Toxic Effects of Glyphosate (Roundup)

Glyphosate is a popular and broadly used herbicide that is effective against weeds, especially in association with transgenic glyphosate-resistant crop systems.  Although glyphosate is considered a low-toxic herbicide, recent studies, such as one conducted by Romano et al., have revealed toxic effects resulting from low-dose commercial formulations.  The aim of the study was to investigate the effect of gestational maternal glyphosate exposure on the reproductive development of male offspring.  Sixty-day-old male rat offspring were evaluated for sexual behavior and partner preference; serum testosterone concentrations, estradiol, follicle-stimulating hormone (FSH) and luteinizing hormone (LH); the mRNA and protein content of LH and FSH; sperm production and the morphology of the seminiferous epithelium; and the weight of the testes, epididymis and seminal vesicles.  The study found that there was an increase in sexual partner preference scores and the latency time to the first mount testosterone and estradiol serum concentrations; the mRNA expression and protein content in the pituitary gland and the serum concentration of LH; sperm production and reserves; and the height of the germinal epithelium of seminiferous tubules.  These results suggest that masculinization processes, behavior, histological processes, and endocrine processes can all be negatively impacted by maternal exposure to glyphosate.
Romano, Marco Aurelio, et al. (2013) “Glyphosate impairs male offspring reproductive development by disrupting gonadotropin expression.” Archives of toxicology 86.4: 663-673. [GSSS romano gonadotropin glyphosate]

                  Sexual differentiation in the brain takes place from late gestation to the early postnatal days.  This process is dependent on the conversion of circulating testosterone into estradiol by the enzyme aromatase.  This process will eventually determine the gener-specific reproductive endrocrinology and behavior in adults.  A reduction in aromatase activity was observed in placental and embryonic human cells treated with low concentrations of a commercial formulation of glyphosate (Benachour et al. 2007).  From previous studies, the authors suspect that the herbicide glyphosate may be characterized as a potential endocrine chemical disruptor.  Endrocrine disruptors are defined as exogenous agents that interfere with the production, release, transport, metabolism, binding, action or elimination of natural hormones responsible for the maintenance of homeostasis and the regulation of developmental processes (Kavlock et al. 1996).
                  Romano et al.investigated the effect of gestational maternal glyphosate exposure on the reproductive development of male offspring.  Sexual differentiation in the brain occurs during the late gestational and the early postnatal days. Multiple factors can influence sexual expression.  First, sexual behavior is influenced by hormones, so the serum concentrations of testosterone, estradiol, FSH and LH were measured.  The pituitary expression of mRNA and protein content of LH and FSH was also analyzed to assess the possible glyphosate-mediated interference with their production.  Sex hormone serum concentrations may also affect sperm production and the morphology of the seminiferous epithelium, which were evaluated by testicular and epididymal sperm counts and the morphometric analysis of histological sections.  Lastly, the weight of the testes, epididymides and the seminal vesicle, the growth of the animals, and the weight and age at puberty were recorded to evaluate the effect of the treatment on these conditions.
First, to verify sexual partner preferences, the males were exposed at PND60 to a sexually mature male and a female in estrous.  The males from dams treated with glyphosate spent significantly more time in contact with female rats than control animals, suggesting a preference for the female gender.  Next, the glyphosate treatment led to an increase in the latency to first mount, latency to first intromission and latency to mount after first ejaculation.  Furthermore, the levels of both testosterone and estradiol were different between the control group and the glyphosate treated group.  Specifically, the group treated with glyphosate showed higher levels of testosterone and estradiol compared to control animals.  Next, the analysis of LH mRNA expression showed increased levels in treated animals, which was accompanied by higher amounts of LH protein in the pituitary and the serum.  In addition to this, the FSH mRNA expression was increased in treated animals, but this was not associated with a rise in the FSH protein in the pituitary or the serum. 
The changes in hormones observed may influence spermatogenesis.  Thus, the researchers monitored total sperm production, daily sperm production, sperm reserves and sperm transit at PND60.  Glyphosate exposure during the perinatal period increased the total and daily sperm production.  Also, an altered morphometry of the seminiferous epithelium was observed in treated animals.  This alteration caused an increase in epithelial height and a reduction in luminal diameter without changes in the tubular diameter.  In addition, the weight of the testes was found to be similar between groups.  The weight of the undrained seminal vesicle was not altered, but the drained seminal vesicle was heavier than the control group.  This finding suggests that this structure contained a smaller amount of fluid.  Interestingly, the glyphosate rats were observed to initiate puberty at an earlier age.  This change was accompanied by a reduction in their body weight.  However, the weights of the animals at the same age were not different, indicating that observed lower weight is merely a function of the younger age at puberty onset.

This experiment indicated changes in most of the parameters evaluated.  The results suggest that maternal exposure to glyphosate disturbed the masculinization processes.  The authors conclude that glyphosate exposure promotes behavioral changes and histological and endrocrine problems.  Further study is suggested to evaluate whether the effects of maternal exposure to glyphosate are dose-dependent.

Works Cited
Benachour, Nora, et al.“Time-and dose-dependent effects of roundup on human embryonic and placental cells.” Archives of Environmental Contamination and Toxicology 53.1 (2007): 126-133.
Kavlock, Robert J., et al. “Research needs for the risk assessment of health and environmental effects of endocrine disruptors: a report of the US EPA-sponsored workshop.” Environmental health perspectives 104.Suppl 4 (1996): 715.

Safety of GM Crops

Genetically modified biotechnology is the fastest-adopted technology in the history of modern agriculture.  In 1996 there were 1.7 million hectares of GM crops; since then that number has increased to 148 million hectares—an 87-fold increase.  But, there is some concern surrounding the potential side effects of randomly inserting exogenous genes in plant genomes.  Mainly, an insertion of exogenous genes could produce modified biochemical processes, new proteins, or other secondary pleiotropic effects.  Evaluating the substantial equivalence of GM groups to traditional crops is therefore essential to guarantee the safe use of GM crops and alleviate the fears consumers have about GM food.  Here, the researchers evaluated the effects of transgenes on rice seed proteomes by 2-D differential in-gel electrophoresis (2D-DIGE) combined with mass spectrometry (MS). The study found that GM events do not substantially alter proteome profiles as compared with conventional genetic breeding and natural genetic variation.  Specifically, mass spectrometry revealed 234 proteins differentially expressed in the 6 materials (BAR68-1, D68, 2036-la, MH86, MH63, ZH10), which are involved in different cellular and metabolic processes.  This finding suggests that metabolism, protein synthesis and destination, and defense response in seeds are important in differentiating rice cultivars and varieties.
Gong, Chun Yan, et al. “Proteomics insight into the biological safety of transgenic modification of rice as compared with conventional genetic breeding and spontaneous genotypic variation.” Journal of Proteome Research 11.5 (2012): 3019-3029. [GSSS gong proteomics insight rice]

                  Studies of diverse plant species have demonstrated that changes in transcript levels are not fully followed by the same changes in protein levels.  Proteins are the key players in gene function and are directly involved in metabolism and cellular development or have roles as toxins, antrinutrients, or allergens.  Therefore, comparisons of GM proteomes and control lines are of necessary importance when trying to determine GM crop safety.  The results of these experiments can reveal molecular differences in varieties produced by conventional genetic breeding and natural genetic variation and help researchers better assess the safety of a GM crop. Here, the researchers evaluated the effects of transgenes on rice seed proteomes by 2-D differential in-gel electrophoresis (2D-DIGE) combined with mass spectrometry (MS).  Two sets of GM indica rice and controls were used: Bar68-1 transformed with herbicide resistant gene bar and its non transgenic control indica variety D68, and 2036-la transformed with insect-resistant genes cry1Ac/sckand its nontransgenic control indicavariety MingHui 86 (MH86).  In addition to these, the researchers used MH63, which is a parental line used for breeding MH86, and japonica rice Zhonghua 10 (ZH10).  In summary, the experimental design included GM rice and controls, different indica varieties (parental and filial), and indica and japonicacultivars.
                  First, the researchers confirmed that their transgenic lines were successfully transformed.  PCR with specific primers revealed BAR68-1 had one detectable DNA fragment with a size of 568 bp, which corresponded to the bar gene for herbicide resistance.  2036-la line had 2 detectable DNA fragments of 1709 bp and 358 bp corresponding to cry1Ac and sck genes, respectively (coding for insect resistance).
                  Next, a 2D-DIGE with pH 4–7 strips analyzed seed proteomes from different rice lines and detected about 2250 protein sports in each image.  A principal component analysis (PCA) was conducted to investigate similarities in the proteomes of the 6 rice lines.   Much less variation was found in the proteomes between transgenic lines and their controls than between different indica varieties or between indica and japonica cultivars.  In addition to this, the researchers analyzed differentially expressed proteins (DEPs) from the seed proteomes of the 6 lines which revealed 423 (Student’s t test) and 443 (ANOVA) protein spots, respectively, with statistically significant differences in expression.  The largest differences of protein expression were found between indica (varieties NH63, D68, and MH86) and japonica (ZH10) cultivars.  A smaller difference was found between the 3 indicavarieties, an even smaller difference between NH63 and MH86, and the least between transgenic lines and controls (Bar68-1 vs D68; 2036-la vs MH86).  Compared with conventional genetic breeding and natural genetic variation, rice seed proteomes were largely unchanged with transgenic modification.
                  In addition to the principal component analysis, mass spectrometry was used to analyze 264 differentially expressed proteins (DEPs) selected on the basis of (1) 1.2-fold changes in expression and (2) significant difference by both Student’s ttest and ANOVA.  These proteins were classified into eight functional categories: metabolism, protein synthesis and destination, defense response, cell growth and division, pyruvate orthophosphate dikinases (PPDKs), signal transduction, transcription, and transporters.  Most of the DEPs were involved in metabolism (31.2%), protein synthesis and destination (25.2%), and defense response (22.4%).  In summary, proteins implicated in central carbon metabolism, starch synthesis, protein folding and modification, and defense response showed altered expression in response to natural genetic variation, conventional breeding, and transgene modification.
                  To examine the identified DEPs in more detail, the authors analyzed the expression patterns of 218 DEPs using GeneCluster 2.0.  The DEPs were grouped into 6 clusters: c0, c1, c2, c3, c4, c5. The 6 clusters were further grouped into three antagonistic pairs (clusters pairs: c1 vs c3, c1 vs c4, c2 vs c5).  Proteins in c0c3 sets were assumed to contribute to the variability between D68/MH63 and MH86/ZH10.  Proteins grouped into c1c4 sets possibily separated MingHui from D68/ZH10.  Furthermore, the c2c5 sets may be the main contributors to the separation between japonica and indica rice.  Although there were large changes in expression of these proteins among nontransgenic varieties, their expression was similar between transgenic lines and their respective controls in all clusters.
                  The authors further performed PCA for the 218 DEPs to estimate the contribution of DEPs to the total variability observed within rice lines and identify proteins responsible for the variability.  The results of the PCA confirmed the authors finidings from cluster analysis that proteins in 3 clusters pairs (c1c4, c2c5, and c0c3) may have a distinct contribution to the entire variability of the data set. 
                  Next, the authors evaluated the distribution on 218 DEPs involved in different function categories and subcategories in cluster pairs c0c3, c1c4, and c2c5.  The results of this analysis clearly showed different functional categories and subcategories-related proteins distributed heterogeneously in these cluster pairs.  For example, metabolism-related proteins were mainly in c1c4 and c2c5, but less so in c0c3.  Protein synthesis and destination-related proteins were more often in c0c3 and c2c5 than in c1c4. Most of the defense response-related proteins were in c0c3 and c1c4.  Thus, the different functional categories in these clusters confirmed differences in biological processes in all analyzed rice lines.
                  To investigate the changes in biological processes as a result of natural genetic variation, conventional breeding, and transgene modification, the authors performed expression profile analysis of protein groups associated with nine functional categories and subcategories showing significant contribution to the differences.  The expression patterns of proteins involved in glycolysis and starch synthesis were similar, with relatively high levels in 2 nontransgenic MinHui varieties and low levels in D68 and ZH10. The opposite relationship was found with proteins involved in the TCA pathway.  All in all, the expression of proteins in transgenic lines and their respective controls was changed, but the changes were similar to those observed between certain non-transgenic varieties.
                  All these data combined suggested that GM does not significantly alter the rice seed proteomes as compared with natural genetic variation and conventional genetic breeding.  Specifically, the integration in rice genomes and expression of bar or cry1Ac/sck do not change the proteome patterns as compared with natural genetic variation and conventional breeding.  Apart from the safety conclusions of this experiment, the results also show that the proteins differentially expressed in nontransgenic rice varieties had functions in central carbon metabolism, starch synthesis, protein folding and modification, and defense response.  For future experiments, these processes can be further investigated to differentiate and explore rice varieties. 

Safety of Transgenic Crops

This paper investigates whether or not the research accumulated over the last 20 years is sufficient to support the safety of transgenic crops. This research  has been focused on the compositional equivalency between genetically modified (GM) crops and their non-transgenic counterparts in order to assess their human health safety. Organizations such as the European Food Safety Authority (EFSA) have prescribed intensive designs for compositional field trials to assess the safety of transgenic food. More specifically, the aims of said trials are centered in evaluating the intended gene products, typically proteins. Here, Herman and Price (2013) argue that there is overwhelming evidence that transgenesis is less disruptive to crop composition compared with traditional breeding. Furthermore, the authors ask if sufficient uncertainty still exists in the safety of transgenic crops to demand extensive safety trails, inherently slowing down the development and progression of the genetically modified food industry. Is it reasonable to expect a greater risk of negative compositional changes in GM crops compared with traditionally bred crops and is it reasonable to continue to uniquely require compositional equivalence studies for GM crops to evaluate safety? There are important questions to explore when taking into account the task of feeding a rapidly growing global population.
 Herman R., Price W. (2013) Unintended Compositional Changes in Genetically Modified (GM) Crops: 20 Years of Research. Journal of Agricultural and Food Chemistry, doi: 10.1021/jf400135r

Compositional equivalence testing for (GM) crops was designed 20 years ago in order to investigate the potential unwanted side effects of genetically engineering crops to exhibit superior and more efficient phenotypes. Traditionally, three different conditions are implemented during the field studies that assess the safety of the GM crop: the GM crop itself, a near-isogenic nontransgenic line, and one or more nontransgenic commercial reference lines. Furthermore, the GM line entries are often supplemented by cultivating them in plots treated with the herbicide to which the crop has tolerance and also in plots where this herbicide is not sprayed. Next, researchers collect plant tissue samples from each entry and analyze them for an array of nutrients and antinutrients (typically 60–80). Statistical comparisons are performed between the GM crop and the non-GM companion. If statistical differences are observed, the biological relevance of the compositional changes is evaluated by determining if the observed levels would be unsafe within the context of how the crop is produced and consumed.
                  Even more intensive compositional field trails exist, one being prescribed by the European Food Safety Authority (EFSA). According to their requirements, at least eight field sites must be used with at least four replicates per site. Furthermore, if the GM line is being tested for herbicide resistance, both sprayed and unsprayed entries must be included. Also, at least six nontransgenic reference lines must be included with at least three lines being represented at each field site. Intensive studies like this are required for both new transgenic events and combined-trait products in which two transgenic events are bred together by traditional means.
                  These expanding requirements have increased composition study costs over 10-fold during the period of 20 years since research started. Initially, a study in the United States would have cost $100,000 per study, but now that cost has skyrocketed to over U.S. $1 million per study. This is becoming an increasingly impeding barrier to the growing GM industry, not to mention that additional tests have been suggested as a requirement to the production of genetically modified foods.
                  The authors argue that traditionally bred crops are inherently safer than GM crops. Nontransgenic breeding includes intraspecific crosses, wide crosses, crosses with wild relatives, tissue culture regeneration, and mutagenesis events. These techniques have been shown to be associated with genetic mutation, deletions, insertions, and rearrangements. The fact that breeders have been purposefully selecting crops that have beneficially random mutations lends justification to transgenic crop production. In other words, transgenic modification is doing what traditional breeding does, but in a shorter amount of time. In fact, the development of recent molecular techniques and our growing understanding of genetics lay fertile ground for a prosperous and safe transgenic crop industry.  Additionally, non-transgenic crop breeding programs generally do not monitor any resulting compositional changes that might accompany a plants improved agronomic characteristic. For example, breeders selected for endogenous insect resistance, which is a process often coupled with the up-regulation of glycoalkaloids, a toxic compound that can cause sickness. Conversely, all GM varieties are routinely screened for glycoalkaloid levels to ensure their safety. Furthermore, much of our knowledge about the compositional variation that occurs in some large-acre crops such as corn, soybean, and cotton has occurred, in part, as consequence of the efforts made to evaluate the compositional safety of GM crops. Thus, this knowledge shows the added benefit of genetically modified crop development and progression.
                  It is important to consider the severity and abundance of unintended genetic effects in GM crops in comparison with those that are traditionally bred. Often, transgenic inserts are sequenced to determine if they have been inserted as intended and to confirm that they encode for the desired gene product. In addition, the portions of DNA upstream and downstream of this segment are sequenced as well to understand if any native genes or regulatory elements are disrupted. Finally, the plant genome is probed to ensure that only one insertion site exists. Conversely, traditional breeding may result in many genes randomly recombining which may lead to unwanted side-mutations. Thus, these two systems pose very different standards of regulation and safety, with genetically modified crops being the most investigated of the two.
                  The compositional safety of genetically modified crops should be considered in the context of the normal composition of the crop. Both vary in their phenotype from the composite line from which they were derived. Here, the question lies with determining whether the changes in the GM crops are more frequent, of higher magnitude, or inherently more dangerous when comparing then with traditionally bred crops. If the compositional difference is expected to differ in both the GM line and its near-isogenic counterpart, then any significant difference found between the two is just a measure of that expected difference. Rather, the authors suggest that a better safety assessment would be to evaluate if the observed level of the compositional analyte differs meaningfully from the normal array of levels observed for the aggregate crop that has a history of safe consumption. Furthermore, collecting data from increasingly larger studies likely only serves to detect small and fleeting differences that are expect and irrelevant to safety.
                  In conclusion, the 20 years of research that has been done on genetically modified crops has confirmed the compositional equivalence between GM crops and their traditionally bred counterparts. Over the past 20 years, the U.S. FDS found all of the 148 transgenic events that they evaluated to be equivalent to their conventional counterparts. Also, Japanese regulators have confirmed the same for 189 submissions. The assessment of GM foods has compiled enough information to feel confident in the future and safety of GM crops.  Unfortunately, the growing amounts of regulation and testing barriers that surround GM research greatly stunt the growth of the industry. There is significance and necessity in removing the negative stigma attributed towards genetically modified foods. With public support and reformed regulations, research on genetically modified organisms can flourish and positively affect our quality of life. 

Relative Effects of Glyphosate on Glyphosate-Tolerant Maize Rhizobacterial Communities is Not Altered by Soil Properties

The rhizosphere is the narrow region of soil that is directly influences by root secretions and soil microorganisms. Much of the nutrient cycling and disease suppression needed by plants occurs immediately adjacent to roots. In this study, researchers compared the effects of glyphosate (Roundup Plus), a post-emergence applied herbicide, a pre-emergence applied herbicide (GTZ), and untreated soil. The effects of these variables was monitored by sequencing the soil DNA encoding 16S rRNA and high-throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region. Previous research suggests that PCR amplification and sequencing of the full-length 16S rRNA genes from soil DNA samples combined with massive parallel pyrosequencing of the SSU rRNA V6 hypervariable region proves to be very useful for studying changes in the diversity of the bacterial communities associates with various habitats (Acosta-Martinez et al. 2010). In total, three different methods were used to analyze the herbicide effect on the rhizobacterial communities of genetically modified NK603 glyphosate-tolerant maize. These results were compared with the diversity data obtained when glyphosate was grown in soil with different characteristics. Barriuso and Mellado found that both herbicide treatments decreased the bacterial diversity in the rhizosphere, especially the Actinobacteria taxonomic group.
Barriuso J., Mellado R. Relative Effects of Glyphosate on Glyphosate-Tolerant Maize Rhizobacterial Communities is Not Altered by Soil Properties. 2011. Journal of Microbial Technology 22 (2), 159–165
                  Previous study has indicated that the absorption of glyphosate by the leaves of glyphosate-resistant plants can alter the root exudation and thereby affect the rhizosphere communities (Kremer et al. 2005). The study by Barrius and Mellado was aimed at testing if the effects of herbicides on the rhizobacterial communities of genetically modified NK603 glyphosate-tolerant maize varies according to different soil locations. First, to determine the chemical properties of the soils, the rhizosphere samples were ground using a mortar and dried at 60°C for 12 hours. The total mean values for the carbon, nitrogen, and hydrogen content from three independent measures were determined using a Leco CHSN-932 autoanalyzer.
                  Rhizosphere samples were taken from commercial maize fields of glyphosate tolerant maize, of the NK603 variety, located in Calera y Chozas in Toledo, Spain. There, plants were treated in pre-emergence with 450 g/l acetochlor or 214 g/l terbuthylazine (4 l/ha), in post emergence with 360 g/l of glyphosate as isopropylamine salt (0.72 kg/ha), or untreated with herbicides. The maize and both herbicides were obtained from Monsanto. The plants were harvested from the three experimental fields at two different growth stages. The first was 7 days after glyphosate application and the second was just before crop harvesting at final growth, approximately six months after seeding. Each plot was divided into subplots, and three samples were taken from each subplot at collection times. Therefore, a total of nine subplots were sampled from each maize field at each collection time and equal amounts of soil were pooled from all 27 samples.
                  The rhizospheres from each collection time were pooled and three independent DNA extractions were conducted. The DNA was then subjected to PCR amplification to amplify the 1.5–kb-long fragment encompassing the 16S rRNA coding sequence. The DNA was then subjected to pyrosequencing, resulting in 750–nucleotide long sequences corresponding to the 16S rRNA protein. A phylogenetic tree showing the highest degree of homology between the 16S rRNA sequences was creating using ClustalX, a multiple sequence alignment computer program.
                  The chemical properties of the soils showed carbon values of 1.81%, 1.78%, and 1.75% and nitrogen values of 0.21%, 0.22%, and 0.22%, respectively corresponding to soils not treated with herbicide, treated with glyphosate, and treated with GTZ for the samples taken 7 days after glyphosate application. Furthermore, carbon values of 2.35%, 2.31%, and 2.35%, and nitrogen values of 0.23%, 0.22%, and 0.22%, were obtained at the final sampling times. There were no significant differences ascribed to the treatment with either of the two herbicides.
                  Pyrosequencing of the V6 region of the 16S rRNA subunit were processed in a taxonomic breakdown and a diversity distribution was obtained. Among the sequences obtained from the untreated soil, 41% belonged to the Proteobacteria phylum, 20% to the Acidobacterium phylum, and 13% belonged to the Actinobacteria phylum. This bacterial distribution was similar to that of the 8,345 sequences analyzed from the glyphosate-treated soil (Proteobacteria was slightly lower, and Acidobacteria and Actinobacteria were slightly higher). Conversely, the Proteobacteria and Actinobacteria were lower in the 11,277 sequences analyzed from the GTZ-treated soil, and the Acidobacteria were slightly higher. At the final sampling time, Proteobacteria represented 38% of the rhizobacterial composition in the untreated soil, whereas Acidobacteria ccounted for 30% and Actinobacterial for 11%. These results were similar for the glyphosate-treated soil, but differed from those of the GTZ-treated soil, where the Proteobacteria and Actinobacteria were lower, whereas the percentage of Acidobacteria was higher.
                  The rhizobacterial communities were more similar within each sampling time than when comparing the herbicide treatments, and the supporting values for all the nodes were above 90%. Furthermore, the species richness was determined using the ACE and Chao1 estimators, the results showed a reduction in diversity in bother herbicide-treated soils at both sampling times. In addition, diversity was lower at the second sampling time when compared to the first sampling time. The relative differences observed at 3%, %%, and 10% dissimilarity levels were qualitatively equivalent for al the soil treatments. In conclusion, Actinobacteria was the phylum most significantly affect by the GTZ herbicide. Actinobacteria are an important component of soil bacterial communities playing a major role in organic matter turnover in soils, and are able to decompose organic materials by means of hydrolytic enzymes they synthesize and secrete. However, the diversity analysis showed a reduction of species richness in both herbicide-treated soils at both sampling times, with the values from GTZ-treated soil at the final sampling time being the lowest.

                  The results of this study unveils the effects of herbicidal treatments on the structure of bacterial communities present in the maize rhizosphere. Although, glyphosate treatment revealed a less aggressive impact than GTZ treated soil, further research is need to determine the extent and rate to which chronic herbicidal use may deteriorate the composition of rhizobacterial communities, depending on the particular soil characteristics and applied herbicide. 

Works Cited
Acosta-Martinez, V., S. E. Dowb, Y. Sun, D. Wester, and V. Allen. 2010. Pyrosequencing analysis for characterization of soil bacterial populations as affected by an integrated livestock cotton production system. Appl. Soil Ecol. 45: 325.
Kremer, R. J., N. E. Means, and S. J. Kim. 2005. Glyphosate affects soybean root exudation and rhizosphere microorganisms. Int. J. Environ. Anal. Chem. 85: 11651174.

A 90-day feeding study of glyphosate-tolerant maize with the G2-aroA gene in Sprague-Dawley rats.

Maize is a staple food crop and important raw material for feed and industry because it is nutritious and historically has been inexpensive to grow, however competing weeds currently threaten the production rate and quality of maize. To combat the growth of weeds, the agricultural industry has implemented genetically modified crops that contain herbicidal resistance. The most dominant herbicidal resistant trait is that for glyphosate (RoundupÔ) tolerance. In this study, the food safety of GM maize with the G2-aroAgene for glyphosate tolerance was assessed in a 90-day feeding study and compared with a non-GM isogenic line. Seventy male and seventy female Sprague-Dawley rats were fed a diet composed of maize with the G2-aroA gene. The researchers conducted multiple tests to assess if there were any significant effects in GM-fed rats. This included a compositional analysis, survival and clinical observations, body weight gain and food consumption, hematology examination, serum chemistry, organ weight, and pathology examination. This sub-chronic toxicological study indicated significant differences in a few of these tests, however the researchers did not attribute this variance to the presence of G2-aroA maize grain. Thus, they concluded that G2-aroA maize grain does not cause adverse effects in Sprague-Dawley rats.
Zhu, Yaxi., He, Xiaoyun., Luo, Yunbo., Zou, Shiying., Zhou, Xin., Huang, Kunlun., Xu, Wentao., 2013. A 90-day feeding study of glyphosate-tolerant maize with the G2-aroA gene in Sprague-Dawley rats. Food and Chemical Toxicology 51, 280­–287.

Glyphosate is an active ingredient in RoundupÒagricultural herbicides inhibits the biosynthesis of aromatic amino acids by suppressing the activity of the SPSPS enzyme. This enzyme is encoded by the aroA gene and its mutant allele was found to express a mutant EPSPS enzyme that is insensitive to glyphosate. Thus, by transforming crops to express the mutant aroA gene, glyphosate can be used to exterminate unwanted weeds while still allowing crops to grow. The G2-aroA gene studied by Zhu et al. is a new mutant strain found to have glyphosate resistance.
In order to test if the consumption of G2-aroA crops is safe, researchers compared Sprague-Dawley rats subjected to a 90-day diet of G2-aroA crops to ones on a non-GM diet. Flours from GM and non-GM maize were formulated into rodent diets at concentrations of 12.5%, 25%, and 50%.  The researchers used seventy weaned male and seventy weaned female rats supplied by Vital River Laboratories Co. Ltd (Beijing, China). The rats had an average body weight of 80–100 g, were kept in groups of 5, at a regulated temperature, light cycle, and humidity. The rats had ad libitum access to water and feed and were observed daily for mortality and signs of toxicity or other notable behaviors. Body weight and food intake were measured once a week. Furthermore, blood tests such as white blood cell counts (WBC), red blood cell counts (RBC), and hemogloblin concentrations (MCHC) were evaluated. In addition, serum chemistry was conducted to observe glucose, protein, and cholesterol levels. Lastly, after the 13-week exposure test, selected organs were weighed and underwent histopathology examination.
The male rats in the 25% GM group had significantly higher body weights than the other male groups. However, the significance was not considered related to the addition of G2-aroA maize in the experimental diet because there were no differences observed in males consuming the 50% GM maize diets. The researchers further dismiss significant differences with the same reasoning. The values of RBC and hematocrit (HCT) of the 12.5% GM male group and the value of mean corpuscular volume (MCV) of the 12.5% GM female group were significantly different from the 12.5% non-GM group. Moreover, the mean values of MCH and MCHC of males fed 50% G2-aroA transgenic maize diets were higher (P < 0.05) than the values observed in the 50% non-GM groups. The mean values of MCV and MCHC of females fed with 25% GM maize were different (P < 0.05) from the values observed in the reference control group and the 25% non-GM group.
The serum chemistry results yielded significant differences as well, but the researchers did not attribute the adverse effects to the experimental diet. Statistically significant differences in the mean value of total cholesterol (CHO) were observed between the 12.5% GM and non-GM group in both genders. However, this change was not found in the 25%, 50%, and reference group and therefore dismissed as non-significant. Furthermore, the values of blood urea nitrogen (BUN) and triglycerides (TG) in the males and the value of LDG in the females in the 25% GM maize were significantly different from those of non-GM groups and reference control group. Other significant differences were found in the creatinine (CREA) values of males, alkaline phosphatase (ALP) in females, and total protein (TP) and albumin (ALB), when comparing between males and females in the 50% GM-group. Lastly, the weights of ovaries and brains of the females consuming the 25% GM maize diet were lower than those observed in the 25% non-GM control diet and the weight of the kidneys were higher in the female 50% GM group than those of the 50% non-GM group. The researchers did not attribute these significant differences to the experimental GM diet because these relationships were not observed at higher concentrations of GM maize diets.
This sub-chronic toxicological study was intended to determine the safety of long-term dietary exposure to grain from GM G2-aroA herbicide-tolerant maize. The researchers concluded that a high concentration of GM maize does not have adverse effects because none were observed in the 50% GM maize experimental groups. None of the significant differences were attributed to the GM maize diet and therefore the consumption of a GM G2-aroA maize diet was concluded to be safe at all concentration levels. 

Up-regulation of a H+- pyrophosphatase (H+-PPase) as a strategy to engineer drought-resistance crop plants

Drought resistant is a desirable trait in agriculture during a period of climate change and water depletion. Transgenic crops over expression of the vacuolar H+-pyrophosphate (H+-PPase) AVP1 in the model plant Arabidopsis thaliana resulted in enhanced performance under soil water deficits, as studied by Park et al.(2005) AVP1 plays a large role in root development through the facilitation of auxin fluxes. Auxins are a class of plant hormones that play a role in the coordination of growth and behavioral processes and are essential for plant body development. Researchers looked to improve crop performance by expressing AVP1 in commercial tomatoes, Lycopersicon esculentum. The study resulted in an increased root biomass, greater pyrophosphate-driven cation transport into root vacuolar fractions, and enhanced recovery of plants from an episode of soil water deficit stress. Transgenic crops engineered to tolerate periods of drought could significantly increase yields for many developing countries and contribute to the fight against famine.
Park S., Li J., Pittman J., Berkowitz G., Yang H., Undurraga S., Morris J., Hirschi K., Gaxiola R. Up-regulation of a H+– pyrophosphatase (H+-PPase) as a strategy to engineer drought-resistance crop plants. 2005. PNAS 102 (52) 18830-18835

The transformation of Lycopersicon esculentum tomatoes was performed by means of the Agrobacterium-mediated transformation method using cotyledon and hypocotyl explants. The AVP1D1 gene is the gain-of-function mutant of the AVP1 gene that has a coordinated increase of both PPi hydrolytic activity and PPi-dependaent H+-translocation. Homozygous T2 XAVP1D lines were selected to use in all of the experiments reported in the study.
A Southern blot analysis indicated the absence of the transgene from control plants and the presence of the 35S AVP1D construct in genomic DNA of the transgenic tomato plants. In all instances, genomic DNA was digested with EcoR1, separated on a 0.9% agarose gel by electrophoresis, and probed with BgIII fragment of the AVP1D open reading frame. In total, five transgenic lines were generated. The relative intensities of the transgenic lines XAVP1D–1, XAVP1D–2, and XAVP1D-3 were observed through a Western blot. A 200%, 230%, and 150% level increase of H+-PPase protein in their root tonoplast membrane fractions was observed when compared to the control line.
Researchers continued on to study the chemical and transport effects of the recombinant protein in the transgenic tomato lines. The results indicated the transgenic lines have a mean 56% increase in H+-PPase hydrolytic activity when compared to the control plants. In addition, kinetics of PPi– and ATP-dependent 45Ca2+uptake into vacuolar membrane vessicles from transgenic and control plants were monitored. It was observed that the PPi-dependent 45Ca2+uptake was 31% greater in vesicles from the XAVP1D lines than the control, whereas ATP-dependent 45Ca2+uptake was unchanged by AVP1D expression.
Park et al. conducted soil water deficit experiments with seeds from T2homozygous vector-control and XAVP1D expressing lines were germinated on Murashige and Skoog (MS) inorganic salts, 3% (wt/vol) sucrose, MS vitamins, and 100 mg/liter kanamycin. Plants were grown for a period of 5 weeks in soil and watered regularly to field capacity. Then, the soil was allowed to dry by withholding water. In every experiment, stress was induced by withholding water until all plants showed severe signs of drought (i.e., visivle loss of turgor and wilting). In total, seven independent experiments were performed with vector controls and T2 XAVP1D lines. Three of them were condicted at the greenhouse facilities of College Station, Texas, and four at the Agricultural Biotechnology greenhouse of the University of Connecticut. Researchers found that the transgenic plants demonstrated recovery after relief of the water deficit stress that was not seen in the control group.
One downside of H+-PPase overexpression could be the accumulation of toxic metals in the fruit of transgenic plants. The researchers evaluated fruit contents both the control and XAVP1D plants and found no significant difference in the levels of Pb2+, Mo2+, Mn2+, Cd2+, Zn2+, Cu2+, Fe2+, or Ca2+.
Leaf water potentials of four control plants and a totally of eight transgenic plants were monitored during the imposed stress period. Researchers observed that transgenic plants maintained greater leaf water potentials and take up greater amounts of water during imposed soil water deficits. Transgenic pants maintained greater leaf water potentials from day 4 onward compared with control plants. During the end of the stress cycle, the leaf water potential of XAVP1D plants was 0.2–0.3 MPa greater than control plants at the same day of stress. It was visually evident that the XAVP1D had more enhanced plant water status during the latter part of the stress trial compared with controls at day 5 of the imposed stress.
Once possible explanation for greater leaf water potentials and enhanced plant performance could be that stomata closure was greater in XAVP1Dplants, thus restricting water usage during stress. This hypothesis was not supported by the gas exchange analysis where stomata conductance was similar in both sets of plants throughout the course of the water stress. Researchers attributed the enhanced plant performance of the transgenic crops to the significantly greater water uptake during stress periods. Water uptake in transgenic plants was significantly greater when compared with control plants between days 3 and 5 (by 14%), days 5 and 6 (by 75%), and days 6 to 8 (by 45%).
Lastly, an increased root growth in the AVP1-expressing plants could be the cause of the water deficit recovery phenotype. A visual observation and root dry weight supported the notion that transgenic tomato plants had significantly more extensive root systems.
Thus, the results of this experiment suggest that AVP1D expression increases root growth, water uptake, leaf water potentials, and plant survival under soil water deficit. Conversely, a loss of function study of the AVP1D gene supported the notion that it is crucial for plant growth and development. The results of this study have exciting implications for agriculturalist. Hopefully, this gene can be engineered into a variety of modern crops in order to improve yields under water deficit conditions. 

Phenotyping transgenic wheat for drought resistance

Drought is one of the largest environmental stress factors that agriculturalist struggle with because it limits plant growth and crop productivity globally. Plant responses to drought often have complex mechanisms which are normally under multigenic control. Time, intensity, duration, plant-soil-atmosphere interactions, and frequency of drought stress can all affect the plants response. St. Pierre et al. (2012) evaluated field performances of 14 transgenic wheat lines previously selected under greenhouse conditions for survival to severe drought and high water use efficiency. The study was conducted under various water regimes in field conditions to compare biomass production and yield performance between transgenic lines and control lines. Researchers over-expressed dehydration-responsive element-binding (DREB) transcription factors that were previously found to enhance drought resistance in transgenic plants like tomatoes, peanuts, rice, barley, and wheat (Kasuga et al., 2009). The paper found some significant conclusions by transforming DREB1A from Arabidopsis thaliana under the stress-inducible promoter rd29A. Researchers found that the survival rate of transgenic plants was increased without growth retardation and a positive association between WUE and total biomass. Moreover, researchers found that some transgenic plants produced a higher yield under well-irrigated field conditions. Finally, the 14 transgenic lines were evaluated in the field and showed no pleiotropic effects or unpredictable unwanted events. This study suggests that plants can be transformed to recover better after severe water stress. Thus, a greater resilience towards droughts could be extremely useful when growing in dry climates or in drought prone areas. —Paloma Medina
Saint Pierre, Carolina, et al., 2012. Phenotyping transgenic wheat for drought resistance. Journal of experimental botany 63, 1799–1808.

Researchers transformed bread wheat (Triticum aestivum), or Bobwhite, with the DREB1A gene from Arabidopsis thaliana under the stress-inducible promoter rd29A. Under this promoter, the target DREB1A gene would only be expressed under stress conditions. The transgenic lines were screened for the DREB1A gene by polymerase chain reaction. The integration of the DREB1A gene was further confirmed by self-pollination and examining the T1 generation to find that DREB1A expression. Thus, the researchers confirmed that DREB1A was integrated into the genome of the transgenic plants.
Fourteen transgenic wheat lines with the DREB1A gene were examined to assess biomass production (BM) and yield performance (YLD) of transgenic plants relative to control lines under different water regimes in field conditions. Three water deficit treatments: severe stress (DEF), terminal water deficit starting at anthesis (ANT), and terminal water deficit starting in grain filling (GF). Anthesis is the point at which a flower is open and functional. These treatments were applied by reducing irrigation starting at different phonological stages. Well-irrigated plots (IRR) received a total of 260 mm of irrigation water throughout the growing season. Conversely, plots under the most severe water deficit treatment (DEF) received a total of 68 mm from irrigation, only 26% of the amount applied to IRR. Plots undergoing the ANT treatment were well-irrigated with a total of 121 mm of water until the booting stage (occurs shortly after flag leaf emergence) of the plant. Lastly, the effect of terminal stress starting in grainfill was evaluated by stopping the irrigation at the early grainfill stage in the GF group.
The evaluation of above-ground biomass and water use efficiency (WUE) took place shortly after anthesis on drought-stressed and well-irrigated plants grown in small pots under greenhouse conditions. On average, drought reduced biomass by 42% after repeated drought cycles imposed from the 5-leaf stage to anthesis. The lowest biomass reduction occurred at 19% in the transgenic line WUE-12 between the well-irrigated and the water-deficit treatments. The control (Bobwhite) line had a biomass reduction of 46%. Moreover, all the transgenic plants selected for high WUE had higher biomass prodction than the null and control line.
In addition to a larger biomass in the transgenic lines, significant differences were observed for water use efficiency (WUE) under water-deficit. All WUE-selected events tended to have higher WUE than controls under well-irrigated conditions, but only one out of the five events was significant. A significant positive correlation was drawn between BM and WUE bother under well-irrigated conditions (r=0.72, P
Plant performance was evaluated under field conditions and found that irrigation regimes had a significant main effect for both biomass (BM) and grain yield (YLD). The total biomass was reduced from 504 g m–2at well-irrigated conditions (IRR) to 325 g m–2 at severe stress conditions (DEF). The transgenic event WUE-11 outperformed the control lines for YLD under IRR conditions. This line had a yield of 366 g m–2under IRR while Bobwheat and the null event yielded 297 g m–2 and 310 g m–2 respectively.
In conclusion, screening genetic lines for drought resistance characteristics can be fundamental to the identification of high performance lines. The researchers of this study suggest that several studies involving transgenic crops may be misleading due to the fact that the plants were grown under artificial stress conditions. The importance of this study is that the plants were tested under field conditions with aims to mimic natural conditions as closely as possible. The results of this experiment support the function of the DREB gene in which transgenic crops were observed to have a higher survival rate and recovery after severe water deficit. Wheat growers can benefit tremendously from the grain yielding potential and drought resistant crop growth under a warming climate. Moreover, the consumer population would also benefit from this transgenic drought-resistant line of crops. Further research is suggested to determine and identify appropriate gene and gene-promoter combinations to maximize drought-resistance and crop yield.

Works Citied
Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. 1999. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcriptionfactor. Nature Biotechnology 17, 287–291.

Analysis of genetically modified red-fleshed apples reveals effects on growth and consumer attributes

With the world population tipping the scale at 7 billion people, the question of food sustainability has been brought to the forefront. Feeding the growing human populace with a finite amount of natural resources is becoming an increasingly difficult task. Genetically engineered foods serve to enhance the health benefits of foods and production rates while retaining consumer expectations of flavor. In apple, the anthocyanin pathway has been shown to be controlled by the MYB transcription factor, MYB10(Espley et al. 2007). Espley et al. (2012) raised the polyphenolic content of apple by genetically engineering the anthocyanin pathway using the apple transcription factor MYB10. The authors did a series of tests that compared genetically modified (GM) apples to their wild type (WT). These tests examined anthocyanin metabolite and transcription levels, flavonoids and proanthocyanin expression, and consumer reaction to MYB10 over-expressed apples. This study aims to enhance novelty and food crop nutritional status, and in a larger sense, produce food that can more adequately sustain our growing populace. Their results indicate that MYB10 over-expressed apples are likely to retain all the current consumer expectations of flavor, with the added appeal of elevated colour and potential health enhancement.—Paloma Medina
Espley, R. V., Bovy, A., Bava, C., Jaeger, S. R., Tomes, S., Norling, C., Crawford, J., Rowan, D., McGhie, T. K., Brendolise, C., Putterill, J., Schouten, H. J., Hellens, R. P. and Allan, A. C. (2012), Analysis of genetically modified red-fleshed apples reveals effects on growth and consumer attributes. Plant Biotechnology Journal. doi: 10.1111/pbi.12017

In the majority of plant species, pigmentation is controlled by the relative amount of anthocyanins, chlorophyll, and carotenoids. These compounds are essential for plant health and function, but are also considered as markers for dietary health. For this reason, plant-breeding programs have targeted their efforts to produce red-fleshed apples, similar to those from Central Asia, with white commercial varieties. Previous examples of GM apples have resulted in obtaining beneficial characteristics such as disease resistance, dwarfing, and extended fruit storage. In apple, the anthocyanin pathway has been shown to be controlled by the MYB transcription factor, MYB10. When MYB10 is fused to a 35S promoter, it is able to drive the accumulation of anthocyanin pigments. The researchers show that a large increase in anthocyanin produces red-fleshed apples. The trees that had an over expression of MYB10 had characteristics of deep pigmentation in the leaves and trunk. A microscopic analysis of the leaves revealed anthocyanin accumulation predominantly in the spongy mesophyll and lower epidermis and in tissue close to the vascular rays.
Though pigmentation increased, anthocyanin accumulation did not appear to compromise photosynthetic electron transport activity (P < 0.05). The electron transport rate (ETR) versus photosynthetically active radiation (PAR) was plotted, and the curves were used to estimate maximum photosynthetic capacity (PSmax) and photosynthetic efficiency (initial slope of the ETR versus PAR). Interestingly, researchers found that photosynthetic capacity was significantly higher in the red MYB10 leaves. Furthermore, firmness of the fruit was measure by a penetrometer, and both MYB10 apples and Royal Gala apples were found to be similar in firmness.
In addition to photosynthetic data analysis of pigmented fruit, anthocyanin concentration of mature fruit was analyzed by high-performace liquid chromatography (HPLC). MYB10 fruit showed a significant increase of five cyanidin glycosides, of which cyanidin-3-galactoside was the most abundant. This glycoside is the leading anthocyanin found in many apple varieties. Futhermore, a qPCR analysis of transcript profiles of genes in the anthocyanin pathway showed significant increases in expression of all the anthocyanin biosynthesis-related genes tested. Not only this but the red-fleshed fruit showed elevated concentrations of flavonols and proanthocyanidins (Pas). Specifically, in MYB10 plants, PA concentrations increased approximately three-fold in all the tissues tested. Flavonols are polyphenolic compounds present in fruits, vegetables, tea, and berries, and have effects similar to antioxidants. Thus, this is positive evidence to support the breeding of MYB10 apples on the grounds of nutritional status.
But there are disadvantages to red-fleshed apples. After storage at 0.5 °C for 12 weeks, researchers observed internal browning in some MYB10 fruit which isn’t normally observed in Royal Gala. In addition, average fruit size for the MYB10 fruit was similar to WT, but average WT fruit were heavier. A variety of apples were tested in a consumer panel of 15 individuals. In total, there was an overall preference for WT fruit due to its overall ‘crisp’ texture and quality.  In addition, metabolite analysis revealed that the cortex of MYB10 fruit contained 1.5-fold and 3.6-fold higher concentrations of fructose and glucose and 0.44-fold lower concentrations of sucrose than WT fruit, although these differences were not significantly different (P > 0.05). The diminished sucrose concentration is reiterated in a consumer panel in which individuals preferred the sweetness of a WT apple to a MYB10 apple. However, when presented with the additional health benefits that red-fleshed apples may have, the consumer panel response towards red-fleshed apples remained positive.
All in all, the changed metabolite profile of the MYB10 fruit did not appear to alter the flavor or aroma qualities of the fruit significantly. If further research is able to find mechanisms to increase sucrose levels, red-fleshed fruit may have more value on the market. Espley et al. presents a surprising multitude of health benefits in altering the transcription factor MYB10. The authors are hopeful that these findings will help plant-breeding programs enhance novelty and food crop nutritional status.
Espley, Richard V. (2007). “Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10 A MYB transcription factor controlling apple fruit colour”. The Plant journal: for cell and molecular biology (0960-7412), 49 (3), p. 414. 

Contrasting patterns of early twenty-first-century glacier mass change in the Himalayas.

Most glaciers around the world have been losing mass. The loss of glacial mass could have serious and negative implications on water resources (ie. hydropower, irrigation, runoff) of the surround communities and is a major contributor to rising sea levels. Kaab et al. 2012 studied glacier thickness changes and estimated mass changes over the Hindu Kush-Karakoram-Himalaya (HKKH) region during 2003 – 2008. To do this, they combined two elevation data sets from the Ice Cloud and land Elevation Satellite (ICESat) and the Digital Elevation Model (DEM) from the Shuttle Radar Topography Mission (SRTM). The authors found that debris-covered ice thins at a similar rate to that of exposed ice, suggesting a reassessment of the role of debris mantles in glacier mass balance. This study can be considered as the first quantitative evaluation of the contribution of glacier imbalance to river runoff. —Paloma Medina

Kaab, A., Berthier, E., Nuth, C., Gardelle, J., Arnaud, Y., 2012. Contrasting patterns of early twenty-first-century glacier mass change in the Himalayas. Nature 488, 495-498

The HKKH is a conglomeration of mountain ranges in Asia spanning 2,000 km and separates the plains of the Indian subcontinent from the Tibetan Plateau. These mountains contain about 60,000 km2 of glaciers, glacierets and perennial surface ice in varying climatic regions. The HKKH accumulates or ablates ice mass in varying areas and in varying times of the year. In the east, glaciers receive most of their accumulation during summer month from the Indian monsoon, whereas in the west they accumulate snow in the winter through westerly atmospheric circulations. Therefore, variability among the glacier geography is large and could lead to different levels of impact of sea-level rise, water resources, and natural hazards. Kaab et al. looks to assess the changes in glacier mass with respect to this variation using ICESat and DEM satellites.
Length changes measured for more than 100 glaciers in HKKH suggest that most Himalayan glaciers have been retreating since the mid-19th century. The SRTM DEM and ICESat data sets provide geographically distinguishable data of glacier thickness. Through these satellite instruments, the researchers were able to distinguish between glacier clean ice, glacier debris cover, glacier firn and snow, open water, and off-glacier. These data were used to map trends of climatological and glaciological patterns between five major subregions of HKKH: the Hindu-Kush south of the Wakhan Corridor (HK), the Karakoram (KK), Jammu-Kashmir (JK), Himachal Pradesh, Uttarakhand and West Napa (HP), and East Nepal and Bhutan (NB). ICESat data showed a +0.14 + 0.06 m yr –1 thickening in the northern and eastern parts of KK. However, ICESat data indicated that HKKH glaciers thinned on average of  –0.21 + 0.06 m yr –1. This thinning is significantly less than the estimated global average from glaciers and ice caps. This difference is mainly attributed to the glacier mass thickening in the Karakoram. ­­
Contrary to previous expectations, the average thinning rates under debris-mantled ice were similar to those of clean ice despite the insulation of the debris covers. This finding suggests that the insulating effect of debris layers with thicknesses exceeding a few centimeters acts on local scales, but not on the larger scale of entire glacial tongues.  The study applied elevation difference trends to the total ice-covered area by using a glacier mask based on a ratio between visible and short-wave infrared Landsat bands and glacier inventories.  The researchers found that there was a mass change of -12.8 + 3.5 gigatonnes per year and a sea level rise contribution of 0.035 + 0.009 mm yr-1for HKKH glaciers, accounting for 3% to 4% of the total contribution from global glaciers and ice caps.
This study suggests using satellite gravimetry to better detect large scale sub-surface mass changes such as from hydrology or tectonics. In addition, this method could better quantify errors in detecting mass changes by relying on studies such as this that estimate glacier thickness changes. Bolch et al. (2012) pushes for more developed and refined remote-sensing methods to estimate glacial changes, draws attention to the climatic and hydrological station network gap, and strongly recommends increasing the number of active research stations on glaciers. They also recommend establishing new programs to cover more climate zones and glacier types. It is important to understand the hydrological impacts of the Himalayan glaciers, and glaciers in general, because many populations rely heavily on glacial river run-off. A negative annual mass budget, or a decrease in glacial mass, yields a surplus of runoff from glacier ice. Conversely, a positive annual budget yields a deficit of runoff because ice has gone into storage on the glacier. The authors hope that their study will improve estimates of groundwater depletion in northern India, which thus far has been difficult to discriminate from glacier loss in satellite gravity observations. Furthermore, glacier melting could have catastrophic consequences on lake or dam areas that cannot accommodate for an influx of water. All in all, the researchers look to redefine and reassess the functionality of debris-covered ice and impact on river run-off. A better understanding of glacier ablation and accumulation gives a more holistic understanding to glacial regulation and preservation.