I would like to acknowledge the many researchers, students, teachers, and citizen scientists who have tested and helped me improve this protocol over the past several years: Dr. Gabriela Bradt, Dr. Robert Steneck, Erica Ferrelli, Ethel Wilkerson, Susan Ayers and the Georgetown Central School 3rd and 4th grade students, Julie Upham and the West Bath Elementary 4th grade students, the GMRI Vital Signs team, and the Idexx Laboratories Community Service Volunteers. I thank Anne Hayden, Caitlin Cleaver, and Hannah Webber for providing comments and suggestions on this manuscript. I thank the following funding sources for supporting the development and execution of this protocol: NOAA Saltonstall-Kennedy Grant Program (Grant #NA18NMF4270194), Maine Sea Grant, Robert and Patricia Switzer Foundation, and the Georgetown Island Education Fund. Finally, this manuscript was greatly improved thanks to comments and edits provided by three anonymous reviewers.

1. Timing of survey work
<ol>
	<li>Conduct surveys between May-November, during the height of productivity in the intertidal zone.</li>
	<li>Schedule surveys around negative or zero tides (generally new and full moon cycles) to allow for adequate time in the low intertidal zone (i.e., at least 2 hours).</li>
</ol>
2. Pre-survey preparation
<ol>
	<li>Locate and print all field guides and data collection sheets (see Appendices) prior to conducting the survey if that is the preferred method. If using the Anecdata app for field guides and data collection, visit the Anecdata website and join the Intertidal Green Crab Project19. The data collection sheet and Anecdata collection categories are identical.</li>
</ol>
3. Site selection and description
<ol>
	<li>Locate a wave-sheltered rocky intertidal site with cobble (i.e., not stable, rolled by wave action) and algal canopy habitat. Ensure that there is at least 100 m of shoreline to accommodate the planned sampling.</li>
	<li>Record the location of the study site using a global positioning system (GPS) unit, or a device such as a smart phone that has GPS capability (e.g., many compass apps are free to download or are already pre-programmed on smart phones). Record site coordinates on the Intertidal Survey Data Sheet (Appendix 3) or directly into the Intertidal Green Crab Project on Anecdata.</li>
	<li>At the predicted low tide time (determined from the NOAA Tide Predications website or an app such as Tides) run a 50 m transect tape vertically from the low intertidal zone (i.e., the splash zone) to the high intertidal zone (i.e., the black microalgal zone that is typically dry at high tide). Divide the resulting distance into three equal sections: high, middle, and low (Figure 1). The low intertidal section, parallel to the shoreline, is the target sampling area.</li>
	<li>Within the low intertidal zone, measure a distance of 100 m parallel to the shoreline (Figure 2) and establish permanent markers delineating this zone using rebar or natural permanent landmarks such as immovable boulders, ledge, dock pilings, etc.</li>
</ol>
4. Conducting survey
<ol>
	<li>Prior to arriving at the survey site, record the following information on the Intertidal Survey Data Sheet (Appendix 3) or directly into the Intertidal Green Crab Project on Anecdata: site name, sampling date, participants, time and height of low tide at the location/date to be sampled (determined using the NOAA Tide Predications website, or an app such as Tides), and lunar phase (determined using a lunar calendar such as www.moongiant.com).</li>
	<li>Upon arriving at the survey site, locate the 100 m section of low intertidal shoreline where the survey will be conducted, unpack gear, and organize data sheets and field guides.</li>
	<li>Optionally, measure water temperature using a waterproof digital thermometer in the shallow water adjacent to the sampling area.
	<ol>
		<li>Measure salinity by placing several drops of water collected adjacent to the sampling area onto the refraction prism of a salinity refractometer.</li>
		<li>Record water temperature in &#176;C and salinity in parts per thousand (ppt) on the Intertidal Survey Data Sheet or directly into the Intertidal Green Crab Project on the Anecdata app.</li>
	</ol>
	</li>
	<li>Begin the survey by haphazardly tossing the 1 m2 quadrat within the predefined low intertidal zone area that runs parallel to the shoreline (a transect tape is not needed to conduct the survey because the sample area has already been defined). Record a visual estimate of the percent of both moveable rock (i.e., cobble/gravel that you can look underneath) and algae canopy cover (e.g. Ascophyllum or Fucus spp.) within the quadrat to the nearest quarter percent (i.e., 0, 25, 50, 75, or 100%). A rocky intertidal habitat is often patchy and can contain areas of sand, mud, ledge, or other habitats where green crabs are not found.
	<ol>
		<li>To avoid skewing density estimates by sampling unsuitable habitat, only sample quadrats with greater than 50% movable rock, or greater than 50% algal canopy. Also avoid sampling areas where boulders or ledges are noticeably elevated above the profile of the shoreline within the low intertidal zone, as this habitat may be more representative of the mid intertidal zone.</li>
	</ol>
	</li>
	<li>Within each quadrat, lift moveable rocks or cobble and carefully move aside algae to look for crabs. Be sure to replace all rocks and algae as they are found. Collect all of the crabs found and store them in a bucket until the entire quadrat has been searched.</li>
	<li>Identify the species of each crab using the Intertidal Crab Field Guide (Appendix 1, or source on Anecdata project platform) and record using the species codes listed on the Intertidal Survey Data Sheet (Appendix 3) or on the Intertidal Green Crab Project on the Anecdata app.</li>
	<li>Measure the carapace width (CW) of each crab across the widest part of the carapace, spanning from tip to tip of the terminal spines, to the nearest 1 mm using Vernier calipers.</li>
	<li>Use the abdomen (or &#8216;apron&#8217;) on the ventral side of the crab to determine sex. Male crabs tend to have a narrow, pointed abdomen and female crabs tend to have a wider, beehive shaped abdomen (Appendix 1). Only record sex for crabs &#8805; 10 mm CW.</li>
	<li>For all crabs, record number of claws, number of legs, shell condition (i.e., hard- or soft-shell as determined by whether the carapace resists (hard) or gives (soft) when finger pressure is applied), and the presence (i.e., ovigerous) or absence of extruded eggs for females.</li>
	<li>Optionally, record the color for green crabs, but not other crab species, using the color protocol developed by Young and Elliot20 (Appendix 2). This protocol should only be used if the actual paint chips can be sourced and brought into the field, as printed versions can vary substantially. Identify pre-molt shell condition for green crabs using external pre-molt indicators (Appendix 4, or source on Anecdata project platform). Pre-molt green crabs are within 3 weeks of molting and are of particular interest to the emerging soft-shell green crab fishery21,22.</li>
	<li>Return all crabs to the habitat within the quadrat once all measurements and characteristics have been recorded.</li>
	<li>Continue haphazardly tossing the quadrat within the predefined low intertidal area until a total of 10 m2 is sampled. Continually move forward along the low intertidal area of shoreline and ensure that quadrats are separated by a minimum of 1 m so that resampling does not occur and a maximum of 10 m so that the survey area does not exceed 100 m.</li>
</ol>
5. Data management and analysis
<ol>
	<li>If using data sheets, check all raw data sheets for errors and legibility post-survey, photocopy, scan, and archive. Use the photocopy for data entry into an excel spreadsheet (see Appendix 5 for example) or into the Intertidal Green Crab Project on Anecdata19. Store scanned data sheets electronically.</li>
	<li>Conduct data analyses appropriate to the design of the study. Useful population metrics include crab density (total number of crabs divided by total number of quadrats sampled), sex ratio, cumulative size frequency, injury rate, shell condition ratio, and overall species encounter rates (e.g., % of native vs. invasive crabs).</li>
</ol>

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M., McKie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economics+of+harmful+invasive+species:+A+review.">Economics of harmful invasive species: A review.</a> Diversity. 6, 500-523 (2014).</li><li>Kamenova, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasions+toolkit:+current+methods+for+tracking+the+spread+and+impact+of+invasive+species.">Invasions toolkit: current methods for tracking the spread and impact of invasive species.</a> Advances in Ecological Research 2017. 56, 85-182 (2017).</li><li>Carlton, J. T., Cohen, A. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+ecology+of+the+green+crab,+Carcinus+maenas+(L.+1758)+in+a+temperate+estuary,+Portugal.">Feeding ecology of the green crab, Carcinus maenas (L. 1758) in a temperate estuary, Portugal.</a> Crustaceana. 79, 1181-1193 (2006).</li><li>Pickering, T., Quij&#243;n, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+effects+of+a+non-indigenous+predator+in+its+expanded+range:+assessing+green+crab,+Carcinus+maenas,+prey+preference+in+a+productive+coastal+area+of+Canada.">Potential effects of a non-indigenous predator in its expanded range: assessing green crab, Carcinus maenas, prey preference in a productive coastal area of Canada.</a> Marine Biology. 158, 2065-2078 (2011).</li><li>Rossong, M. A., Williams, P. J., Comeau, M., Mitchell, S. C., Apaloo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agonistic+interactions+between+the+invasive+green+crab,+Carcinus+maenas+(Linnaeus)+and+juvenile+American+lobster,+Homarus+americanus+(Milne+Edwards).">Agonistic interactions between the invasive green crab, Carcinus maenas (Linnaeus) and juvenile American lobster, Homarus americanus (Milne Edwards).</a> Journal of Experimental Marine Biology and Ecology. 329, 281-288 (2006).</li><li>Rossong, M. A., Quijon, P. A., Williams, P. J., Snelgrove, P. V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foraging+and+shelter+behavior+of+juvenile+American+lobster+(Homarus+americanus):+the+influence+of+a+non-indigenous+crab.">Foraging and shelter behavior of juvenile American lobster (Homarus americanus): the influence of a non-indigenous crab.</a> Journal of Experimental Marine Biology and Ecology. 403, 75-80 (2011).</li><li>Matheson, K., Gagnon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+temperature,+body+size,+and+chela+loss+on+competition+for+a+limited+food+resource+between+indigenous+rock+crab+(Cancer+irroratus+Say)+and+recently+introduced+green+crab+(Carcinus+maenas+L).">Effects of temperature, body size, and chela loss on competition for a limited food resource between indigenous rock crab (Cancer irroratus Say) and recently introduced green crab (Carcinus maenas L).</a> Journal of Experimental Marine Biology and Ecology. 428, 49-56 (2012).</li><li>Davis, R. C., Short, F. T., Burdick, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+effects+of+green+crab+damage+to+eelgrass+transplants.">Quantifying the effects of green crab damage to eelgrass transplants.</a> Restoration Ecology. 6, 297-302 (1998).</li><li>Garbary, D. J., Miller, A. G., Williams, J., Seymour, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drastic+decline+of+an+extensive+eelgrass+bed+in+Nova+Scotia+due+to+the+activity+of+the+invasive+green+crab+(Carcinus+maenas).">Drastic decline of an extensive eelgrass bed in Nova Scotia due to the activity of the invasive green crab (Carcinus maenas).</a> Marine Biology. 161, 3-15 (2014).</li><li>Congleton, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+Maine+softshell+clam+landings.">Trends in Maine softshell clam landings.</a> Journal of Shellfish Research. 25, 475-480 (2006).</li><li>Pershing, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+adaptation+in+the+face+of+rapid+warming+leads+to+collapse+of+the+Gulf+of+Maine+cod+fishery.">Slow adaptation in the face of rapid warming leads to collapse of the Gulf of Maine cod fishery.</a> Science. 350, 809-812 (2015).</li><li>Young, A. M., Elliott, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+history+and+population+dynamics+of+green+crabs+(Carcinus+maenas).">Life history and population dynamics of green crabs (Carcinus maenas).</a> Fishes. 5, (2019).</li><li>Young, A. M., Elliott, J. A., Incatasciato, J. M., Taylor, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seasonal+catch,+size,+color,+and+assessment+of+trapping+variables+for+the+European+green+crab+Carcinus+maenas+(Brachyura:+Portunoidea:+Carcinidae),+a+nonindigenous+species+in+Massachusetts,+USA.">Seasonal catch, size, color, and assessment of trapping variables for the European green crab Carcinus maenas (Brachyura: Portunoidea: Carcinidae), a nonindigenous species in Massachusetts, USA.</a> Journal of Crustacean Biology. 37, 556-570 (2017).</li><li>St-Hilaire, S., Krause, J., Wright, K., Poirier, L., Singh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Break-even+analysis+for+a+green+crab+fishery+in+PEI,+Canada.">Break-even analysis for a green crab fishery in PEI, Canada.</a> Management of Biological Invasions. 7, 297-303 (2016).</li><li>Poirier, L. 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The author has nothing to disclose.

This protocol describes a survey method for assessing spatial and temporal trends of crab populations in the rocky intertidal zone that is accessible to multiple users including researchers, educators, students, and citizen scientists. The benefits of this protocol include the following: it does not require specialized or expensive equipment, the methodology is approachable for a wide range of skill levels (e.g., 3rd and 4th grade students have successfully used it), and it can easily be adapted to suit the needs of the investigator. In addition, the optional use of Anecdata allows for observations to be recorded directly into a data storage platform and accessed by a broad array of users. While the species of interest here is the invasive European green crab, this protocol could be used for any number of intertidal species. However, it is important to carefully consider unique characteristics and behavior of each species targeted when generalizing this protocol.
Many citizen scientist and education initiatives that conduct shoreline surveys for green crabs do not utilize a standard unit of measurement (e.g., quadrat) or target a specific area of the shoreline (e.g., low intertidal zone), making spatial and temporal comparisons difficult. This protocol utilizes simple techniques to target a standardized area along the shoreline. However, this is also a limitation of the protocol in that more rigorous methods or specialized equipment would yield more exact tidal height measurements within the survey area. The survey area is defined as 100 m of shoreline in the low intertidal, but the site should further be defined as a section of shoreline delimited by natural or manmade boundaries that are easy to distinguish, or by a minimum boundary distance of 300 m from the survey area. This is an important delineation if investigators are considering surveying multiple sites in close proximity.
The protocol also includes collecting detailed measurements that can be useful for researchers and managers. It is important to note that not all measurements will be appropriate for all users, such as identifying pre-molt green crabs, which takes a considerable amount of training. Furthermore, sex identification is only recommended for crabs &#8805; 10 mm CW, as the sex of smaller juveniles can be difficult to determine. Although it is not part of the protocol, it is also recommended that citizen scientists, students, educators, and others who may not be familiar with the target species utilize a digital camera to document uncertainties and allow for troubleshooting outside of the survey timeframe or to upload to the Anecdata platform for review.
As ocean temperatures continue to warm23,24 and become more favorable for green crab populations in the Gulf of Maine and Atlantic Canada15,16, assessing their distribution and abundance will be critical for timely mitigation and adaptation strategies. For example, recent efforts to develop a soft-shell green crab fishery that would allow coastal communities to benefit from green crabs have relied on population monitoring data to determine when and where to target pre-molt and soft-shell crabs21,22. Bounty programs and other removal efforts may also benefit from distribution and abundance data that can aid in determining how best to allocate time and resources. Similarly, municipal shellfish management programs often conduct yearly conservation activities such as replenishing local mudflats with hatchery raised clam seed and local trends of green crab distribution and abundance could inform best practices for these conservation measures.
Finally, this protocol has proven to be an effective outreach and education tool for students and citizen scientists. In addition to the 3rd and 4th grade students who have been using the protocol for several years, it has also been used with undergraduate students from the University of Southern Maine, and community service volunteers from Idexx Laboratories. Furthermore, an adapted version of this protocol is now being used by the Gulf of Maine Research Institute&#8217;s Vital Signs Program, which partners with Maine students and citizen scientists to monitor invasive species. Ultimately, the data collected using this protocol has a wide range of uses, including to inform ecological research, conservation efforts, mitigation strategies, and fishery development, as well as for educational outreach purposes.

Biological invasions can potentially disrupt species interactions and ecological processes, and may have far reaching ecological1,2,3 and economic consequences4. The ability to successfully predict, mitigate, and adapt to invasions strongly depends on characterizing spatiotemporal population dynamics5. While a range of tools exist (e.g., population genetics, stable isotopes) and are emerging (e.g., eDNA) for tracking invasive species, traditional in situ monitoring techniques continue to be widely utilized for assessing invasive species distribution and abundance.
The European green crab (Carcinus maenas) is an invasive species that was first detected in North America in 1817 and has successfully invaded ecosystems worldwide6,7. Green crabs have a multitude of negative impacts on local ecosystems, including reducing native bivalve populations through predation8,9, competing with native crustaceans for food and shelter10,11,12 and destruction of eel grass habitat and subsequent changes to fish community structure12,13,14. Compounding these issues is the link between increasing temperature and increasing green crab abundance and/or range expansion15,16, which has had severe ecological and socio-economic consequences in areas such as the Gulf of Maine, where warming is occurring faster than 99% of the world&#8217;s other oceans17.
On the eastern seaboard of North America, green crabs range from Virginia to Newfoundland. They are most commonly found on wave-protected shorelines, estuaries, and embayments in depths ranging from the high tide level to 5-6 m18. Their presence in the intertidal zone makes them an ideal marine species for shoreline surveys. The most distinguishing characteristic used to identify green crabs is the pattern of five spines or &#8216;teeth&#8217; on each side of the eyes and three spines between the eyes (see Appendix 1). Their carapace (dorsal side) is typically a mottled dark green and brown, but ventral color patterns can vary greatly (see Appendix 2).
There are many organizations, researchers, citizen scientist groups, and educators currently conducting green crab population monitoring. However, the lack of a standardized protocol makes it difficult to compare datasets and to ultimately understand green crab populations on both a local and regional scale. This protocol is designed to quantify spatiotemporal population dynamics of green crabs in the rocky intertidal zone in New England and Atlantic Canada. Ideally, the development of a standardized, inexpensive, and easily adaptable survey will promote long-term monitoring efforts by a wide range of users, including researchers, citizen scientists, educators, and students.
Although green crabs are the target species of interest in this protocol, data is also collected for native Jonah and rock crabs (Cancer borealis and Cancer irroratus), as well as the invasive Asian shore crab (Hemigrapsus sanguineus). These are crab species commonly found in the rocky intertidal zone in northern New England, and trends in their population distribution and abundance have ecological and economic significance. An Intertidal Crab Field Guide was developed alongside this protocol to aid in crab identification (Appendix 1) specific to northern New England. A data entry and storage platform called &#8220;Intertidal Green Crab Project&#8221; was also developed for this protocol using Anecdata19. Anecdata is a free online citizen science platform that provides web-based and mobile solutions for gathering and accessing observations, and provides a user-friendly platform to easily collect, manage, and share data.

<table>
	<tbody>
		<tr>
			<td>1 m2 PVC quadrat (1/2&#34; PVC)</td>
			<td>Any hardware/home improvement store</td>
			<td></td>
			<td>PVC can be sourced at any hardware/home improvement store and cut into 1m lengths to form quadrat (4 1/2&#34; PVC elbows will also be needed to connect 1 m lengths into square)</td>
		</tr>
		<tr>
			<td>1/2&#34; rebar</td>
			<td>Home depot</td>
			<td>5152</td>
			<td>*optional (for marking low intertidal area)</td>
		</tr>
		<tr>
			<td>40 m Fiberglass Transect Tape</td>
			<td>Grainger</td>
			<td>3LJX1</td>
			<td></td>
		</tr>
		<tr>
			<td>5 gal bucket</td>
			<td>Home depot</td>
			<td>05GLHD2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ade Advanced Optics Salinity Refractometer</td>
			<td>Amazon</td>
			<td></td>
			<td>*optional</td>
		</tr>
		<tr>
			<td>Clip board</td>
			<td>Any office supply store or Amazon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uei Waterproof Digital Thermometer</td>
			<td>Amazon</td>
			<td></td>
			<td>*optional</td>
		</tr>
		<tr>
			<td>Vernier calipers</td>
			<td>Bel-Art</td>
			<td></td>
			<td>Many companies make calipers, however our preferred brand is Bel-Art which can be sourced on Amazon</td>
		</tr>
	</tbody>
</table>

assessing intertidal populations of the invasive european green crab

Invasive species have caused major disruptions to ecosystems worldwide. The European green crab invaded North America in the 1800s and is considered one of the world&#8217;s 100 worst invaders by the IUCN. Observations of spatiotemporal green crab population dynamics are essential for predicting and managing the ecological and economic impacts of this harmful invasive species. This protocol was developed in an effort to create a standardized method for assessing green crab population dynamics in the rocky intertidal zone of New England and Atlantic Canada. The protocol was designed to be accessible to multiple users including researchers, educators, students, and citizen scientists. Although it was designed for surveying crab populations, this protocol is easy to adapt and could be used for any number of intertidal species. The resulting data collected using this protocol has a wide range of uses, including to inform ecological research, conservation efforts, mitigation strategies, and fishery development, as well as for educational outreach purposes.

In 2019, this protocol was used to conduct monthly intertidal green crab surveys at three locations from May-November (Sandy Point, Yarmouth, ME (43&#176;46&#39;17.92&#34;N, 70&#176; 8&#39;45.52&#34;W), Robinhood Cove, Georgetown, ME (43&#176;48&#39;13.80&#34;N, 69&#176;44&#39;50.97&#34;W), and New Meadows River, West Bath, ME (43&#176;51&#39;17.84&#34;N, 69&#176;51&#39;55.20&#34;W)), and at one location from May-August (Damariscotta River, Walpole, ME (43&#176;56&#39;9.42&#34;N, 69&#176;34&#39;52.75&#34;)). The data collected indicated wide variations in spatial and temporal green crab population density (Figure 3) and sex ratio (Figure 4), as well as significant differences in cumulative size frequency among sites (e.g., Damariscotta crabs were significantly smaller and Sandy Point crabs were significantly larger than the other populations sampled (Kolmogorov-Smirnov test, p &#60; 0.05 for all comparisons), Figure 5). Shell condition changed seasonally within each site, with peak pre-molt and soft-shell phases for males in the spring and for females in the spring and fall (Figure 6).
The protocol was also used in 2018 and 2019 by Georgetown Central School 3rd and 4th grade students who surveyed the same site in Georgetown, ME each year in October. They observed a shift in the population from being dominated by 5-15 mm CW sized crabs in 2018 to 15-30 mm CW sized crabs in 2019 (Figure 7). They also observed an increase in the percent occurrence of the invasive Asian shore crab from 3.5% in 2018 to 9% in 2019 (Figure 8).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61667/61667fig01.jpg" /> 
Figure 1: Tidal height zonation.&#160;Diagram of tidal height zonation along the shoreline. Surveys are conducted in the low intertidal zone (i.e., the lower third of the intertidal zone that runs parallel to the shoreline). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61667/61667fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61667/61667fig02.jpg" /> 
Figure 2: Defining survey area.&#160;Image of a survey site in Yarmouth, Maine with tidal zonation displayed in yellow and survey area in green. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61667/61667fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61667/61667fig03.jpg" /> 
Figure 3: Green crab density.&#160;Average monthly density (#/m2) of green crabs at Damariscotta River, New Meadows, Robinhood Cove, and Sandy Point intertidal survey sites. Error bars represent &#177; 1 standard error. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61667/61667fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61667/61667fig04.jpg" /> 
Figure 4: Green crab sex ratio.&#160;Sex ratio by month for green crabs at Damariscotta River, New Meadows River, Robinhood Cove, and Sandy Point intertidal survey sites. Sex ratio is expressed as the percent of the population that is male. The green &#8220;0&#8221; appearing in July indicates 0% male crabs and 100% female crabs at Robinhood Cove in that month. Blue &#8220;NA&#8221; indicates no data collected at Damariscotta River from September-November. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61667/61667fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61667/61667fig05.jpg" /> 
Figure 5: Green crab size frequency.&#160;Cumulative size frequency of green crabs at Damariscotta River, New Meadows River, Robinhood Cove, and Sandy Point intertidal monitoring sites for all months combined (May-November for Damariscotta River, New Meadows River, Robinhood Cove, and May-August for Damariscotta River). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61667/61667fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61667/61667fig06.jpg" /> 
Figure 6: Shell condition ratio.&#160;Monthly shell condition ratio of A) female and B) male green crabs at New Meadows River intertidal monitoring site. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61667/61667fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61667/61667fig07.jpg" /> 
Figure 7: Student-collected size frequency.&#160;Size frequency of green crabs surveyed by Georgetown Central School 3rd and 4th grade students in 2018 and 2019. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61667/61667fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61667/61667fig08.jpg" /> 
Figure 8: Species occurrence.&#160;Percent occurrence of crab species surveyed by Georgetown Central School 3rd and 4th grade students in A) 2018 and B) 2019. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61667/61667fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Assessing Intertidal Populations of the Invasive European Green Crab

Understanding spatiotemporal patterns in green crab population dynamics is essential for predicting and managing the ecological and economic impacts of this harmful invasive species. This protocol was developed in an effort to create a standardized method for assessing green crab populations in the rocky intertidal zone of the Northwest Atlantic.

Invasive species have caused major disruptions to ecosystems worldwide. The European green crab invaded North America in the 1800s and is considered one ...

assessing-intertidal-populations-of-the-invasive-european-green-crab

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5.9K Views.  Manomet, Inc.. Understanding spatiotemporal patterns in green crab population dynamics is essential for predicting and managing the ecological and economic impacts of this harmful invasive species. This protocol was developed in an effort to create a standardized method for assessing green crab populations in the rocky intertidal zone of the Northwest Atlantic.

Video: Assessing Intertidal Populations of the Invasive European Green Crab

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to inorganic arsenic through diet, information about distribution of arsenic species in various food types must be generated. A method, previously validated in a collaborative trial, has been applied to determine inorganic arsenic in a wide variety of food matrices, covering grains, mushrooms and food of marine origin (31 samples in total). The method is based on detection by flow injection-hydride generation-atomic absorption spectrometry of the iAs selectively extracted into chloroform after digestion of the proteins with concentrated HCl. The method is characterized by a limit of quantification of 10 &#181;g/kg dry weight, which allowed quantification of inorganic arsenic in a large amount of food matrices. Information is provided about performance scores given to results obtained with this method and which were reported by different laboratories in several proficiency tests. The percentage of satisfactory results obtained with the discussed method is higher than that of the results obtained with other analytical approaches.

The usefulness of an analytical method to determine inorganic arsenic in a wide range of food matrices is demonstrated. The method consists of selective extraction of inorganic arsenic into chloroform with a final determination by hydride generation-atomic absorption spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to ...

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which promotes growth of large weaponry and body size. Populations of the same species display each phenotype depending on environmental conditions. Although antler and body size of male white-tailed deer (Odocoileus virginianus) varies by physiographic region in Mississippi, USA and is strongly correlated with regional variation in nutritional quality, the effects of population-level genetics from native stocks and previous re-stocking efforts cannot be disregarded. This protocol describes how we designed a controlled study, where other factors that influence phenotype, such as age and nutrition, are controlled. We brought wild-caught pregnant females and six-month-old fawns from three distinct physiographic regions in Mississippi, USA to the Mississippi State University Rusty Dawkins Memorial Deer Unit. Deer from the same region were bred to produce a second generation of offspring, allowing us to assess generational responses and maternal effects. All deer ate the same high-quality (20% crude protein deer pellet) diet ad libitum. We uniquely marked each neonate and recorded body mass, hind foot, and total body length. Each subsequent fall, we sedated individuals via remote injection and sampled the same morphometrics plus antlers of adults. We found that all morphometrics increased in size from first to second generation, with full compensation of antler size (regional variation no longer present) and partial compensation of body mass (some evidence of regional variation) evident in the second generation. Second generation males that originated from our poorest quality soil region displayed about a 40% increase in antler size and about a 25% increase in body mass when compared to their wild harvested counterparts. Our results suggest phenotypic variation of wild male white-tailed deer in Mississippi are more related to differences in nutritional quality than population-level genetics.

Phenotypic differences among cervid populations may be related to population-level genetics or nutrition; discerning which is difficult in the wild. This protocol describes how we designed a controlled study where nutritional variation was eliminated. We found that phenotypic variation of male white-tailed deer was more limited by nutrition than genetics.

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

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 ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...

Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular substrates offer key advantages over cell-based aAPC, including precise control of signal presentation parameters and physical properties of the aAPC surface to modulate its interactions with T cells. aAPC constructed from anisotropic particles, particularly ellipsoidal particles, have been shown to be more effective than their spherical counterparts at stimulating T cells due to increased binding and larger surface area available for T cell contact, as well as reduced nonspecific uptake and enhanced pharmacokinetic properties. Despite increased interest in anisotropic particles, even widely accepted methods of generating anisotropic particles such as thin-film stretching can be challenging to implement and use reproducibly.
To this end, we describe a protocol for the rapid, standardized fabrication of biodegradable anisotropic particle-based aAPC with tunable size, shape, and signal presentation for T cell expansion ex vivo or in vivo, along with methods to characterize their size, morphology, and surface protein content, and to assess their functionality. This approach to fabricating anisotropic aAPC is scalable and reproducible, making it ideal for generating aAPC for &#34;off-the-shelf&#34; immunotherapies.

Here, we present a protocol to quickly and reproducibly generate biologically inspired, biodegradable articifical antigen presenting cells (aAPC) with tunable size, shape, and surface protein presentation for T cell expansion ex vivo or in vivo.

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular ...

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

Assessing Intertidal Populations of the Invasive European Green Crab

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Chapters in this video

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry.

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

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

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR