The support from the National Key Research and Development Program of China (No. 2016YFC1401601), the Postgraduate Research &#38; Practice Innovation Program of Jiangsu Province (No. SJKY19_0415) supported by the Fundamental Research Funds for the Central Universities (No. 2019B62814), the National Natural Science Foundation of China (Nos. 41890805, 41806026 and 41730536) and Joint Advanced Marine and Ecological Studies in the Bay of Bengal and the eastern equatorial Indian Ocean were greatly acknowledged. The authors appreciate the provision of data from sources including the National Aeronautics and Space Administration (NASA), the European Centre for Medium-Range Weather Forecasts (ECMWF), the Copernicus Marine and Environment Monitoring Service (CMEMS) and the National Oceanic and Atmospheric Administration (NOAA).

1. Dataset acquisition
<ol>
	<li>SST and CHL
	<ol>
		<li>Download a dataset of satellite observations for SST and CHL from MODIS-Aqua (podaac-tools.jpl.nasa.gov/), where the spatial resolution of both datasets is approximately&#160;4.5 km at daily intervals. 
		NOTE: Structure the directories&#160;and data following the example scripts folder available in the Supplemental Files. Store the .nc files of the satellite data in the &#39;Data&#39; folder. Add the path to the NetCDF toolbox&#160;in the analysis software (i.e., MATLAB). Select Add with subfolders to enclose the paths of the &#39;UTILITIES&#39; folder and its subfolders.</li>
		<li>Determine the time span. To maintain consistency among different datasets, use the same time span for all parameters. Adjust the time span based on the temporal coverage and use the longest observation period among different datasets. For this protocol, download 15 years of data from October 2002 to September 2017.</li>
		<li>Determine the spatial coverage. 
		NOTE: The designed study region is between 105&#176;E and 123&#176;E and between 0&#176; and 25&#176;N.</li>
		<li>Check preprocessing instructions. Read instructions in the .nc files regarding the preprocessing requirements of the SST and CHL data (e.g., whether scaling is needed). 
		NOTE: The downloaded dataset already exclude&#160;data over land and within 5 km of the coastline, as well as those contaminated by clouds.</li>
		<li>Load SST and CHL data into the analysis software. Type Read_MODIS_SST in the command window to read the SST data. Similarly, type Read_MODIS_CHL in the command window to read the CHL data. Transform the CHL data logarithmically because they have a log-normal distribution31. 
		NOTE: Loaded variables include SST and CHL in three dimensions, representing meridional location, zonal location, and time in days, respectively. The range of SSTs is between -2 and 44, and the range of CHL is between 0.01 and 20.</li>
	</ol>
	</li>
	<li>Sea level anomaly (SLA)
	<ol>
		<li>Download daily SLA data with a 25 km spatial resolution from 2002&#8211;201732. 
		NOTE: SLAs&#160;describe&#160;the difference between observed sea surface heights and the mean sea surface height over 20 years (1993&#8722;2012) for a&#160;corresponding pixel. The SLA data are processed by SSALTO/DUACS and distributed by Archiving, Validation, and Interpretation of Satellite Oceanographic Data (AVISO, https://www.aviso.altimetry.fr).</li>
		<li>Load data into the analysis software. Load single-day SLA data by typing Read_SLA in the command window. 
		NOTE: The &#39;Data&#39; folder in the Supplemental Files only includes one sample datum in the script for illustration.</li>
	</ol>
	</li>
	<li>Wind speed
	<ol>
		<li>Obtain the wind information from an ERA-Interim reanalysis product, which is a global atmospheric reanalysis dataset developed by the European Center for Medium-Range Weather Forecasts (ECMWF)33. Download wind data for the same period (October 2002&#8211;September 2017) to maintain consistency with the CHL and SST data. 
		NOTE: The wind dataset has a spatial resolution of approximately 25 km and was interpolated from the original dataset with a spatial resolution of approximately 0.7&#176;.</li>
		<li>Load data into the analysis software. Type Read_WindVector in the command window to read the one-month wind data. Calculate the monthly mean by averaging the original data, which is at 6 h intervals.</li>
	</ol>
	</li>
	<li>Topography
	<ol>
		<li>Download the high-resolution topography data from the National Centers for Environmental Information website (NCEI, https://maps.ngdc.noaa.gov/viewers/wcs-client/). The spatial resolution is ~2 km. Obtain the ETOPO1 data for bedrock in XYZ format for the selected study region.</li>
		<li>Load data into the analysis software. Type Read_topography in the command window to load the topography data into the analysis software.</li>
	</ol>
	</li>
</ol>
2. Data preprocessing
<ol>
	<li>Temporal average
	<ol>
		<li>Due to the large cloud coverage in the SST and CHL data, replace the original data with 3-day average data. To do this, after running the Read_MODIS_SST.m and Read_MODIS_CHL.m scripts (step 1.1.5), type Temporal_average in the command window to run the script.</li>
	</ol>
	</li>
	<li>Interpolation into the same grid
	<ol>
		<li>Because the spatial resolution is not consistent for different datasets, interpolate the SST and CHL data into a spatial grid that is the same as the wind and SLA spatial grid before making comparisons. After running the Temporal_average.m and Read_WindVector.m scripts, type Interpolation_grid in the command window to run the script.</li>
	</ol>
	</li>
	<li>Wind stress and wind stress curl
	<ol>
		<li>Type Wind_stress_curl in the command window to calculate the wind stress (WS) and wind stress curl (WSC) using the following equations: 
		<img alt="Equation 1" src="/files/ftp_upload/61172/61172equ01.jpg" /> 
		<img alt="Equation 2" src="/files/ftp_upload/61172/61172equ02.jpg" /> 
		where <img alt="Equation 10" src="/files/ftp_upload/61172/icon01.jpg" /> is the wind speed vector; <img alt="Equation 10" src="/files/ftp_upload/61172/icon02.jpg" /> is the WS in the same direction as the wind vector; <img alt="Equation 10" src="/files/ftp_upload/61172/icon03.jpg" /> and <img alt="Equation 10" src="/files/ftp_upload/61172/icon04.jpg" /> are the WS in the east and north directions, respectively; <img alt="Equation 10" src="/files/ftp_upload/61172/icon05.jpg" /> is the air density (equal to 1.2 kg/m3); and C is the drag coefficient (a value of 0.0015 is used) under neutral stability conditions34.</li>
	</ol>
	</li>
	<li>Monthly averages
	<ol>
		<li>Calculate the monthly SST, wind, and SLA time series as 30-day averages in each pixel by typing Monthly_average to run the script. Due to the high cloud coverage rate, use a 60-day average as the monthly time series for CHL, including 30 days before to 30 days after the 15th day of the month.</li>
	</ol>
	</li>
</ol>
3. SST front detection
<ol>
	<li>Spatial smoothing
	<ol>
		<li>Type Spatial_smoothing to run the script to average the three-day SST data in each pixel. 
		NOTE: A large amount of noise was identified in the SST data. Thus, the data were&#160;smoothed with a 3 x 3 spatial average. When no data were available in the original 3-day averaged data, the spatial averaged data were set as unavailable.</li>
	</ol>
	</li>
	<li>SST gradient
	<ol>
		<li>Type SST_gradient to run the script to calculate the zonal and meridional SST gradients (i.e., Gx and Gy, respectively) as the SST difference between the nearest two pixels divided by the corresponding distance via equation (3). Use the obtained gradient vector to calculate the total gradient, G, as a scalar following equation (4). 
		<img alt="Equation 3" src="/files/ftp_upload/61172/61172equ03.jpg" /> 
		<img alt="Equation 4" src="/files/ftp_upload/61172/61172equ04.jpg" /></li>
	</ol>
	</li>
	<li>Local maximum
	<ol>
		<li>Identify a front by testing a&#160;SST gradient value: label a&#160;pixel as a potential frontal pixel if the value is larger than a designated threshold. Only maintain the local maximum pixel in the same direction perpendicular to the gradient direction if there are connected pixels with values larger than the threshold. Here, define the threshold as 0.035 &#176;C/km following former studies10,28. 
		NOTE: The corresponding script &#8216;Local_maximum.m&#8217; is available in the&#160;Supplemental Files.</li>
	</ol>
	</li>
	<li>Monthly frontal probability (FP) 
	NOTE: The frontal probability (FP) describes the probability of observing a front.
	<ol>
		<li>Calculate the FP for a certain time span (in this case, a&#160;monthly interval), by typing Monthly_FP to run the script. Divide the occurrence of fronts at each pixel during a time window by the number of days that are&#160;free of clouds.</li>
	</ol>
	</li>
</ol>
4. Spatial and temporal variability
<ol>
	<li>Seasonal cycle
	<ol>
		<li>Calculate the seasonal cycles of different factors as the averages of different seasons. Define the seasons as follows: winter is from December to February, spring is from March to May, summer is from June to August, and fall is from September to November. 
		NOTE: The seasonal cycle is not shown in this study; the following method is used to explain the spatial and temporal variability instead.</li>
	</ol>
	</li>
	<li>Empirical orthogonal function (EOF)
	<ol>
		<li>Remove the temporal average and unavailable pixels. Before conducting the EOF, subtract the overall mean at each pixel and exclude the locations where missing observations exceed 20% because of cloud coverage. Load data by typing load(&#39;Monthly_data_for_EOF.mat&#39;) in the command window.</li>
		<li>Apply an EOF to describe the spatial and temporal variabilities of&#160;different parameters. Type Empirical_orthogonal_function.m to run the script to calculate the magnitude (Mag), eigenvalues (Eig), and amplitude (Amp) of the EOFs for the dataset (i.e., time series of monthly averaged SST, wind stress, wind stress curl, CHL, and FP). 
		NOTE: The function decomposes the monthly time series into different modes, which are composed of spatial and temporal patterns and the variance explained by each mode decreases with increasing mode number.</li>
	</ol>
	</li>
</ol>
5. Intercorrelation
<ol>
	<li>Correlation at the seasonal scale
	<ol>
		<li>Calculate the correlations between two factors using their time series at each pixel by typing Seasonal_correlation to run the script. Because the seasonal cycle is not removed, check the significance of the correlation for all correlations.</li>
	</ol>
	</li>
	<li>Correlation of an anomalous field
	<ol>
		<li>Calculate the correlations between the monthly CHL anomalies and other factors, such as SST, WS, fronts, and SLAs. Obtain the monthly anomalies (i.e., the deviation from the mean status) by subtracting the overall average for a&#160;corresponding month from the monthly time series. Type Anomalous_correlation to run the script and obtain the correlations.</li>
	</ol>
	</li>
</ol>
6. Displaying information and calculating relationships
<ol>
	<li>Display satellite information.
	<ol>
		<li>Type Sat_SCS_Fig3457 to run the script to generate a showcase of satellite information, including SST, CHL, and frontal distributions. Set the current folder as &#8216;scripts&#8217; where the data &#8216;Sat_SCS_data.mat&#8217; are located. 
		NOTE: Figure 1, Figure 2, Figure 3, and Figure 4 show SST, CHL, fronts, wind, and topography for the selected date as an example.</li>
	</ol>
	</li>
	<li>Display the EOF result by typing Sat_SCS_Fig890.m to run the script. 
	NOTE: Figure 5, Figure 6, and Figure 7 describe the spatial magnitude, monthly average, and time series of first two modes for CHL, SST, and fronts, respectively.</li>
	<li>Calculate the relationship between CHL&#160;and other factors at seasonal timescales and for anomalous fields by typing Sat_SCS_Fig1112.m to run the script. Obtain the correlation map for seasonal variabilities&#160;(Figure 8) and anomalies&#160;(Figure 9).</li>
</ol>

The authors have nothing to disclose.

In this study, the major features of marine systems are described using satellite observations. The CHL, which can be used to represent ocean production, is selected as an indicator factor. Factors related to CHL variability were investigated using monthly averaged time series, e.g., SST, WS, WSC, FP and SLA. Three critical steps are described in this study: acquiring satellite data for different parameters, describing their spatial and temporal variabilities via EOF, and determining interrelationships among different factors by calculating correlation coefficients. A detailed procedure showing the identification for daily frontal distribution, which is derived from the SST observations, is included. Two major approaches have been developed for SST front detection: the gradient method10,38 and the histogram method39,40. The histogram method is based on a similar range of values for SST, which can be used to divide the water masses into different groups. The pixels with values between different groups representing the pixel in a transitional band are defined as fronts. On the other hand, the gradient method separates several relatively uniform water bodies as pixels with large gradient values. A comparison study was conducted, and they found lower false rates using the histogram method and fewer missed fronts using the gradient method41. In this study, the gradient-based method38 was adopted following former studies10,28. The algorithm can avoid front break-up into multiple edge fragments by allowing the magnitude to decrease to a level below a smaller threshold. In addition to the dataset included here, other satellite observations, such as the aerosol index, can also be used with a similar approach.
Most of the procedures can be directly applied in other regions or datasets. Modification may take place to change the threshold of front detection. Because the SST gradient in the SCS is comparable with the Eastern Boundary Current System28, the same thresholds were implemented for the current study. A previous study revealed that the SST gradient from different datasets can vary as much as three times42, which makes the method somehow less objective. Substantial studies have investigated frontal activities around the global oceans28,43. The best approach to validate fronts is to compare them with in situ observations. Yao44 described the monthly frontal distribution for the SCS. Their results agreed well with the in situ measurements. The overall gradient should be checked and adjusted since its value may vary depending on the spatial resolution and instruments. In particular, the threshold should be updated when another SST dataset is used. A basic understanding of the regional dynamics is fundamental to understanding frontogenesis45,46,47. The front detection script can be developed by individual authors based on the description in this paper.
Satellite information offers a comprehensive understanding of surface features, and a results comparison with in situ observations can aid in evaluating credibility. However, satellite observations are limited to the ocean surface, which limits the application for understanding the vertical structure of the water column. In a recent study, satellite observations revealed that the surface CHL increased by 15 times, but the vertical integrated value only increased by 2.5 times48. This difference was because the surface value was impacted by the coeffects of phytoplankton growth and shoaling of MLD, resulting in an unrealizable value at the surface. Thus, the surface feature may not offer an accurate description for the entire water column. Additionally, the influence of cloud coverage limits the continuous observations of satellites. Thus, monthly time series are calculated for different factors over the same region and same period. This will guarantee the credibility of calculating the correlations among different factors. However, the short-period events, e.g., typhoons that last for a few days to a week, will not be resolved.
Compared with former studies, the proposed method can offer spatial information at the pixel level, which can help to evaluate the dynamics in a more detailed manner. Some former studies averaged the entire SCS as a single number and obtained a time series. They found that an unusually strong WS and high SST can induce anomalously high CHL16, which is consistent with the current result. However, the spatial variation in the relationships was not resolved. In this study, the basin-scale correlation between WS and CHL was weak in the anomalous field. A large significant correlation was only identified for certain areas, e.g., in the center of the SCS (Figure 9B). Thus, the current method offers a comprehensive description for investigating spatial variations. Similarly, observations from two Bio-Argo floats were used and revealed that WSC did not correlate with CHL variability20. However, the trajectories of the two floats are only located in certain regions. In this case, it was exactly within the band where the correlation between the CHL level and the WSC was not significant (Figure 8D). The proposed method is very helpful for resolving the spatial dependence among factors, which is a fundamental characteristic of the global ocean.
In summary, the method used here can accurately describe the spatial distribution and temporal variability in ocean surface features using satellite observations. With the increasing resolution of satellite datasets, more detailed features can be identified and investigated, which enables a general understanding of regional features, including CHL, SST, and SSH. The correlation of monthly time series among different factors can aid in understanding their dynamic relationships and potential impact on an ecosystem49. Because the correlation can largely vary at different spatial locations, the proposed method offers a detailed and comprehensive description. A similar approach can be applied to any ocean basin worldwide, which will be greatly helpful to improve the understanding of marine dynamics and ecosystems.

Remote sensing technology offers great datasets with large spatial scales and long periods for describing marine environments. With the increasing spatial resolution of satellites, detailed features are now resolved from the regional scale to a few hundred meters1,2. An improved understanding of marine dynamics can be achieved with most updated satellite observations3.
By incorporating multiple sensors on a remote sensing platform, a comprehensive description of different parameters is possible. Sea surface temperature (SST) is the basic parameter that has been observed for more than half a century4. Recently, observations for sea surface chlorophyll-a (CHL) have become available and can be used to describe marine productivity5. Altimetry satellites are used for measuring sea surface height6,7, which is strongly related to mesoscale eddy activities in the global ocean8,9. In addition to eddies, frontal activities are also important for impacting regional dynamics and primary production10.
The major focus of the current study is to find a standard procedure to describe the spatial distribution and temporal variabilities of different ocean factors. In this method,&#160;SST, CHL, SSH, and front-data, which are derived from SST gradients, are analyzed to determine patterns. In particular, the CHL is used to represent the productivity of the ocean, and a method is introduced to investigate the relationship between CHL and other ocean parameters. To validate the method, the time period between October 2002 and September 2017 in the South China Sea was used to examine all parameters. The method can be easily used for other regions around the globe to capture major ocean patterns and explore how marine dynamics impact the ecosystem.
The South China Sea (SCS) was designated as the study region because of its relatively high coverage rate of satellite observations. The SCS is abundant in solar radiation; thus, the CHL is mainly determined by the availability of nutrients11,12. With more nutrients being transported into the euphotic layer, CHL levels can increase13. Mixing, induced by wind, can introduce nutrients into the ocean surface and enhance CHL14. The SCS is uniquely dominated by a monsoon wind system, which determines the dynamics and ecosystem in the region. The monsoon wind is strongest during winter15. In summer, the winds change direction and the wind speeds are much weaker than those in winter16,17. The wind intensity can determine the strength of vertical mixing, such that the mixed layer depth (MLD) deepens as the wind increases in winter and becomes shallower as the wind decreases in summer18. Thus, more nutrients are transported into the euphotic layer during winter when the wind is strong19 and CHL reaches its highest point of the year20,21.
In addition to the wind, the MLD can also be determined using other factors, such as SST and sea level anomalies&#160;(SLAs), which ultimately impact nutrient content and CHL22. During winter, the weak vertical gradient is associated with low temperatures at the surface20. The corresponding MLD is deep and more nutrients can be transported upward; thus, the CHL in the surface layer is high17. An increasing variation in CHL levels can be attributed to mesoscale eddies, which induce vertical transport and mixing23. Upwelling is usually found in cyclonic eddies associated with depressed SLAs8,9 and elevated CHL concentrations24. Downwelling is usually found in anticyclonic eddies associated with elevated SLAs8,9 and depressed CHL concentrations24. For other seasons, the MLD becomes shallow, and mixing becomes weak; thus, low CHL can be observed over the majority of the basin25. The seasonal cycles of CHL levels are subsequently predominant for the region26.
In addition to mixing, fronts and their associated coastal upwelling can further modulate the CHL. The front, which is defined as a boundary of different water masses, is important to determine the regional circulation and ecosystem responses27. Frontogenesis is usually associated with coastal upwelling and convergence28,29, which can induce nutrients and elevate the growth of phytoplankton30. Different algorithms have been developed to automatically identify fronts from satellite observations, including histogram and SST gradient methods. The latter approach is adopted in this study28.
The correlation of time series between CHL and different factors offers great insights for quantifying their relationship. The current study offers a comprehensive description of how to use satellite observations to reveal regional marine dynamics related to productivity. This description can be used as a guide for investigating the surface processes in any part of the ocean. The structure of this article includes a step-by-step protocol, followed by descriptive results in the text and figures. The applicability in addition to the pros and cons of the method are subsequently discussed.

<table>
	<tbody>
		<tr>
			<td>Matlab</td>
			<td>MathWorks</td>
			<td>Matlab R2016</td>
			<td>https://www.mathworks.com/products/matlab.html; referred to analysis software in the protocol</td>
		</tr>
		<tr>
			<td>Sea surface chlorophyll</td>
			<td>NASA</td>
			<td>MODIS</td>
			<td>mg/mg3 (podaac-tools.jpl.nasa.gov)</td>
		</tr>
		<tr>
			<td>Sea surface height</td>
			<td>AVISO</td>
			<td>AVISO</td>
			<td>meter (www.aviso.altimetry.fr)</td>
		</tr>
		<tr>
			<td>Sea surface temperature</td>
			<td>NASA</td>
			<td>MODIS</td>
			<td>&#176;C (podaac-tools.jpl.nasa.gov)</td>
		</tr>
		<tr>
			<td>Topography</td>
			<td>NOAA</td>
			<td>NGDC</td>
			<td>meter (maps.ngdc.noaa.gov/viewers/wcs-client/)</td>
		</tr>
		<tr>
			<td>Wind</td>
			<td>ECMWF</td>
			<td>ERA-interim</td>
			<td>m/s (www.ecmwf.int/en/forecasts/datasets)</td>
		</tr>
	</tbody>
</table>

investigating the relationship between sea surface chlorophyll and major features of the south china sea with satellite information

Satellite observations offer a great approach to investigate the features of major marine parameters, including sea surface chlorophyll (CHL), sea surface temperature (SST), sea surface height (SSH), and factors derived from these parameters (e.g., fronts). This study shows a step-by-step procedure to use satellite observations to describe major parameters and their relationships in seasonal and anomalous fields. This method is illustrated using satellite datasets from 2002&#8211;2017 that were used to describe the surface features of the South China Sea (SCS). Due to cloud coverage, monthly averaged data were used in this study. The empirical orthogonal function (EOF) was applied to describe the spatial distribution and temporal variabilities of different factors. The monsoon wind dominates the variability in the basin. Thus, wind from the reanalysis dataset was used to investigate its driving force on different parameters. The seasonal variability in CHL was prominent and significantly correlated with other factors in a majority of the SCS. In winter, a strong northeast monsoon induces a deep mixed layer and high level of chlorophyll throughout the basin. Significant correlation coefficients were found among factors at the seasonal cycle. In summer, high CHL levels were mostly found in the western SCS. Instead of a seasonal dependence, the region was highly dynamic, and factors correlated significantly in anomalous fields such that unusually high CHL levels were associated with abnormally strong winds and intense frontal activities. The study presents a step-by-step procedure to use satellite observations to describe major parameters and their relationships in seasonal and anomalous fields. The method can be applied to other global oceans and will be helpful for understanding marine dynamics.

Watch this Scientific Journal Video about Investigating the Relationship between Sea Surface Chlorophyll and Major Features of the South China Sea with Satellite Information at JoVE.com

Investigating the Relationship between Sea Surface Chlorophyll and Major Features of the South China Sea with Satellite Information

Sea surface chlorophyll, temperature, sea level height, wind, and front data obtained or derived from satellite observations offer an effective way to characterize the ocean. Presented is a method for the comprehensive study of these data, including overall average, seasonal cycle, and intercorrelation analyses, to fully understand regional dynamics and&#160;ecosystems.

Satellite observations offer a great approach to investigate the features of major marine parameters, including sea surface chlorophyll (CHL), sea surface ...

investigating-relationship-between-sea-surface-chlorophyll-major

Research

JoVE Journal

Environment

5.6K Views.  Hohai University. Sea surface chlorophyll, temperature, sea level height, wind, and front data obtained or derived from satellite observations offer an effective way to characterize the ocean. Presented is a method for the comprehensive study of these data, including overall average, seasonal cycle, and intercorrelation analyses, to fully understand regional dynamics and ecosystems.

Video: Investigating the Relationship between Sea Surface Chlorophyll and Major Features of the South China Sea with Satellite Information

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and organiclastic sulfate reduction (OSR) in marine sediments. However, unravelling the complex pyritization sequence is a challenge because of the coexistence of different sequentially formed pyrite phases. This manuscript describes a sample preparation procedure that enables the use of secondary ion mass spectroscopy (SIMS) to obtain in situ &#948;34S values of various pyrite generations. This allows researchers to constrain how SO4-AOM affects pyritization in methane-bearing sediments. SIMS analysis revealed an extreme range in &#948;34S values, spanning from -41.6 to +114.8&#8240;, which is much wider than the range of &#948;34S values obtained by the traditional bulk sulfur isotope analysis of the same samples. Pyrite in the shallow sediment mainly consists of 34S-depleted framboids, suggesting early diagenetic formation by OSR. Deeper in the sediment, more pyrite occurs as overgrowths and euhedral crystals, which display much higher SIMS &#948;34S values than the framboids. Such 34S-enriched pyrite is related to enhanced SO4-AOM at the sulfate-methane transition zone, postdating OSR. High-resolution in situ SIMS sulfur isotope analyses allow for the reconstruction of the pyritization processes, which cannot be resolved by bulk sulfur isotope analysis.

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Analyses of the sulfur isotopic composition (&#948;34S) of pyrite from methane-bearing sediments have typically focused on bulk samples. Here, we applied secondary ion mass spectroscopy to analyze the &#948;34S values of various pyrite generations to understand the diagenetic history of pyritization.

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and ...

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

Techniques for Investigating the Anatomy of the Ant Visual System

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye anatomy of insects. These include traditional histological techniques optimized for work on ant eyes and adapted to work in concert with other techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM). These techniques, although vastly useful, can be difficult for the novice microscopist, so great emphasis has been placed in this article on troubleshooting and optimization for different specimens. We provide information on imaging techniques for the entire specimen (photo-microscopy and SEM) and discuss their advantages and disadvantages. We highlight the technique used in determining lens diameters for the entire eye and discuss new techniques for improvement. Lastly, we discuss techniques involved in preparing samples for LM and TEM, sectioning, staining, and imaging these samples. We discuss the hurdles that one might come across when preparing samples and how best to navigate around them.

This article outlines a suite of techniques in light and electron microscopy to study the internal and external eye anatomy of insects. These include several traditional techniques optimized for work on ant eyes, detailed troubleshooting, and suggestions for optimization for different specimens and regions of interest.

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye ...

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf mottling, vein yellowing, defoliation, root decay, and ultimately, the death of the citrus plant. When infected, the citrus plants have stunted growth and produce flowers out of season. These flowers rarely yield fruit, and those that do yield small, bitter, irregularly shaped citrus fruit that are not desirable. This disease is spread by the Asian citrus psyllid, Diaphorina citri, and by the grafting of infected citrus tissue. The pathogen has a long and variable incubation period within the citrus plant&#8212;sometimes years, before symptoms appear. Attempts to culture this pathogen in vitro have been unsuccessful, possibly due to the low and uneven concentration of the pathogen within infected citrus tissue, or because it is difficult to replicate the environmental conditions conducive to growth of the pathogen. It is very difficult to identify the disease before it has spread, due to its long incubation period and researchers&#39; inability to culture the pathogen. As a result, the disease only becomes apparent after suddenly destroying a citrus farmer&#39;s entire yield. Presented here is a method for the accurate and specific detection of the citrus greening pathogen, Candidatus liberibacter spp. using a genomic DNA extraction kit and PCR. This method is simple, efficient, cost effective, and adaptable for quantitative analysis. This method can be adapted for use on any citrus tissue; however, it is potentially limited by the amount of pathogen present in the tissue. Nevertheless, this method will allow citrus farmers to identify infected citrus plants earlier, and curb the spread of this destructive disease before it can further spread.

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus Greening is a particularly destructive disease affecting citrus crops globally. Presented here is a simple method using PCR and genomic DNA extraction of citrus leaf tissue for the accurate and precise identification of the citrus greening pathogen, Candidatus liberibacter spp.

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf ...

Methods for Image-based Surveys of Benthic Macroinvertebrates and Their Habitat Exemplified by the Drop Camera Survey for the Atlantic Sea Scallop

Underwater imaging has long been used in the field of marine ecology but decreasing costs of high-resolution cameras and data storage have made the approach more practical than in the past. Image-based surveys allow for initial samples to be revisited and are non-invasive compared to traditional survey methods that typically involve nets or dredges. Protocols for image-based surveys can vary greatly but should be driven by target species behavior and survey objectives. To demonstrate this, we describe our most recent methods for an Atlantic sea scallop (Placopecten magellanicus) drop camera survey to provide a procedural example and representative results. The procedure is divided into three critical steps that include survey design, data collection, and data products. The influence of scallop behavior and the survey goal of providing an independent assessment of the U.S. sea scallop resource on the survey procedure are then discussed in the context of generalizing the method. Overall, the broad applicability and flexibility of the University of Massachusetts Dartmouth School for Marine Science and Technology (SMAST) drop camera survey demonstrates the method could be generalized and applied to a variety of sessile invertebrates or habitat focused research.

Image based surveying is an increasingly practical, non-invasive method to sample the marine environment. We present the protocol of a drop camera survey that estimates the abundance and distribution of the Atlantic sea scallop (Placopecten magellanicus). We discuss how this protocol can be generalized for application to other benthic macroinvertebrates.

Underwater imaging has long been used in the field of marine ecology but decreasing costs of high-resolution cameras and data storage have made the ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Experimental Study of the Relationship Between Particle Size and Methane Sorption Capacity in Shale

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard for evaluating the mining value of shale gas. Currently, studies on the correlation between particle size and methane adsorption are controversial. In this study, an isothermal adsorption apparatus, the gravimetric sorption analyzer, is used to test the adsorption capacity of different particle sizes in shale to determine the relationship between the particle size and the adsorption capacity of shale. Thegravimetric method requires fewer parameters and produces better results in terms of accuracy and consistency than methods like the volumetric method. Gravimetric measurements are performed in four steps: a blank measurement, preprocessing, a buoyancy measurement, and adsorption and desorption measurements. Gravimetric measurement is presently considered to be a more scientific and accurate method of measuring the amount of adsorption; however, it is time-consuming and requires a strict measuring technique. A Magnetic Suspension Balance (MSB) is the key to verify the accuracy and consistency of this method. Our results show that adsorption capacity and particle size are correlated, but not a linear correlation, and the adsorptions in particles sieved into 40 - 60 and 60 - 80 meshes tend to be larger. We propose that the maximum adsorption corresponding to the particle size is approximately 250 &#181;m (60 mesh) in the shale gas fracturing.

We use an isothermal adsorption apparatus, the gravimetric sorption analyzer, to test the adsorption capacity of different particle sizes of shale, in order to find out the relationship between particle size and the adsorption capacity of shale.

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard ...

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

Many insects live above the ground as larvae and adults and as pupate below the ground. Compared to the above-ground stages of their life cycles, less attention has been paid on how environmental factors affect these insects when they pupate within the soil. The tea looper, Ectropis grisescens Warren (Lepidoptera: Geometridae), is a severe pest of tea plants and has caused huge economic losses in South China. The protocols described here aim to investigate, through multiple-choice bioassays, whether mature last-instar E. grisescens larvae can discriminate soil variables such as the substrate type and moisture content, and determine, through no-choice bioassays, the impact of the substrate type and moisture content on pupation behaviors and the emergence success of E. grisescens. The results would enhance the understanding of the pupation ecology of E. grisescens and may bring insights into soil-management tactics for suppressing E. grisescens populations. In addition, these bioassays can be modified to study the influences of various factors on the pupation behaviors and survivorship of soil-pupating pests.

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

Here, we present a protocol to investigate the pupation preference of mature larvae of Ectropis grisescens in response to soil factors (e.g., substrate type and moisture content) using choice bioassays. We also present a protocol of no-choice bioassays to determine the factors that affect the pupation behaviors and survivorship of E. grisescens.

Many insects live above the ground as larvae and adults and as pupate below the ground. Compared to the above-ground stages of their life cycles, less ...

Design and Use of an Apparatus for Quantifying Bivalve Suspension Feeding at Sea

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves (i.e., mussels, oysters, and clams) presents new challenges. Quantitative data on the feeding behavior of suspension-feeding mollusks is necessary to determine important ecosystem interactions of offshore shellfish farms, including their carrying capacity, the competition with the zooplankton community, the availability of trophic resources at different depths, and the deposition to the benthos. The biodeposition method is used to quantify feeding variables in suspension-feeding bivalves in a natural setting and represents a more realistic proxy than laboratory experiments. This method, however, relies upon a stable platform to satisfy the requirements that water flow rates supplied to the shellfish remain constant and the bivalves are undisturbed. A flow-through device and process for using the biodeposition method to quantify the feeding of bivalve mollusks were modified from a land-based format for shipboard use by building a two-dimensional gimbal table around the device. Planimeter data reveal a minimal pitch and yaw of the chambers containing the test shellfish despite boat motion, the flow rates within the chambers remain constant, and operators are able to collect the biodeposits (feces and pseudofeces) with sufficient consistency to obtain accurate measurements of the bivalve clearance, filtration, selection, ingestion, rejection, and absorption at offshore shellfish aquaculture sites.

A flow-through device for using the biodeposition method to quantify filtration and feeding behavior of bivalve mollusks was modified for shipboard use. A two-dimensional gimbal table built around the device isolates the apparatus from boat motion, thereby allowing the accurate quantification of bivalve filtration variables at offshore shellfish aquaculture sites.

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves ...