current of the instrument, can be weakened by calculating the reflectance from the spectral measurements of a reference white panelFoliar reflectance in the VIS and NIR regions (including the red edge) has often been considered a good candidate for representing the biochemical or biophysical parameters of vegetationThe exact relationship between LNC and the reflectance spectra remains unclear. With this consideration, a SVM, capable of constructing both linear and nonlinear inversion, was employed in this study. Different from an artificial neural network, a SVM has excellent generalization performance with a strong theoretical foundation in statistical learning theoryWe consider a set of training points, The dual problem isAfter solving problem (2), the approximate function isα*−α is the result. Using MATLAB , where relevant inherent functions and the library LIBSVMThe model output R2), root mean square error (RMSE), and relative error (RE) were calculated as shown below to evaluate the performance of the estimation models:Altogether 220 rice samples were collected in 2014 and 2015. They were divided randomly into two datasets: 80% (176) as the training dataset and the remaining 20% (44) as the validation dataset for predicting LNC. The coefficient of determination  was similar to that of Eitel, et al.R2 =  0.71–0.76)..82 mg/g . Compareet al.W in t ha−1) estimates in winter wheat decreased when the actual W was large. The saturation effect described above with increasing LNC still exists even when using the complete range of reflectance instead of red light-dependent or NIR-based indicesThe scatter of the predicted and observed rice LNC compared with the 1:1 line  suggesteet al.R2 ranging from 0.45 to 0.69 for narrow bands). Chen, et al.R2 of 0.72 for corn and 0.44 for wheat. Erdle, et al.et al.1510) −log (1/R1680)]/[log (1/R1510) + log (1/R1680)]) correlated with the foliar N concentration of native shrub vegetation Spectral indices have been widely applied detect crop N status. For example, Eitel, Based on a systematic analysis of the quantitative relationships between LNC and reflectance characteristics, the capability of an HSL system with 32 channels to estimate rice LNC was evaluated through comparisons with the passive hyperspectral sensor ASD FieldSpec Pro FR (using the reflectance spectra from 400 to 1000 nm) and an MSL system with four bands. Through the regression results of SVM, rice LNC was best predicted by HSL. ASD provided comparable results with HSL in this study. Limited by the number of wavelengths, the MSL provided a moderate regression.HSL demonstrated its potential as a rapid and non-destructive tool for assessing rice LNC, which can facilitate real-time N management decisions. Additional studies should be carried out to test the monitoring relationships further using independent datasets, which can help to test the reliability under a range of conditions. In addition, the HSL system shows promise for other agronomic applications, such as examining other crop biochemical properties and soil parameters, which merits exploration in future research.How to cite this article: Sun, J. et al. Evaluation of hyperspectral LiDAR for monitoring rice leaf nitrogen by comparison with multispectral LiDAR and passive spectrometer. Sci. Rep.7, 40362; doi: 10.1038/srep40362 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


An. stephensi and the human malaria parasite, P. falciparum to conduct a comprehensive evaluation of the thermal performance curves of a range of mosquito and parasite traits relevant to transmission. We show that biting rate, adult mortality rate, parasite development rate, and vector competence are temperature sensitive. Importantly, we find qualitative and quantitative differences to the assumed temperature-dependent relationships. To explore the overall implications of temperature for transmission, we first use a standard model of relative vectorial capacity. This approach suggests a temperature optimum for transmission of 29°C, with minimum and maximum temperatures of 12°C and 38°C, respectively. However, the robustness of the vectorial capacity approach is challenged by the fact that the empirical data violate several of the model’s simplifying assumptions. Accordingly, we present an alternative model of relative force of infection that better captures the observed biology of the vector–parasite interaction. This model suggests a temperature optimum for transmission of 26°C, with a minimum and maximum of 17°C and 35°C, respectively. The differences between the models lead to potentially divergent predictions for the potential impacts of current and future climate change on malaria transmission. The study provides a framework for more detailed, system-specific studies that are essential to develop an improved understanding on the effects of temperature on malaria transmission.Malaria transmission is known to be strongly impacted by temperature. The current understanding of how temperature affects mosquito and parasite life history traits derives from a limited number of empirical studies. These studies, some dating back to the early part of last century, are often poorly controlled, have limited replication, explore a narrow range of temperatures, and use a mixture of parasite and mosquito species. Here, we use a single pairing of the Asian mosquito vector,  Anopheles stephensi, and the human malaria parasite, Plasmodium falciparum, to thoroughly characterize the influence of temperature on key transmission-related traits. The results reveal a number of novel insights and challenge some longstanding assumptions regarding the nature of mosquito and parasite thermal responses. This study provides an experimental blueprint for further system-specific studies necessary to more fully understand the implications of changing temperatures on malaria transmission.Many of the mosquito and parasite life history traits that combine to influence the transmission intensity of malaria  are strongly temperature sensitive. Yet, in spite of decades of research, the precise relationships between individual traits and temperature remain poorly characterized. As a consequence, the majority of studies exploring the influence of local environmental conditions, or prospective impacts of climate change, draw on a combination of studies that utilize different experimental methods and a range of mosquito and parasite species. Here, we use the Indian malaria mosquito,  P. falciparum in Africa utilized data from a Latin American malaria vector for biting rate, a North American vector infected with P. vivax for vector competence, a mixture of 6 malaria vector species from Asia, Africa, and North America for parasite development rate, and even a nonmalaria vector  for fecundity [Numerous studies show the transmission of malaria to be strongly influenced by environmental temperature –10. Thisecundity . Many otecundity , 24–26. P. falciparum inside the mosquito vector [An. maculipennis). Furthermore, many other historical experiments that explore temperature-sensitive parasite development rate are very poorly replicated (data points sometimes based on single mosquitoes), contain little or no information about the sources of infectious blood or blood infection levels, and explore a limited temperature range [Similarly, the iconic degree-day model developed in the 1960s to define the EIP  of o vector , 28 has re range –33. WhetAn. stephensi and P. falciparum across a range of temperatures relevant to malaria transmission. Specifically, we measured adult mosquito mortality rate, the duration of the EIP (the time for parasites to reach their infectious stage), vector competence , and biting rate across several temperatures from 21°C to 34°C. We then use these data to generate temperature-dependent models of relative vectorial capacity (rVC) and relative force of infection. Our data and the contrasting models highlight the need to improve empirical understanding of the effects of temperature on malaria transmission in addition to providing an experimental framework for conducting future species-specific research across a range of vector-parasite pairings.Here, we present a detailed investigation of how temperature affects key mosquito and parasite traits for a single species pairing of An. stephensi mosquitoes on human blood infected with P. falciparum. We then transferred these mosquitoes to temperature-controlled incubators set to 21, 24, 27, 30, 32, and 34°C  and across temperature and block . Upon pairwise log-rank comparison analysis, we found several instances of significant effects of block x temperature interactions , which is the maximum sporozoite prevalence and provides a measure of vector competence, k (a rate constant), which describes the instantaneous change in proportion of infectious mosquitoes through time, and t (the inflection point), the time at which 50% of maximum proportion infectious is reached [Dissection of mosquitoes revealed an increase in the prevalence of sporozoite-infected mosquitoes over time in all temperatures . To desc reached ,46.At 21°C, 24°C, and 27°C, sporogony was well described by the logistic function applied to the full data series. However, at 30°C, 32°C, and 34°C, the raw data showed an initial increase in prevalence of infectious mosquitoes followed by a decline, which was not accurately described by the logistic function alone . For sub10, EIP50, EIP90, and vector competence  values change with temperature in each experimental block , taking the mean of the first 2 gonotrophic cycles for each temperature. We found that gonotrophic cycle length declined with increasing temperature, although with differences between cycle lengths diminishing as temperature increased . The perOur empirical data enable us to generate thermal performance curves for biting rate, vector competence, mosquito mortality rate, and parasite development rate.An. gambiae and An. funestus, have been shown to take multiple blood meals per gonotrophic cycle [An. stephensi.For estimates of daily biting rate, we followed convention by taking the reciprocal of the mean gonotrophic cycle length ,52,54–57ic cycle . Howeverg) of our logistic curves, while for parasite development rate we used the reciprocal of the median EIP (EIP50).For vector competence, we used values of the asymptote .Having defined the effects of temperature on biting rate, mortality rate, EIP, and vector competence, it is possible to characterize the overall effects of temperature on transmission potential using a metric such as rVC. The rVC is the daily rate at which mosquitoes can transmit parasites to humans  , 56, 59,0 [In 0 , 51, 60,0   to our data points for mean transmission potential at each temperature . The resP. falciparum and a key mosquito vector conducted to date. The results yield several important insights that challenge classical assumptions. We also provide an experimental blueprint for future species-specific explorations of temperature-mediated changes in malaria transmission, and such data will be essential for future predictive and theoretical modeling studies.This study represents one of the most detailed empirical investigations of the effects of temperature on Most conventional malaria transmission models assume constant rate of adult mortality per day –55, 59. Aedes mosquitoes indicated that age-dependent mortality is more likely to be found in the laboratory as external mortality factors such as predation, encountering insecticides, and nutritional stress tend to be more prevalent in the field, confounding the effects of senescence [A. aegypti in Southeast Asia and Latin America suggests that age-dependent mortality is observable in the field, and that older mosquitoes die at quicker rates than younger mosquitoes in the same cohort [Anopheles spp. under field conditions.A recent analysis of survival data for nescence . Howevere cohort . WhetherP. falciparum sporogony, either the classic degree-day models [10), the median value (EIP50), or the time to maximum prevalence (approximating our EIP90). Our results clearly show potential for substantial variation between these measures, particularly at cooler temperatures. In turn, these discrete, single-value measures of EIP can yield markedly different estimates of transmission potential (such as rVC) for the same mosquito population . Mopulation .A number of recent studies describe parasite development rate  as a unimodal function, suggesting a decline in growth rate as temperatures increase above the optimum , 25, 66.P. falciparum has no lethal effect on naturally occurring mosquito hosts [P. falciparum might impact mosquito survival at higher temperatures , or that infectious mosquitoes exhibit differential mortality and die at a quicker rate compared with noninfectious mosquitoes. Our current experimental design does not enable us to determine the mechanism explicitly, but when we compare overall mosquito survival with the rate of decline in the proportion of infectious mosquitoes over time, we see no significant difference in instantaneous hazard rates . We areFinally, we show that the predicted effects of temperature on overall transmission potential differ between a standard vectorial capacity model and an alternative model of force of infection, with further differences within the vectorial capacity model that are dependent on which estimate of EIP is used See . One reaOur model of rVC derives from the standard formulation developed by Garret-Jones , which iIn general, we demonstrate that a detailed, system-specific examination of temperature sensitivity yields quantitatively and qualitatively different estimates of temperature-dependent life history traits, compared to the currently accepted relationships that integrate data from diverse studies and a mixture of mosquito and parasite species. For some traits, the differences appear small . For other traits, the differences are substantial; our observed mortality rates are much higher, our parasite development rates are greater than those predicted by standard models at cool temperatures, and unlike contemporary unimodal thermal performance curves , we see We acknowledge that malaria transmission is not determined by temperature alone , 69–71. P. falciparum [am to ensure temperature and humidity were stable throughout the duration of the experiment.Temperatures were selected to capture key transmission range for lciparum , 28, 30.P. falciparum strain NF54  were maintained in RPMI 1640 medium , supplemented with 50 μM hypoxanthine and 10% human A+ serum . Culture was maintained in an atmosphere of 5% CO2, 5% O2, and 90% N2. Parasite cells were then subcultured into O+ human erythrocytes . Gametocyte initiation occurred at 5% haematocrit and 0.8% to 1.0% mixed-stage parasitemia. The culture was maintained for 17 days and parasite cells were provided fresh media daily.In vitro cultures of On the day of the blood meal, gametocyte cultures  were briefly centrifuged, and the supernatant was removed and discarded. Pelleted erythrocytes were diluted to 40% haematocrit using fresh A+ human serum and O+ human erythrocytes. The mixture was pipetted into glass bell jars fixed with a Parafilm membrane and connected by plastic tubing with continuously flowing water heated to 37°C. Each bell jar was filled with 2 mL of blood culture, which feeds approximately 200 females. Mosquitoes were given 20 minutes to fully engorge, after which the bell jars were removed, as the parasites in culture are no longer viable after 20 minutes. In each cup, >95% of females were observed to have engorged fully. Immediately following the blood meal, mosquitoes were transferred to their respective temperature treatments and maintained by providing cotton balls soaked with 10% glucose and 0.05% para-aminobenzoic acid in water, which were replaced daily.An. stephensi Liston adult females were from our laboratory colony  maintained at standard insectary conditions . Three- to 5-day-old females were aspirated into cardboard cups (475 mL), with approximately 150 per cup. Four cups were allocated to each of the 6 incubators set at the different experimental temperatures , totalling approximately 600 females per temperature. Each cup was provided a human blood meal infected with in vitro cultured P. falciparum strain NF54 .Salivary gland sampling began on day 10 post-blood meal for 21°C and 24°C, day 8 for 27°C, day 6 for 30°C, and day 5 for 32°C and 34°C, based on results from a pilot experiment . For eacDead mosquitoes were counted daily in each cup. For survival analysis, mosquitoes removed for dissections each day and those that remained alive at the end of the experiment (day 25) were considered censored cases. We compared a range of plausible mortality curves including exponential, log-logistic, log-normal, Weibull, and Gompertz distributions for each block x temperature combination individually by using the R package flexsurv and selected the best-fit model using AIC  Tables.n = 85 per temperature treatment) were then transferred to individual 50 mL plastic tubes covered with mesh and filled with 5.0 mL of distilled water. Each tube was provided a cotton ball moistened with 10% glucose solution that was replenished daily. Daily, tubes were checked for presence of eggs between 9:00 am and 10:00 am. Females in tubes that had laid eggs were then transferred to a clean tube and sugar was withheld for 6 hours, after which these females were offered a second human blood meal on the membrane feeding system .To estimate effects of temperature on the length of the gonotrophic cycle, 3- to 5-day-old females were fed to repletion on a membrane feeder using pork intestine sausage casing filled with human blood heated to 37°C. Fully engorged females Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigDynamics of survival (purple line) and the proportion of mosquitoes alive and infectious (orange line) for each replicate cup and block in the 30°C treatment used to mathematically analyze the possibility of differential mortality.(TIF)Click here for additional data file.S4 FigDynamics of survival (purple line) and the proportion of mosquitoes alive and infectious (orange line) for each replicate cup and block in the 32°C treatment used to mathematically analyze the possibility of differential mortality. Replicate cup 4 in experimental block 1 was discarded due to sugar pad being replaced with a water pad after blood feeding, so unusually high mortality due to starvation occurred during the first 24 hours post-blood meal.(TIF)Click here for additional data file.S5 FigDynamics of survival (purple line) and the proportion of mosquitoes alive and infectious (orange line) for each replicate cup and block in the 32°C treatment used to mathematically analyze the possibility of differential mortality. Replicate cup 4 in experimental block 1 was discarded due to sugar pad being replaced with a water pad after blood feeding, so unusually high mortality due to starvation occurred during the first 24 hours post-blood meal.(TIF)Click here for additional data file.S1 TableData for proportion of infectious mosquitoes on each day of salivary gland dissections during pilot experiment.(XLSX)Click here for additional data file.S2 TableResults of post-hoc pairwise log-rank statistics across all temperatures, comparing temperatures between block.(XLSX)Click here for additional data file.S3 TableResults of post-hoc pairwise log-rank statistics across both blocks, comparing all temperatures to each other across blocks.(XLSX)Click here for additional data file.S4 TableResults of post-hoc pairwise log-rank statistics across temperature in experimental block 1, comparing all temperatures to each other. Bold italics represent groups not statistically different from each other.(XLSX)Click here for additional data file.S5 TableResults of post-hoc pairwise log-rank statistics across temperature in experimental block 2, comparing all temperatures to each other.(XLSX)Click here for additional data file.S6 TableResults of post-hoc pairwise log-rank statistics across temperature and block, comparing all temperatures to each other between each block. Bold italics represent groups not significantly different from each other.(XLSX)Click here for additional data file.S7 Tableflexsurv using 3 survival distributions  commonly used in studies of mosquito mortality across block and temperature for 21°C through 27°C. Bolded models represent the best fit model for each block x temperature combination.Comparison of survival models built using the R package (XLSX)Click here for additional data file.S8 Tableflexsurvusing 3 survival distributions  commonly used in studies of mosquito mortality across block and temperature for 30°C through 34°C. Bolded models represent the best fit model for each block x temperature combination.Comparison of survival models built using the R package (XLSX)Click here for additional data file.S9 TableNonlinear exponential models used for each block and temperature combination for instances where a decrease in proportion of infectious mosquitoes was observed. Models represent the description of the curve only for data points past the point of truncation (day of peak proportion of infectious mosquitoes) used for binary logistic models.(XLSX)Click here for additional data file.S10 Tableg, k, tm) for best fit model listed for and each block separately. Bolded numbers are predicted values, with 95% confidence intervals in parentheses.Parameter values ((XLSX)Click here for additional data file.S11 Table10, EIP50, and EIP90 from the binary logistic regression model for each block and temperature combination as parameterized in Predicted values and 95% confidence intervals (in parentheses) for EIP(XLSX)Click here for additional data file.S12 TableData for number of females laying the first clutch of eggs and the mean gonotrophic cycle length for each temperature for the first clutch.(XLSX)Click here for additional data file.S13 TableData for number of females still alive after laying the first clutch of eggs, the number that laid a second clutch of eggs, and the mean gonotrophic cycle length for each temperature for the second clutch of eggs.(XLSX)Click here for additional data file.S14 TableMating success across each temperature in gonotrophic cycle/biting rate experiment. A successful mating was considered either successful oviposition or the presence of sperm in dissected spermathecae, in the case a female did not lay eggs over the course of the experiment.(XLSX)Click here for additional data file.S15 Table10, or time to 10% of maximum proportion infectious) and the maximum estimate for EIP . EIP, extrinsic incubation period.Values for relative vectorial capacity using minimum estimate for EIP (defined here as EIP(XLSX)Click here for additional data file.S16 Tableet al. 2013. The R2 refers to our empirical data and the corresponding best fit models.Comparison of nonlinear models for performance of traits across temperature for the data presented in this paper and the data used in Mordecai (XLSX)Click here for additional data file.S17 TableMathematical analysis for the possibility of differential mortality or recovery. Each temperature x block x cup combination was analyzed using a Gompertz distribution with ‘surviving’ and ‘surviving and infectious’ as categorial covariates. Each combination was analyzed beginning at the day of peak prevalence and ending at the day of lowest prevalence. In the instance that two days in a row had the same peak prevalence, analysis began at the second day with the same value. Overlapping hazard rates (signified by “yes”) indicate that it is most likely that the decrease in the proportion of infectious mosquitoes in higher temperatures over time scales with mortality rates.(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Triticum aestivum L.) grain production. However, overly dense plant populations have an increased risk of lodging. We examined lignin deposition during wheat stem development and the regulatory effects of plant density using the wheat cultivars shannong23 and weimai8. Plants were cultivated at densities of 75, 225 and 375 plants per m2 during two growing seasons. Our results showed that decreasing plant density enhanced culm quality, as revealed by increased culm diameter, wall thickness and dry weight per unit length, and improved the structure of sclerenchyma and vascular bundles by increasing lignification. In addition, more lignins were deposited in the secondary cell walls, resulting in strong lodging resistance. The guaiacyl unit was the major component of lignin and there was a higher content of the syringyl unit than that of the hydroxybenzyl unit. Furthermore, we hypothesised that the syringyl unit may correlate with stem stiffness. We describe here, to the best of our knowledge, the systematic study of the mechanism involved in the regulation of stem breaking strength by plant density, particularly the effect of plant density on lignin biosynthesis and its relationship with lodging resistance in wheat.Increasing plant density is one of the most efficient ways of increasing wheat ( Triticum aestivum L.) is a major agronomic problem that has far-reaching economic consequences1Stem lodging in wheat  analysis of stalk strength in four Maize populations. They showed that candidate genes with overlapping QTL confidence intervals included those involved in lignin synthesisTriticum aestivum L.) and common buckwheat (Fagopyrum esculentum M.)1213The plant cell wall provides mechanical support to cells, tissues and the entire plant body8sex1 mutant, which exhibits impaired in starch turnoversex1 mutants, with an increased proportion of syringyl as compared with guaiacyl and p-hydroxyphenylLignin deposition in the plant cell walls is not only developmentally regulated but can also be affected by environmental conditions such as biotic and abiotic stresseso-methytransferase (COMT)Mechanical strength is controlled by quantitative trait loci (QTL)202123Increasing plant density is the main ways of improving the grain yield of winter wheat. However, overly dense populations may lead to weak stem characteristics and a consequent lodging with strong wind and rain. Studies of the crop lodging response to plant density have reported inconsistent results. Most studies have demonstrated that lodging is more severe at higher plant densities. For example, one study reported that increasing of the plant density caused significant decreases in the stalk crushing strength, dry weight per unit length, and the diameter of the internode decreased significantly3538In both cultivars, from anthesis to the hard dough stage, the breaking strength gradually decreased. Plant density significantly (P < 0.01) influenced the breaking strength of the second basal internodes, which gradually increased with decreasing densities. Compared with the D3 treatment, the D2 treatment significantly increased the breaking strength of the second basal internode by 46.41%, 54.39% and 112.5% from 2012 to 2013 and by 29.97%, 81.64% and 88.14% from 2013 to 2014 (means of the two cultivars) at the anthesis, milk and hard dough stages, respectively. Similarly, the D1 treatment significantly increased the breaking strength by 85.02%, 90.43% and 178.24% and by 65.08%, 163.18% and 178.53% (means of two cultivars) for the respective parameters. The characteristics of the stem second basal internodes of the WM8 plants were increased compared with the SN23 plants in all treatments in both growing seasons .To evaluate the differences in stem mechanical strength that respond to plant density, the morphological characteristics of the second basal internode were analysed. The second basal internode characteristics were strongly (P < 0.01) affected by plant density . CompareTo further identify the factors responsible for the effects of plant densities on the breaking strength, the anatomical structure of the plants was analysed for each of the treatments. In the transverse sections, lignin was observed mainly in the epidermis, sclerenchyma and vascular bundles . Stain iFurthermore, to determine any anatomical changes among the plant density treatments, we analysed basal second internode cross-sections by scanning electron microscopy. Plant densities strongly affected the sclerenchyma and the structure of vascular bundles . As planBased on the different effects that the different plant densities had on the degree of lignification, lignin accumulation during wheat stem development was determined. A significant difference in lignin concentration was observed between treatments; lignin accumulation increased with decreasing plant density. The total lignin accumulation of the SN23 plants that received the D1 or D2 treatment was increased by 18.69% and 12.26% (means of two years), respectively, compared with the D3 plants, whereas these values were 18.74% and 10.24%, respectively, in WM8 plants. Furthermore, lignin accumulation during wheat stem development was inconstant . A relatWe successfully separated the three lignin monomers using UPLC-MS/MS . In bothThe total lignin monomer contents of the WM8 plants were higher than those of the SN23 plants. In addition, the plants that received the D3 treatment showed the lowest total released monomers. Compared with the D3 treatment, the total monomer contents of the SN23 plants that received the D1 and D2 treatments were 17% and 10% higher, respectively, and the total monomer contents of the WM8 plants were 13% and 8% higher, respectively ,b. Both TaPAL gene gradually decreased. Ta4CL gene expression reached its peak 14 days after the second internode formation followed by a second peak at 28 days. The relative expression of the TaCCR gene was highest at 0 to 14 days and then gradually decreased. The relative expression of the TaCAD gene decreased to its minimum at 21 days after the formation of the second internode, then slightly increased at 28 days, and decreased again at 42 days. The relative expression of the TaCOMT gene was highest at 0 to 14 days after the formation of the second internode, decreased sharply and was minimally detectable at the late growth stage. The highest expression levels in the second basal internode samples were concomitant with the increase in lignin content during stem development. Plant density has strong regulating effects on the expression of lignin biosynthesis genes. Such genes were expressed at relatively higher levels in the D1 treatment, which might explain the higher levels of lignin monomers in the D1 treatment. Overall, the gene expression levels of the WM8 plants were higher than those of the SN23 plants at all stem developmental stages. The lower expression levels of lignin biosynthesis genes approximately at maturity could be due to their preferential expression in young tissues.To illuminate the genes that were likely to be involved in wheat stem lignification, we compared the transcript abundance of the phenylpropanoid gene during wheat stem development by quantitative PCR . SeveralThere was a significantly positive correlation between the breaking strength and lignin content of the second basal internode at the hard dough stage . CorrelaLodging control in wheat is mainly determined by genetic factors, but it can also be modulated by plant density3434Plant density affects several processes during wheat culm development, such as morphological characteristics, anatomical structure, and the lignin biosynthesis inherent to these structures. In this study, we combined physiology, biochemistry, anatomy and molecular biology to clarify the mechanism through which plant density regulates the breaking strength of wheat culms, focusing on lignin biosynthesis. Our results allowed us to conclude that there is a relationship between plant density and the breaking strength of wheat culms. To understand the mechanism through which plant mechanical strength is controlled in wheat, we performed an in-depth analysis of different plant densities.Culm morphological traits are related to lodging resistanceAnatomical stem characteristics affect lodging in wheat. Significant correlations were detected between lodging resistance and several anatomical features, including the width of the mechanical tissue layers and the width of stem wallsA deficiency in the lignin biosynthesis in the secondary cell walls often leads to globally altered plant morphology, cell structure and even sterility43441247Lignin accumulation and its composition  are important factors influencing the breaking strength of wheat culm. It has been suggested that syringyl biosynthesis provides significant mechanical advantages to angiosperm species. However, the guaiacyl function in these plants involves conduction, not mechanical supportTaPAL, Ta4CL, TaCCR, TaCAD and TaCOMT genes were maintained at relatively high levels at approximately the same stage of fast lignin accumulation. Interestingly, the majority of the maximum expression occurred in young internodes followed by a decrease during the later stages of development, whereas lignin content increased at the latest maturity stages. A possible explanation is that a higher level of gene expression occurs in the early stages of stem development and that, even with subsequent down-regulation, lignification is maintained due to the stability of the lignin biosynthesis enzymesTo further clarify the regulation mechanism of lignin and lignin monomer accumulation according to plant density, transcriptomic data were used to determine several genes that are most likely to be involved in lignin biosynthesis in wheat stems at different developmental stages. Previous studies have reported that PAL, 4CL, CCR, CAD and COMT play key roles in lignin biosynthesis in wheat49505152Based on our results, we suggest that a drastic reduction in lignin deposition caused by overly large populations normally impacts plant growth and development. The compression strength and pull strength of wheat stems would be about to change under different plant densities. The mechanical stress would increase under lower plant density. On one hand, this can present as increased the precursor products forming the lignin which provide the stiffness for plant body. On the other hand, due to the responses of plant to stress (e.g. wind stress), plant body would be induced to form defensive substances (lignin). This type of regulatory function in cultivation actually means that induced effect in morphology. A reasonable plant density significantly decreased the risk of lodging occurring, not only by altering the basal stem morphological traits but also by modifying lignin metabolism, especially the mRNA relative expression of genes in the basal internode. It also contributed to lignin accumulation and increasing stem stiffness. It should be noted that this study has examined only a few genes, and other middle pathway enzymes and genes  involvedIn conclusion, we have described here, to the best of our knowledge, the first systematic study of the mechanisms regulated by plant density, especially regarding the effect of plant density on lignin biosynthesis and its relation to lodging resistance in wheat. The above mentioned results are valuable for enhancing lodging resistance in wheat. Therefore, we believe that this work can be considered as a reference study for future wheat cultivation.−1, and the total nitrogen determined by kjeldahl mentod (N). the rapidly available phosphorous determined by colorimetry method (P) and the rapidly available potassium contents determined by flame photometry (K) were 1.23 g kg−1, 17 mg kg−1 and 113 mg kg−1, respectively−1, 100 kg of P2O5 ha−1 and 120 kg of K2O ha−1 were applied as a basal fertiliser before planting. In addition, 120 kg of N ha−1 was applied at the jointing stage (DC 30)−2) corresponding to low (D1), medium (D2) and high (D3) density levels were established for the field experiment. The experimental plots were arranged in a factorial scheme, in a completely randomised block design with three replications in each treatment. The plot size was 3 × 3 m with 10 rows (0.25-m line spacing).Experiments were performed during two growing seasons from October 2012 to June 2013 and from October 2013 to June 2014 at the Shandong Agricultural University Farm, Tai’an, Shandong Province, China . The average content of the organic matter in the tillage layer was 18.11 g kgSampling was performed after the formation of the second basal internode (DC 32). Representative samples of the second basal internode were collected every 7 days from plants growing in the middle of each plot; there were a total of eight collections. The stem sheath was removed from the samples before the samples were plunged directly into liquid nitrogen for at least 30 min, and then stored at −80 °C until analysis.et al.Fifteen representative main stems were chosen for the measurement of the culm diameter, wall thickness, and dry weight of the second basal internode, according to the methods described by Wei et al.et al.N).Breaking strength was measured at anthesis (DC 62), and at the milk (DC 72) and hard dough stages (DC 82) following the methods of Chen Ten 10 days after anthesis, the middle section of the second basal internodes (approximately 0.5 cm in width) from each treatment were fixed in FAA  for 24 h, dehydrated with ethanol, and embedded in paraffin. Cross-sections of approximately 4-μm thick were obtained using a microtome . To visualise the degree of lignification among the treatments, sections were stained with 1% safranin-O for 1.5 h, washed with distilled water, discoloured in an ethyl series and then counterstained with 0.5% fast green for 1 min. Stained cross-sections were mounted on microscope slides and visualised using a Nikon DS-V3 microscope . Lignified zones were stained red.−1 phosphoric acid buffer for 24 h at 4 °C and post-fixed in 1% OsO4 in a 0.1 mol L−1 phosphoric acid buffer for 30 min. After critical point drying, cross-sectioned samples were sputter-coated with gold and viewed using a scanning electron microscope .The middle sections of the second basal internodes were also fixed in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in a 0.1% mol L2, washed five times with 80% ethanol to remove soluble metabolites, washed with acetone and dried in a drying oven. Acetyl bromide analysis was conducted following Peng et al.280 mL−1 g−1 fresh weight (FW).To assess lignin content, 0.3 g of fresh stem samples free of leaf sheaths were ground in liquid Ng for 2 min. The water phase on the top was transferred to a 10-mL centrifuge tube and then extracted with ethyl acetate (2 mL × 3). The organic phase was combined and dried with anhydrous Na2SO4 and the solvent was removed using a Termovap Sample Concentrator. The sample obtained was resuspended with 1.6 mL of the mobile phase  and then subjected to analysis with an Acquity ultra-performance liquid chromatography-electrospray tandem mass spectrometry system (UPLC-MS/MS) . Hydroxybenzaldehyde (H), vanillin (G) and syringaldehyde (S) were used as standards to quantify the monomers in the samples. Chromatographic separation was performed on a Waters Acquity BEH C18 column  with a gradient elution , which were determined by alkaline nitrobenzene oxidation. The conventional nitrobenzene oxidation method was used with some modifications elution . The mas−∆∆Ct.The gene expression encompasses three independent biological replicates of each treatment. For each biological replicate, three technical replicates of each PCR reaction were performed. Briefly, total RNA from the second basal internode was isolated using a modified Trizol extraction method and treated with DNase I to remove any contaminant genomic DNA. First-strand cDNA was synthesised from 1 μg of total RNA using the Prime Script RT Reagent Kit . Quantitative real-time PCR was performed using the SYBR Green Master Mix kit in a Step One Plus instrument . The primers used in the present study are presented in Statistical analysis was performed using Data Processing System software 7.05 (DPS). The means and significant differences between treatments were separated using the least significant differences (LSD) test at 5% probability.How to cite this article: Zheng, M. et al. Manipulation of lignin metabolism by plant densities and its relationship with lodging resistance in wheat. Sci. Rep.7, 41805; doi: 10.1038/srep41805 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


For improvement, we propose: (1) substantial expansion of research and development on native seed quality, viability, and production; (2) open‐source knowledge transfer between sectors; and (3) creation of supportive policy intended to stimulate demand for biodiverse seed.Globally, annual expenditure on ecological restoration of degraded areas for habitat improvement and biodiversity conservation is approximately $18bn. Seed farming of native plant species is crucial to meet restoration goals, but may be stymied by the disconnection of academic research in seed science and the lack of effective policies that regulate native seed production/supply. To illustrate this problem, we identified 1,122 plant species important for European grasslands of conservation concern and found that only 32% have both fundamental seed germination data available and can be purchased as seed. The “ ER targets aim to restore 150 million ha or 15% of degraded ecosystems by 2020 , or pool of species available from these seed suppliers, thus imposes a critical biodiversity filter in ER projects. Where native supply lacks, easily available agronomic or horticultural seeds are used as a substitute, which is ecologically unacceptable. A RSP of native species, which has been systematically sourced between and within populations and species distribution ranges, is necessary for the support of genetic diversity in seed supplies and restored ecosystems ; calcareous alpine grasslands (6170); and acidic alpine grasslands (6150).The European initiative Natura 2000 aims to establish a network of diverse, representative high‐quality protected habitats of conservation concern, much of which will require intensive ER within these habitats, regulated by EU legislation that affects strategies of seed quality and use (Table S2). This includes 116 protected species subjected to legal protection, in most cases endangered or narrow endemic species; 929 indicator species, which are indirectly protected when occurring in protected habitats but unregulated in seed production; and 77 fodder species controlled for quality as domestic stock feed , each species was assigned as data being available (1) or not (0). Similarly, species were either Commercially Available (CA) (1) or not (0).Data were collected as binomial variables. To assess CA and with GDA were calculated for each plant family represented in the target species list to elucidate taxonomic representation as a surrogate of phylogenetic variation. A Generalized Linear Model (GLM) was fitted to assess the variation of CA as a function of GDA and species groups. The GLM was computed with binomial error distribution and logit link function in order to assess the influence of policy groups and GDA (explanatory variables) on CA . All analyses were performed in R Statistical Computing Language and Platform version 3.2.2  of species with  Wickham  and yarrPhillips . The pacects Fox  was used Pohlert .ER within European grassland habitats are spread across 59 plant families, with highest representation in Compositae (146 species) and with the top 5 and 10 families comprising 43% and 62% of the species list, respectively. Information on GDA and CA alone extended to 49%  and 39%  of target species, respectively  of species on the target list .The 1,122 target species with potential interest for GDA are also completely lacking CA, although the sample size is small in these cases  lie within the lower quartile of CA, covering 158 species .The majority of taxonomic families completely lacking GLM are exhibited for the estimate of CA of target species across all variables , and fodder species 0.54  , but this does not necessarily indicate availability from many suppliers. Seed availability and use is compounded by the origin of the seed, as some supplies may not be appropriate for use in every region , that is, RSP to better support ER, industry also requires cooperative market sharing, improved provision and storage strategies. In Australia, United Kingdom, and the United States, there are examples of government, community, or nonprofit groups working cooperatively with seed suppliers to enable the inclusion of species that have challenging seed traits in the commercial RSP supply chain. The U.S. Native Plant Program  should be required at project inception to allow time for realistic production. New policies should be designed to embrace consultation with the native seed industry and restoration professionals.Current legislation relating to et al. ex situ plant conservation programme globally, the Millennium Seed Bank Partnership (MSBP), managed by the Royal Botanic Gardens, Kew, UK, has successfully banked seeds of 13% of the world's wild species, aiming to bank 25% by the year 2020  of suppliers  with commercial availability of seed with and without germination data availability.RDI plots  show jittered points of raw data, center bars indicate the mean of the data, beans outline the smoothed density of the data, whiskers mark the 10% and 90% quantiles of the data, and inference bands show the Bayesian 95% high‐density interval inferential statistics for each group. Letters show statistical differences between groups (Table S4).Click here for additional data file.Table S1 Simplified seed dormancy types (adapted from Baskin & Baskin 2014)Table S2 Full species list, associated category, and associated dataCA = commercial availability , GDA = germination data availability .Table S3 Seventeen seed suppliers across 17 countries used for data collectionTable S4 Statistics representing differences between variables in the percentage of suppliers with seed of each species commercially available compared across species groups χ2 test and post hoc pairwise Tukey and Kramer (Nemenyi) χ2 test, P‐value statistics, indicating significance between group variables. Germination data available = “+GDA,” germination data not available = “–GDA.”Kruskal‐Wallis Table S5 The complete data set summarized by taxonomic family in descending order of percentage of commercial availability (CA)CA = commercially availability, GDA = germination data availability.# = number, % = percentage, Sp. = species, Click here for additional data file.


We used a battery of genes encoding transcription factors  and neuropeptides to study the extended amygdala in developing zebra finches. We identified different components of the central extended amygdala comparable to those found in mice and chickens, including the intercalated amygdalar cells, the central amygdala, and the lateral bed nucleus of the stria terminalis. Many cells likely originate in the dorsal striatal domain, ventral striatal domain, or the pallidal domain, as is the case in mice and chickens. Moreover, a cell subpopulation of the central extended amygdala appears to originate in the prethalamic eminence. As a general principle, these different cells with specific genetic profiles and embryonic origin form separate or partially intermingled cell corridors along the extended amygdala, which may be involved in different functional pathways. In addition, we identified the medial amygdala of the zebra finch. Like in the chickens and mice, it is located in the subpallium and is rich in cells of pallido-preoptic origin, containing minor subpopulations of immigrant cells from the ventral pallium, alar hypothalamus and prethalamic eminence. We also proposed that the medial bed nucleus of the stria terminalis is composed of several parallel cell corridors with different genetic profile and embryonic origin: preoptic, pallidal, hypothalamic, and prethalamic. Several of these cell corridors with distinct origin express FoxP2, a transcription factor implicated in synaptic plasticity. Our results pave the way for studies using zebra finches to understand the neural basis of social behavior, in which the extended amygdala is involved. Taeniopygia guttata; order Passeriformes) are a highly gregarious species of songbirds, that learn and use song for social communication  embryos from embryonic day 14 . Chicken probe information and percentage of homology with zebra finch are given below and in Table cIslet1 . Homology percentage of chicken probe with zebra finch gene: 91.8 % . The complete chicken gene has 94.9 % of homology compared to the zebra finch gene.cLhx6 . Homology percentage of chicken probe with zebra finch gene: 94.9 % . The complete chicken gene has 94 % of homology compared to the zebra finch gene.cLhx9 . Homology percentage of chicken probe with zebra finch gene: 95.7 % . The complete chicken gene has 97 % of homology compared to the zebra finch gene.cNkx2.1 . Homology percentage of chicken probe with zebra finch gene: 90.6 % . The complete chicken gene has 90.5 % of homology compared to the zebra finch gene.somatostatin precursor . Homology percentage of chicken probe with zebra finch gene: 93 % . The complete chicken gene has 90 % of homology compared to the zebra finch gene.Frontal or sagittal brain sections were processed for in situ hybridization using digoxigenin-labeled riboprobes, following a procedure previously described  protocols for the genes mentioned above.http://blast.ncbi.nlm.nih.gov/Blast.cgi) and used the Vector NTI program to construct the predicted zebra finch “Lhx5” and “Mesotocin”sequence. We designed primers to amplify part of the coding region of zebra finch “Lhx5” and “Mesotocin” (Mes). Primers were as follows: forward zLhx5: TTCTCCAGGGAAGGGAAACT; reverse zLhx5: CTAAGCGGACACCACTCCTC; forward zMes: CTCTCCTCCGCTTGCTACAT; reverse zMes: TGACCAGGAGATGCTGTTTG. The resulting PCR products (923 base pairs for zLhx5 and 378 base pairs for zMes) were examined on a TAE agarose gel, cleaned from nucleotides with the Nucleo spin purification kit , and cloned into pGEMTeasy vector . Inserts from three independent “zLhx5” and “zMes” clones were then sequenced on both strands. Consensus sequence was built using the Vector NTI program. The cloned fragments encompass the sequence spanning from positions bp 1 to 923 of zLhx5 and bp 36 to 358 of zMes. The probes were generated from PCR-amplified sequences using M13 primers and T7 or SP6 RNA polymerase to drive the transcription of the mRNA sense and anti-sense probes.To obtain zebra finch Lhx5 and Mesotocin we blasted the corresponding chicken sequences against the zebra finch data base , prehybridized in hybridization buffer (HB) for 2 h at 58 °C  or 65 °C (for the embryos cases), and then hybridized in HB containing the riboprobe overnight at 58 or 65 °C, as explained before . The hybridization buffer contained 50 % of deionized formamide, 1.3X standard saline citrate , 5 mM ethylene-diamine-tetraacetic acid , 1 mg/ml of yeast tRNA (Sigma-Aldrich), 0.2 % Tween-20, 100 µg/ml of heparin (Sigma-Aldrich), completed with water . Following hybridization, the sections were washed with a mix 1:1 of MABT 1X  and HB at 58 or 65 °C during 20 min and washed abundantly at room temperature with MABT 1X (about 2 h). Following this, the sections were blocked with a solution containing blocking reagent (Roche), MABT 1X and sheep serum (Sigma) for 4 h at room temperature, then incubated in an antibody against digoxigenin  overnight at 4 °C, later washed with MABT 1X and finally revealed with BM purple (Roche Diagnostics). Sections were then mounted on glycerol gelatine (Keisers Glycerol) or were processed for immunofluorescence (as explained in next section) and then mounted on immuMount (Thermo Scientific). This mounting media is suitable for the immunofluorescence cases, but not for the in situ hybridization, as it decays the signal.Alternative series of sections and some previously hybridized sections were processed for immunofluorescence to detect FoxP2 . The specificity of this antibody has been checked in zebra finch brain by Western blot and by preincubating the antibody with the FoxP2 protein prior the immunohistochemistry . Selected hybridized sections were re-taken on a Leica microscope (DMR HC) equipped with a Zeiss Axiovision digital camera. For fluorescence image acquisition, a Zeiss Axiovert S 100 microscope equipped with a Zeiss AxioCam HRc camera was used in a first approach. Later, selected sections processed for FoxP2 immunofluorescence and/or in situ hybridization for other transcription factors were analyzed and photographed using an Olympus BX51 microscope equipped for fluorescence and a digital camera (Olympus DP70), at Dr. Agustín González’s lab (University Complutense of Madrid). Selected digital images to be used for the figures were adjusted for brightness/contrast using Adobe PhotoShop and figures were prepared and labeled using FreeHand.http://www.zebrafinchatlas.org/, and The Zebrafinch Brain Architecture Project (http://zebrafinch.brainarchitecture.org/introduction/).For identification of forebrain cell masses during development, we used the atlas of developing chicken brain  and mature neurochemical features (expression of different neuropeptides). The genes selected for this study include many of those previously used in chicken  and quasi-horizontal  brain sections at the level of the central extended amygdala (EAce), hybridized for cIslet1, cPax6, cNkx2.1, cpENK, and cSOM Figs. , 4, and The transcription factors analyzed showed moderate or intense expression during late embryonic stages  and in early post-hatchlings, but declined afterwards. For example, Islet1 expression was stronger in late embryos Fig. a–c than Notably, the combinatorial expression of the transcription factors Pax6, Islet1, Nkx2.1, Lhx5, Lhx6, and Lhx9 Figs. , analyzeFor determining the location and extension of the different areas of the EAce in zebra finch, we analyzed the combinatorial expression patterns of the transcription factors Pax6, Islet1 and Nkx2.1, which allowed the distinction of dorsal striatal , ventral striatal , and pallidal  derivatives. For better identification of specific cell groups, we added FoxP2, pENK and SOM to the analysis, as explained below. Based on the analysis of these genetic markers within the topological framework, the zebra finch EAce is located in the caudal subpallium and can be understood as a structure divided into two major parts: lateral and medial. The lateral component is fundamentally formed by striatal derivatives (from both Std and Stv), and includes the central amygdala and other subdivisions. The medial division is formed within the dorsal pallidal domain and encompasses the dorsal part of the lateral bed nucleus of the stria terminalis (BSTLd). Although these two major parts of EAce contain a majority of either striatal or pallidal cells, they also include subpopulations of immigrant cells, as described below.Two intercalated (ITC)-like areas, rich in expression of Pax6, pENK, and FoxP2, and apparently derived primarily from the dorsal striatal embryonic domain. These areas are located along the dorsolateral margin of the striatum and capsular central amygdala, and include a dorsal part named striatal capsule (StC) and a ventral, patchy part  or ventral  striatal domains. These include the capsular central amygdala (CeC), the central oval nucleus (Ceov) and the peri-intrapeduncular nucleus (pINP). The CeC is located in the caudolateral part of the radial striatal division, and contains Pax6 and pENK expressing cells Fig. f, l. TheWe tentatively identified the subpallial amygdaloid rostral area (SpAr) in Fig. The perioval zone (Pov) is a component of the EAce located in the pallidal domain, as a lateral extension of the BSTLd, with a high content of enkephalinergic (pENK) cells Fig. e. The PoWithin the lateral part of the finch EAce (late embryos and post-hatchlings), we have identified seven subdivisions comparable to those found in chicken . The EMT-derived Pax6 cells in zebra finch can be easily followed into the extended amygdala region , and such expression was seen from embryonic stages Fig.  until juIn addition, a subpopulation of cells expressing somatostatin is seen in parts of the extended amygdala in juvenile zebra finch , pallidal (Nkx2.1 and Lhx6), preoptic , hypothalamic  and prethalamic .Our first objective was to identify the medial amygdala of zebra finch. In other vertebrates such as mice and chickens, this is complex nuclear structure located in the caudolateral and ventral aspect of the subpallium, rich in cells of pallidal and preoptic origins, but also containing subpopulations of immigrant cells of ventral pallial (minor), hypothalamic SPV, and EMT origins  of zebra finch develops in the caudal ventral pallium, as part of the arcopallial complex Fig. ”. DuringRegarding the medial bed nucleus of the stria terminalis (BSTM), based on the expression of different genes we identified in this nucleus several parallel cell corridors or stripes with different genetic profile and perhaps different embryonic origin. Based on Lhx6, we observed a cell corridor of pallidal cells  and neuropeptide genes to study the extended amygdala in zebra finches. Zebra finches are a highly gregarious species of songbirds, that learn and use song for social communication  and medial (EAme) extended amygdala in zebra finches, comparable to many of those described in mice  Laterally: the intercalated-like cells , the capsular central amygdala (CeC), the oval central nucleus (Ceov), the peri-INP, the perioval zone (pOv), and the rostral SpA (SpAr). 2) Medially, the BSTLd, which includes at least four subdivisions: medial, intermediate, lateral, and extratelencephalic. The EAce subdivisions of zebra finches were already visible at prehatching stages , the earliest we analyzed. At these prehatching stages, the zebra finch brain still showed a relatively immature aspect, comparable to E12-E14 of chicken  and the ventral intercalated-like patches (ITCv) with topological location and genetic profile similar to those in the chicken  or in both Pax6 expressing and Islet1 expressing cells (peri-INP or pINP), comparable to similar subdivisions found in chicken  and Nkx2.1 . This area was located in a topological location similar to that in the chicken , and also had a similar cell composition  and the BSTM, was previously identified in different birds, including galliformes , columbiformes (pigeon), psittaciformes (budgerigars) and oscine passeriformes (songbirds)  and enkephalin (pENK), which appear to come from the Pavc and Padv, respectively. This is based on the observations discussed above for the EAce. A cell corridor of pENK cells is seen extending from the dorsoventral pallidal progenitor domain, Padv, laterally through the Pov. Some of these pENK may populate the medial amygdalar nucleus, although other pENK cells of the medial amygdala may originate elsewhere. This may be similar in chicken (unpublished observations). Regarding the SOM cells, as in mammals and chicken, in the zebra finch the SOM-expressing cells of the extended amygdala may originate in the Pavc, since a stream of such cells is visible extending from this domain laterally until reaching the medial amygdala; this stream of SOM cells is parallel but ventral to that of the ENK-expressing cells mentioned before. Laterally, the SOM cells aggregate in the medial amygdalar nucleus, in a more ventral location with respect to the enkephalinergic cell subpopulation  A preoptic component of the BSTM, BSTMpo, which is seen as a stream of cells crossing from ventral to dorsal the anterior commissure. These cells express Lhx5 and likely come from the commissural preoptic subdivision, as in mice . In addition to these, the BSTM also contains EMT derived cells, but it is unknown whether they overlap with the other cells, or occupy a different space within the nucleus. The BSTM in chicken  and vasotocin (AVT) cells, probably coming from this hypothalamic SPV domain, which play a very important role in sex-specific gregariousness and pair-bonding (Kelly and Goodson


The aim of this study was to do determine the efficacy of the topical anaesthetic Tri-Solfen® in the amelioration of the pain of castration in piglets. The trial was conducted over a three day period, and blocked across six litters with 12 piglets treated on days one and two, and 16 on day three. The piglets were randomly allocated by weight and litter to 1 of 4 treatment groups: (i) sham castration ; (ii) surgical castration with no anaesthetic intervention ; (iii) surgical castration with post-operative topical anaesthesia ; (iv) surgical castration with a pre-operative intra-testicular lignocaine hydrochloride injection . Wound sensitivity testing involved von Frey monofilaments of weights 4g and 300g, and an 18 gauge needle, used to stimulate the wound and surrounding skin respectively, at various pre-determined sites. Neonatal piglets receiving topical anaesthesia (Tri-Solfen®) spray into castration wounds had significantly lower wound sensitivity responses for up to 4h, compared to those castrated following intra-testicular lignocaine injection or those with no treatment. The use of topical anaesthetic is suggested as a practical and affordable method of improving piglet welfare during castration. Castration of male piglets is a routine husbandry procedure commonly performed within the first few days of life in commercial piggeries globally. Extensive research to assess the effect of castration on the welfare of the piglets indicates that castration causes significant stress, pain and discomfort that can persist for up to 4 days –4.Castration of piglets is usually performed without anaesthesia or analgesia, presumably because the anaesthetic techniques commonly used for comparable surgeries in human and veterinary medicine  are either too complex, costly and time consuming to be practical or affordable for use on-farm. A number of publications have described the effective use of analgesic and anaesthetic interventions to address the pain associated with piglet husbandry procedures –9 althouThe European Union (EU) legislated that as of January 2012, surgical castration of pigs must utilize prolonged local anaesthesia and/or analgesia, with the aim of removing the practice altogether by 2018. Whilst cost-benefit analyses conducted in relation to this legislation indicate that general and local anaesthesia strategies are beneficial, in other jurisdictions such as Australia the costs of lsuch procedures are considered excessive, particularly where veterinarians are required by law to administer restricted drugs such as lignocaine .Tri-Solfen®  is a commercially available topical anesthetic spray-on wound dressing with haemostatic and antiseptic properties, registered in Australia for the alleviation of pain of castration in sheep and cattle. It contains lignocaine (40.6 g/L), bupivacaine (4.5 g/L), adrenalin (24.8 mg/L) and cetrimide (5.0 g/L) in a gel base, and has been reported to be effective in ameliorating wound pain and improving healing during mulesing, castration and tail docking in sheep and castration in calves –14. Due All experimental protocols were approved by The University of Sydney Animal Ethics Committee .The trial was performed using 40 Landrace x Large white piglets sourced from a commercial herd at The University of Sydney Farms near Cobbitty, NSW. Piglets were from mixed litters and aged 3–5 days old when castrated. The piglets were housed in farrowing stalls with the sow and their littermates, before and upon completion of the trial. Piglets were removed for the procedure and subsequent wound sensitivity testing, and retained in large crates containing a sawdust floor cover for the 5 hour duration of the study, with an overhead heat lamp providing warmth. The piglets were returned to their sows on conclusion of the observation period.The trial was conducted over a three day period using piglets from six litters with 12 piglets treated on days one and two, and 16 on day three. On each day, piglets were weighed and numbered 1–12 (day one and day two) or 1–16 (day three) with marker crayon for identification.n = 10); (ii) surgical castration with no anaesthetic intervention ; (iii) surgical castration with post-operative topical anaesthesia ; (iv) surgical castration with a pre-operative (5mins) intra-testicular lignocaine hydrochloride (1ml per testis) injection .The piglets were randomly allocated by weight and litter to 1 of 4 treatment groups: (i) sham castration  incision with a sterile scalpel to express the testes. The exposed testes were then excised following incision of the spermatic cord. The whole procedure including TRI treatment, took less than 1 minute per animal. Sham-castrated piglets were handled and restrained similarly to the castrates, with the scrotum and testes manipulated, but no incision made. Piglets in the TRI group received 1mL of Tri-Solfen® spray to each scrotal incision upon exposure of the testis using a 1mL pipette to apply 0.5mL to the cut skin edge and spermatic cord prior to excision. The pipette end was then inserted along the spermatic cord inside the wound to apply a further 0.5mL to ensure that all retracted tissue was coated (12).WST involved von Frey monofilaments of weights 4g and 300g, and an 18 gauge needle, used to stimulate the wound and surrounding skin respectively, at various pre-determined sites. These procedures were applied as per previous studies ,12,14. AWound sensitivity was measured at 5 locations, presenting from left to right: left cut skin edge, left wound body, the skin in the middle of the incisions (Middle), right wound body and right cut skin edge. Site and von Frey weight /needle were randomized between each measurement and piglet to avoid confounding of results Involuntary motor responses were scored using an adjusted customized numerical rating scale , 12, 14.For the analysis, body weight was coded into three categories (WeightCat): (1) 0.9–2.28 kg; (2) 2.3–3.38 kg; and (3) 3.3–4.7 kg. Score data was analysed using ordinal logistic regression in AsREML® . Treatment, time, WeightCat, stimulation type (Stim) and site were included as fixed effects, with piglet and litter as the random effects. Z values were determined using logit and SED arrays, and P-values calculated. Data is presented as cumulative odds ratios with the statistical probabilities of piglets in each treatment group displaying a response score of Y = 0, 1, 2 and 3. In effect, these probabilities are the averages of the scores from the 2 von Frey weights and the 18g needle. For all statistical calculations, P values < 0.05 were considered statistically significant.Mean piglet weight was 2.7 ± 0.45kg. There was a significant effect of weight on piglet response (P = 0.004). Smaller piglets (WeightCat 1) had the greatest probability of more severe response scores, with the larger piglets (WeightCat 3) least likely to respond .There was a significant Treatment x Stim interaction (P < 0.001). Severity of response increased significantly with weight of stimulation from the 4g von Frey to the 18G needle in all treatment groups . There wThere was a significant Treatment x Time interaction (P < 0.001). There was a general increase in response severity over the 4h in castrated piglets . At 1 miLocal anesthetics have been found to reduce acute pain associated with castration in piglets when injected into the scrotum , 7, 8 HoTopical anesthesia, applied during and immediately after castration has previously been found to be practical and effective for reducing post-operative pain associated with surgical husbandry procedures in lambs , 18, 19 Local anaesthetic agents impede nociception, acting directly on nerve tissue to inhibit the conduction of nerve impulses responsible for the sensation of pain. They are absorbed through mucosal surfaces and damaged skin, and can induce rapid and profound local anaesthesia when applied to open wounds , 21–23. Local anaesthesia via injection of has been shown to reduce pain accompanied by significant reductions in the cortisol response of surgical castration , 7. Our In this study, piglets had the greatest response to needle pin prick stimulation of the tissue within and surrounding the wound. This was expected as this stimulus is known to induce a detectable pain response –17. ThisCastrated piglets that did not receive topical anesthetic had significantly greater pain responses from 2 to 4 hours following castration, demonstrating the recruitment of the pain cascade in CAST piglets not receiving any pain management. It appears likely that the pain management from the injected lignocaine in LIG piglets had diminished by the 2 h time point, as there was a rise in sensitivity from 30min to 1.5 h, and the responses of LIG piglets did not differ from CAST piglets after 2 h. This is to be expected as the lignocaine is reported to have effect for 1.5–2 hrs. In addition, it is possible that the injected lignocaine had little effect on the skin and peri-testicular tissue, leaving them vulnerable to nociception.TRI piglets displayed apparent wound anaesthesia up to 4 h, with significantly lower pain scores than CAST and LIG piglets. The inclusion of long acting Bupivicaine in the topical anaesthetic may explain this extended duration. This indicates that topical anaesthetic spray is effective at reducing wound pain for a prolonged period following castration.Response scores of castrated piglets were not significantly different immediately following castration (1min). It is hypothesized that this was due to the up-regulation of sensory receptors following the incision, resulting in a release of natural pain mediators including endorphins to mitigate immediate pain associated with the incision and tissue removal. From 30min following castration TRI treated piglets had the lowest pain response scores, indicating effective anaesthesia to wound stimulation for at least 4 h.These findings suggests that the global pig industries currently using piglet castration, may benefit from consideration of these findings as they offer a possible means of improving the welfare of male piglets during this routine but painful husbandry procedure.S1 FileThis is the dataset used for analysis in this trial.(XLSX)Click here for additional data file.