Name: field collection and laboratory maintenance of canopy-forming giant kelp to facilitate restoration
Uploaded: 2024-06-07T13:00:00
Duration: 14 min 44 s
Description: Canopy-forming kelps are essential foundation species, supporting biodiversity and providing ecosystem services valued at more than USD$500 billion ...

This work was funded by the California Sea Grant Kelp Recovery Research Program R/HCE-17 to JBL and MESB, a National Science Foundation Research Traineeship award DGE-1735040 to PDD, The Nature Conservancy, Schmidt Marine Technology Partners, Sustainable Ocean Alliance, Tinker Foundation to AP-L, and The Climate Science Alliance Baja Working Group to RBL and JL. We thank Steven Allison, Cascade Sorte, Samantha Cunningham, Sam Weber and Caitlin Yee at the University of California, Irvine; Mark Carr, Peter Raimondi, Sarah Eminhizer, Anne Kapuscinski at the University of California, Santa Cruz; Walter Heady and Norah Eddy at The Nature Conservancy; Filipe Alberto and Gabriel Montecinos at the University of Wisconsin, Milwaukee; Jose Antonio Zertuche-Gonz&#225;lez, Alejandra Ferreira-Arrieta, and Liliana Ferreira-Arrieta at the Universidad Aut&#243;noma de Baja California; Luis Malpica-Cruz, Alicia Abad&#237;a-Cardoso, and Daniel D&#237;az-Guzm&#225;n from MexCal; the MexCalitos divers Alejandra Reyes, Monica Peralta, Teresa Tavera, Julia Navarrete, Ainoa Vilalta, Jeremie Bauer, and Alfonso Ferreira; and Nancy Caruso for technical advice. We thank the Instituto de Investigaciones Oceanol&#243;gicas, Universidad Aut&#243;noma de Baja California for providing facilities used to develop the water bath system. We thank Ira Spitzer for underwater and drone video content.

The authors have nothing to disclose.

Anthropogenic climate change is a growing threat to the health of the world&#39;s oceans44,45,46,47,48, resulting in major disturbances and biodiversity loss49,50,51,52. To accelerate the restoration of degraded ecosystems, the United Nations has declared 2021 through 2030 the &#34;UN Decade on Ecosystem Restoration,&#34; coinciding with the &#34;UN Decade of Ocean Science for Sustainable Development&#34;, which aims to reverse the deterioration in ocean health53. In line with this global call to action, the Kelp Forest Alliance has launched the Kelp Forest Challenge to restore 1 million hectares and protect 3 million hectares of kelp forest by the year 204054. Marine restoration is undervalued55, and kelp ecosystems receive considerably less attention than habitats such as coral reefs, mangrove forests, and seagrass meadows56. Restoration of degraded ecosystems has been shown to be effective in re-building marine ecosystems but can cost on average between $80,000 - $1,600,000 per hectare, with median total costs likely to be two to four times higher57. Current and projected losses call upon developing scalable, feasible, and cost-effective kelp restoration methodologies as urgent conservation interventions.
Current kelp restoration efforts use a combination of methodologies to address site-specific drivers of kelp loss, including transplantation of adult kelps, direct seeding of zoospores and/or gametophytes, grazer control, and installation of artificial reefs11. However, these methods require substantial resources and have limited scalability. Typical transplantation of adult kelps requires the laborious deployment of artificial materials or structures on the benthos, by divers. Bottom-up interventions to re-establish coastal rocky reefs, such as controlling competitors and grazers, are also restricted by labor costs as they rely on the manual underwater removal or exclusion of these biotic stressors11. The &#39;green gravel&#39; technique overcomes these limitations with simple deployment from the surface, requiring no underwater installment or technical knowledge and scalability at relatively low costs28. This innovative approach provides a promising restoration tool, urging extensive trials across diverse locations and environments to unlock its full potential32.
While successful restoration efforts with &#39;green gravel&#39;&#160;have been documented in sheltered fjords in Norway using the sugar kelp, Saccharina latissima26, this technique is still in the piloting phase for Macrocystis pyrifera in the Eastern Pacific. Additional trials are needed to address the expected survivorship of M. pyrifera outplants within its range. In wave-exposed conditions typical of M. pyrifera growth, smaller gravel may be more prone to movement and abrasion, leading to damaged outplants. Furthermore, positive buoyancy provided by gas-filled pneumatocysts of M. pyrifera may lead to &#39;green gravel&#39; outplants being effectively carried away from the restoration site, and thus, gravel size and weight are important factors to explore for this species. In a recent pilot study (May 2022; Ensenada, Baja California, Mexico), preliminary success in the field with M. pyrifera has been observed, indicated by haptera attachment to surrounding substrate and growth of juveniles reaching 1.2 m in length after two months in the field (Figure 4). This demonstrates a clear opportunity that has yet to be explored in utilizing &#39;green gravel&#39;&#160;for M. pyrifera in the Eastern Pacific. This video showcases the &#39;green gravel&#39; technique with M. pyrifera and is a valuable resource that simplifies and centralizes existing practices in the culturing phase of restoration to support studies that address successes and limitations in different field settings.
With the &#39;green gravel&#39; technique, many smaller, individual gravel units can be seeded at a scale that may increase the probability of success compared to more common transplantation approaches with adult plants. However, the key scalable aspect of this technique is its simple deployment from the surface, which can facilitate the restoration of large areas by boat. For field settings where the deployment of small gravel is not suitable, this protocol can be adapted to transplant M. pyrifera on a wide range of substrates, including larger gravel or even small boulders, string that can be tied to natural or deployed underwater anchors, or tiles that can be bolted or glued using marine epoxy to the seafloor in more exposed conditions. These deployment adaptations will not change the facilities needed for M. pyrifera culturing but will subsequently increase the cost of deployment.
Anthropogenic disturbances and climate change are currently overcoming the capacity for natural populations to adapt. This poses significant challenges to traditional conservation efforts that restore ecosystems to their historic states58,59,60,61,62,63. Thus, conservation frameworks have expanded to include anticipatory management considering resilience and adaptive capacity64. Anticipatory management to address climate change is being implemented for tree species in forest ecosystems65&#160;and has been proposed for further restoration efforts to enhance the evolutionary potential of outplants66,67. Although these strategies are inherently easier to manipulate in terrestrial environments, several studies are beginning to explore their application in marine environments62,68,69,70. For example, coral reefs are threatened by numerous anthropogenic stressors that have resulted in unprecedented declines71,72. In response to the losses of these important foundation species, active restoration and assisted adaptation techniques are increasingly advocated to conserve remaining coral reefs and their associated functions62,73,74. One technique involves translocating individuals within their current species distribution range to increase tolerance to heat stress75. Regarding the restoration of canopy-forming kelps, &#39;green gravel&#39; has a customizable framework to explore assisted adaptation techniques such as translocation of resilient genotypes to vulnerable areas, non-genetic manipulation such as hybridization, or acclimatization of individuals to environmental stress62&#160;with outcomes aimed towards obtaining more resistant strains for restoration programs76,77.
Harnessing local support to enhance restoration endeavors is crucial to sustain kelp ecosystem conservation success. Engaging local stakeholders can increase local buy-in for restoration needs6,50&#160;and promote coastal stewardship that could subsequently result in increased funding and longevity of kelp ecosystem protection. As with all other kelp restoration methodologies, structured decision-making frameworks integrating diverse ecological, socio-economic, and conservation objectives will help achieve optimal outcomes for kelp ecosystems and the communities they support11.

Canopy-forming kelps (brown macroalgae in the order Laminariales) are globally important foundation species, dominating coastal rocky reefs in temperate and Arctic seas1. These kelps form structurally complex and highly productive biogenic habitats known as kelp forests that support taxonomically diverse marine communities2. Kelp forests worldwide provide many ecosystem services to humans, including commercial fisheries production, carbon and nutrient cycling, and recreational opportunities, with a total estimated value of USD $500 billion per year3.
Despite their substantial value, kelp forests face growing anthropogenic pressures in many regions3. Climate change presents one of the most significant threats to kelps due to long-term ocean warming combined with the increasing frequency of temperature anomalies3,4,5,6,7. Increased ocean temperatures are associated with nutrient limitation8, while exposure to heat stress above physiological thresholds can result in mortality9. In combination with variable regional local stressors7, kelp populations are globally declining by approximately 2% per year10 with significant losses and persistent shifts to alternate community states in certain regions6,11,12,13,14. Natural recovery of kelp populations alone may not be sufficient to reverse the extent of current and projected losses15,16,17,18, underscoring the importance of active restoration.
Current kelp restoration efforts can use a combination of methodologies to re-establish these important foundation species on coastal rocky reefs3,19. Methodologies chosen to address site-specific concerns depend on geographic context, the specific impediments to kelp recovery, and the social-ecological context11. Understanding the connections and interdependence of social-ecological systems is the key, and interventions that engage local institutions and garner support from local communities enhance the likelihood of successful restoration efforts20.
In addition to climate change, herbivore pressure or interspecific competition drives, declines, or suppresses the recovery (e.g., by sea urchins13, herbivorous fish21,22, turf algae9,23, or invasive algae24). Restoration may focus on the removal of these biotic stressors25, although these methods require substantial resources and continuous maintenance11. To catalyze kelp species recovery, there have been efforts toward a direct seeding approach, for example, weighing mesh bags filled with fertile kelp blades to the benthos that release zoospores into the environment26. This method, however, is time-intensive and requires technical underwater installation and removal. Other cases focus on transplanting large quantities of whole adult donor plants, which may compromise closely associated and vulnerable donor populations and are often limited to small scales due to reliance on continual transplantation27.
For regions, where kelp spore limitation may be impeding kelp forest recovery due to habitat fragmentation, a relatively new kelp restoration approach called the &#39;green gravel&#39; technique, was introduced. The technique was successfully trialed at theFl&#248;devigen Research Station in southern Norway28&#160;and represented a promising option for restoration due to cost-effectiveness and scalability. The workflow of this technique is as follows: (1) a spore solution is created from fertile tissue collected from reproductive adult kelps in the field and then seeded onto small substrates, such as gravel; (2) early-stage kelps are reared in laboratory-controlled abiotic conditions on substrates; (3) substrates with visible sporophytes are deployed in the field on specific reefs as &#39;green gravel&#39;, where sporophytes continue to grow. Note that typical transplantation efforts of adult individuals require laborious and cost-inhibitive underwater installation by divers, and the &#39;green gravel&#39; technique uses simple deployment from the surface28.
The &#39;green gravel&#39; technique is currently being tried out by members of numerous international working groups29 across different environments and several laminarian kelp species. This protocol describes the required facilities, materials, and methods for tissue collection, sporulation, seeding, rearing conditions, regular maintenance, and monitoring of early-stage kelp prior to deploying this restoration technique in the field using the giant kelp, Macrocystis pyrifera. This protocol is a valuable resource for researchers, managers, and stakeholders seeking to provide insight into the successes and limitations of this method with M. pyrifera in different field settings.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>0.22 &#181;m filters</td>
			<td>Milipore</td>
			<td>SCGPS05RE</td>
			<td>Natural seawater sterilization</td>
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		<tr>
			<td>1 L glass bottles</td>
			<td>Amazon</td>
			<td>B07J6JP4D1</td>
			<td>Natural seawater sterilization</td>
		</tr>
		<tr>
			<td>1 &#181;m filters (water + air)</td>
			<td>Amazon</td>
			<td>B01M1VWUWL</td>
			<td>Natural seawater sterilization</td>
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		<tr>
			<td>1&#39;&#39; PVC 90-Degree Elbow</td>
			<td>Home Depot</td>
			<td>203812125</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>10 &#181;m filters</td>
			<td>Amazon</td>
			<td>B00D04BG56</td>
			<td>Natural seawater sterilization</td>
		</tr>
		<tr>
			<td>20 &#181;m filters</td>
			<td>Amazon</td>
			<td>B082WS9NPH</td>
			<td>Natural seawater sterilization</td>
		</tr>
		<tr>
			<td>3x5mm tubing</td>
			<td>Amazon</td>
			<td>B0852HXPN6</td>
			<td>Option 1 Small scale - Incubator</td>
		</tr>
		<tr>
			<td>4x4&#39;&#39; Sterile Gauze</td>
			<td>Amazon</td>
			<td>B07NDK8XM3</td>
			<td>Sporulation</td>
		</tr>
		<tr>
			<td>4x6mm tubing</td>
			<td>Amazon</td>
			<td>B08BCRV1FY</td>
			<td>Option 1 Small scale - Incubator</td>
		</tr>
		<tr>
			<td>5 &#181;m filters</td>
			<td>Amazon</td>
			<td>B082WS9NPH</td>
			<td>Natural seawater sterilization</td>
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		<tr>
			<td>50 mL falcon tubing</td>
			<td>Amazon</td>
			<td>B01M04HGPJ</td>
			<td>Sporulation</td>
		</tr>
		<tr>
			<td>8x10mm tubing</td>
			<td>Amazon</td>
			<td>B01MSM3LLZ</td>
			<td>Option 1 Small scale - Incubator</td>
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		<tr>
			<td>Air filters</td>
			<td>Thermo Fisher</td>
			<td>MTGR85010</td>
			<td>Option 1 Small scale - Incubator</td>
		</tr>
		<tr>
			<td>Alcohol lamp</td>
			<td>Amazon</td>
			<td>B07XWD9WWC</td>
			<td>Sporulation</td>
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		<tr>
			<td>Ammonium iron(II) sulfate hexahydrate ACS reagent, 99%</td>
			<td>Sigma</td>
			<td>215406-100G</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Aquarium Grade Gravel</td>
			<td>Amazon</td>
			<td>B07XRCKFBJ</td>
			<td>Option 1 Small scale - Incubator</td>
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		<tr>
			<td>Biotin powder, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, 99%</td>
			<td>Sigma</td>
			<td>B4639-100MG</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Boric Acid, 99.8%, 10043-35-3, MFCD00011337, BH3O3, 61.83, 500g</td>
			<td>Thermo Fisher</td>
			<td>5090113707</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Calcium D-Pantothenate,ge98.0% (T),C9H17NO5,137-08-6,25g,D-Pantothenic Acid Calcium Salt, P0012-25G 1/EA</td>
			<td>Thermo Fisher</td>
			<td>P001225G</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Check valves</td>
			<td>Amazon</td>
			<td>B08HRZR4MM</td>
			<td>Option 1 Small scale - Incubator</td>
		</tr>
		<tr>
			<td>Clear tubing 3/8&#39;&#39; - 10 ft</td>
			<td>Amazon</td>
			<td>B07MTYMW13</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>COBALT(II) SULFATE HEPTAH-100G, WARNING - California - Cancer Hazard, 93-2749-100G 1/EA</td>
			<td>Thermo Fisher</td>
			<td>5090114752</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Compound microscope with camera</td>
			<td>OMAX</td>
			<td>M83EZ-C50S</td>
			<td>Monitoring</td>
		</tr>
		<tr>
			<td>Culture flask</td>
			<td>Thermo Fisher</td>
			<td>07-250-080</td>
			<td>Option 1 Small scale - Incubator</td>
		</tr>
		<tr>
			<td>Culture light</td>
			<td>Amazon</td>
			<td>B07RRRPJ63</td>
			<td>Option 1 Small scale - Incubator</td>
		</tr>
		<tr>
			<td>Culture stoppers</td>
			<td>Amazon</td>
			<td>B07DX6J7QD</td>
			<td>Option 1 Small scale - Incubator</td>
		</tr>
		<tr>
			<td>Drainage connector</td>
			<td>Amazon</td>
			<td>B00GUZ6CV0</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>EDTA CAS Number: 6381-92-6 Molecular Formula: C10H14N2O8Na2- 2H2O Molecular Weight: 372.2</td>
			<td>Thermo Fisher</td>
			<td>50213299</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Eisco Safety Pack Graduated Cylinder Sets Class A, ASTM, Capacity: 10 mL, 25 mL, 50 mL, Graduations: 0.2 mL, 0.5 mL, 1.0 mL, Borosilicate 3.3 Glass, Autoclavable: Yes, Class: Class A, Graduated: Yes, Tolerance: 0.10 mL, 0.17 mL, 0.25 mL</td>
			<td>Thermo Fisher</td>
			<td>S81273</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Eisco Safety Pack Graduated Cylinder Sets Class A, ASTM, Capacity: 50 mL, 100 mL, 250 mL, Graduations: 1.0 mL, 1.0 mL, 2.0 mL, Borosilicate 3.3 Glass, Autoclavable: Yes, Class: Class A, Graduated: Yes, Tolerance: 0.25 mL, 0.50 mL, 1.0 mL</td>
			<td>Thermo Fisher</td>
			<td>S81275</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Eisco Safety Pack Volumetric Flask Sets - Class A, ASTM, Capacity: 10 mL, 25 mL, 50 mL, Borosilicate 3.3 Glass, Autoclavable: Yes, Class: Class A, Closure Material: Glass, Closure Size: Stopper Number: 9, 9, 13, Closure Type: Penny Stopper, Graduated: Ye</td>
			<td>Thermo Fisher</td>
			<td>S81271</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Filter holder</td>
			<td>Amazon</td>
			<td>B07LCKBKCT</td>
			<td>Natural seawater sterilization</td>
		</tr>
		<tr>
			<td>Fisherbrand Graduated Cylinders, Capacity: 500 mL, Graduations: 5 mL, Borosilicate Glass, Autoclavable: Yes, Limit of Error: +/-4.0 mL, Recommended Applications: Education, Subdivision: 5 mL, S63460 1/EA</td>
			<td>Thermo Fisher</td>
			<td>S63460</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>FLEXACAM C1 Camera</td>
			<td>Leica</td>
			<td>FLEXACAM C1</td>
			<td>Monitoring</td>
		</tr>
		<tr>
			<td>Folic acid, C19H19N7O6, CAS Number59303, vitamin m, pteroylglutamic acid, vitamin b9, folvite, folacin, folacid, pteroyllglutamic acid, pteglu, folic acid, folate, 25g, 100781, CHEBI:27470, Yellow to Orange, 2004190, 441.41, OVBPIULPVIDEAOLBPRGKRZSAN</td>
			<td>Thermo Fisher</td>
			<td>AAJ6083314</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Free Standing 20 Gallon Utility Sink</td>
			<td>Amazon</td>
			<td>B094TLH19L</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>GERMANIUM DIOXIDE 99.99 10GR</td>
			<td>Thermo Fisher</td>
			<td>AC190000100</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Glass Graduated Cylinders, Class A Round Base, Eisco, For Use With: Measuring liquids, Capacity: 1000 mL, Graduations: 10 mL White, CH0344OWT 1/EA</td>
			<td>Thermo Fisher</td>
			<td>S88442</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Amazon</td>
			<td>B00L1S93PS</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>Glycerol phosphate disodium salt hydrate isomeric mixture</td>
			<td>Sigma</td>
			<td>G6501-100G</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Growth containers -3.4 Qt- 3.25 Lt transparent containers with transparent lid</td>
			<td>Container store</td>
			<td>#10014828</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>Growth light</td>
			<td>Amazon</td>
			<td>B086R14MFW</td>
			<td>Option 1 Small scale - Incubator</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Amazon</td>
			<td>B07TJQDKLJ</td>
			<td>Sporulation</td>
		</tr>
		<tr>
			<td>HEPES 99.5% (titration)</td>
			<td>Sigma</td>
			<td>H3375-500G</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Hinged plastic jars</td>
			<td>SKS Bottle &#38; Packaging</td>
			<td>40280125.01S</td>
			<td>Option 1 Small scale - Incubator</td>
		</tr>
		<tr>
			<td>Inositol research grade, USP/NF For bacteriology. Optically inactive. Tested for its suitability in tissue culture. Size - 100G Storage Conditions - +15 C TO +30 C Catalog Number - 26310.01 CAS 87-89-8</td>
			<td>Thermo Fisher</td>
			<td>50247745</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Instant Ocean - 50 G</td>
			<td>Amazon</td>
			<td>B000255NKA</td>
			<td>Option 1 Small scale - Incubator</td>
		</tr>
		<tr>
			<td>Inverted Microscope Leica DMi1</td>
			<td>Leica</td>
			<td>DMi1</td>
			<td>Monitoring</td>
		</tr>
		<tr>
			<td>Iron(III) chloride hexahydrate ACS reagent, 97%</td>
			<td>Sigma</td>
			<td>236489-100G</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Licor Ligth Meter Data Logger</td>
			<td>Licor</td>
			<td>LI-250A</td>
			<td>Monitoring</td>
		</tr>
		<tr>
			<td>Light/temperature HOBO data logger</td>
			<td>Amazon</td>
			<td>B075X2SWKN</td>
			<td>Monitoring</td>
		</tr>
		<tr>
			<td>Lights 150W</td>
			<td>Amazon</td>
			<td>B0799DQM9V</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>Manganese sulfate monohydrate meets USP testing specifications</td>
			<td>Sigma</td>
			<td>M8179-100G</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Medium size rocks 2-3 inch, 20 pounds</td>
			<td>Home Depot</td>
			<td>206823930</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>Nicotinic Acid, 99%, C6H5NO2, CAS Number59676, daskil, apelagrin, acidum nicotinicum, akotin, 3carboxypyridine, niacin, 3pyridinecarboxylic acid, nicotinic acid, pellagrin, wampocap, 250g, 109591, CHEBI:15940, 1.4, 2004410, 293 deg.C (559 deg.F), 123.11,</td>
			<td>Thermo Fisher</td>
			<td>AAA1268330</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>p-Aminobenzoic acid 99.82% 4-aminobenzoic acid, C7H7NO2, CAS Number: 150-13-0, 25g, 0210256925 1/EA</td>
			<td>Thermo Fisher</td>
			<td>ICN10256925</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>PCV cement</td>
			<td>Amazon</td>
			<td>B001D9WRWG</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>Plastic water valve</td>
			<td>Amazon</td>
			<td>B0006JLVE4</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>Plastic water valve</td>
			<td>Amazon</td>
			<td>B07G5FY7X1</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>Precision scale 1mg</td>
			<td>Amazon</td>
			<td>B08DTH95FN</td>
			<td>Materials to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Pump for filtered air</td>
			<td>Amazon</td>
			<td>B0BG2BT9RX</td>
			<td>Option 1 Small scale - Incubator</td>
		</tr>
		<tr>
			<td>PVC tubing 1x24&#39;&#39;</td>
			<td>Home Depot</td>
			<td>202300505</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>Quantum Light meter</td>
			<td>Apogee Instruments</td>
			<td>MQ-510</td>
			<td>Monitoring</td>
		</tr>
		<tr>
			<td>Refrigerated Incubator</td>
			<td>Thermo Fisher</td>
			<td>15-103-1566</td>
			<td>Option 1 Small scale - Incubator</td>
		</tr>
		<tr>
			<td>Rubber Grommets</td>
			<td>Amazon</td>
			<td>B07YZD22ZP</td>
			<td>Option 1 Small scale - Incubator</td>
		</tr>
		<tr>
			<td>Salinity refractometer</td>
			<td>ATC</td>
			<td>B018LRO1SU</td>
			<td>Monitoring</td>
		</tr>
		<tr>
			<td>Shade mesh 6x50 ft</td>
			<td>Home depot</td>
			<td>316308418</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>Sodium Nitrate ge 99.0% Nitric Acid, Sodium Salt, NNaO3, CAS Number: 7631-99-4, 500g, 1/EA</td>
			<td>Thermo Fisher</td>
			<td>BP360500</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Soldering for aeration opening</td>
			<td>Amazon</td>
			<td>B08R3515SF</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>Spray isporopyl alcohol</td>
			<td>Amazon</td>
			<td>&#8206; B08LW5P844</td>
			<td>Sporulation</td>
		</tr>
		<tr>
			<td>Stainless steel sissors</td>
			<td>Amazon</td>
			<td>B07BT4YLHT</td>
			<td>Sporulation</td>
		</tr>
		<tr>
			<td>Stainless steel tray</td>
			<td>Amazon</td>
			<td>B08CV33YXG</td>
			<td>Sporulation</td>
		</tr>
		<tr>
			<td>Stainless steel twizzers</td>
			<td>Amazon</td>
			<td>B01JTZTAJS</td>
			<td>Sporulation</td>
		</tr>
		<tr>
			<td>Stir Bars</td>
			<td>Amazon</td>
			<td>B07C4TNKXB</td>
			<td>Materials to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Submersible circulation pump 400 GPH</td>
			<td>Amazon</td>
			<td>B07RZKRM13</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>Submersible Spherical Quantum Sensor</td>
			<td>Waltz</td>
			<td>US-SQS/L</td>
			<td>Monitoring</td>
		</tr>
		<tr>
			<td>Temperature gun</td>
			<td>Infrared Thermometer 749</td>
			<td>B07VTPJXH9</td>
			<td>Monitoring</td>
		</tr>
		<tr>
			<td>Thiamine hydrochloride BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture</td>
			<td>Sigma</td>
			<td>T1270-25G</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Thymine 99% 2, 4-Dihydroxy-5-methylpyrimidine, C5H6N2O2, CAS Number: 65-71-4, 25g, 157850250 1/EA</td>
			<td>Thermo Fisher</td>
			<td>AC157850250</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Transparent Acrylic sheet 24x48 inch</td>
			<td>Home Depot</td>
			<td>202038048</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>Tubing water circulation 1&#39;&#39;x10 ft</td>
			<td>Amazon</td>
			<td>B07ZC1PSF3</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>UV light for natural seawater sterilization</td>
			<td>Amazon</td>
			<td>B018OI7PYS</td>
			<td>Natural seawater sterilization</td>
		</tr>
		<tr>
			<td>Vacum pump</td>
			<td>Amazon</td>
			<td>B087XBTPVW</td>
			<td>Natural seawater sterilization</td>
		</tr>
		<tr>
			<td>Vitamin B12 BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, 98%</td>
			<td>Sigma</td>
			<td>V6629-100MG</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Volumetric Flasks, Class A Glass, Eisco, with Polypropylene Stopper, Graduated, White printed markings, Capacity: 1000 mL, CH0446IWT 1/EA</td>
			<td>Thermo Fisher</td>
			<td>S89446</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Volumetric Flasks, Class A Glass, Eisco, with Polypropylene Stopper, Graduated, White printed markings, Capacity: 500 mL, CH0446HWT 1/EA</td>
			<td>Thermo Fisher</td>
			<td>S89445</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
		<tr>
			<td>Water Chiller 200-600GPM</td>
			<td>Amazon</td>
			<td>B07BHHP71C</td>
			<td>Option 2 - Medium scale - Water bath systems</td>
		</tr>
		<tr>
			<td>Y-splitters for 4x6mm tubing</td>
			<td>Amazon</td>
			<td>B08XTJKFCH</td>
			<td>Option 1 Small scale - Incubator</td>
		</tr>
		<tr>
			<td>Zinc sulfate heptahydrate BioReagent, suitable for cell culture</td>
			<td>Sigma</td>
			<td>Z0251-100G</td>
			<td>Chemicals to create Provasoli&#8217;s Enriched Seawater (PES) and vitamins for media enrichment</td>
		</tr>
	</tbody>
</table>

field collection and laboratory maintenance of canopy-forming giant kelp to facilitate restoration

Canopy-forming kelps are essential foundation species, supporting biodiversity and providing ecosystem services valued at more than USD$500 billion annually. The global decline of giant kelp forests due to climate-driven ecological stressors underscores the need for innovative restoration strategies. An emerging restoration technique known as &#39;green gravel&#39; aims to seed young kelps over large areas without extensive underwater labor and represents a promising restoration tool due to cost-effectiveness and scalability. This video article illustrates a protocol and tools for culturing giant kelp, Macrocystis pyrifera. It also provides a resource for further studies to address the successes and limitations of this method in field settings. We outline field and laboratory-based methods for collecting reproductive tissue, sporulating, inoculating, rearing, maintaining, and monitoring substrates seeded with early life stages using the &#39;green gravel&#39;&#160;technique. The protocol simplifies and centralizes current restoration practices in this field to support researchers, managers, and stakeholders in meeting kelp conservation objectives.

The &#39;green gravel&#39; restoration technique is still in the piloting phase, with limited outplant survival data for other species28, and no published data yet for Macrocystis pyrifera. Using the field collection and laboratory maintenance outlined in this protocol, we tested the significance of site-specific rearing conditions for two distinct donor kelp populations before hypothetical &#39;green gravel&#39; deployment (Figure 5). Reproductive kelp tissue was collected in California (USA) from cooler K1 (Santa Cruz 36.60167&#176;N, 121.88508&#176;W) and warmer K4 (San Diego, 32.85036&#176;N, -117.27600&#176;W) populations and reared at two temperatures: (1) 12 &#176;C (the standard culturing temperature for seaweed aquaculture, and the mean winter SST for K1), and (2) 20 &#176;C (the mean summer SST for K4, and a 4 &#176;C heatwave for K1). All glass slides used for monitoring kelp life stage development were marked with a standardized grid, and high-resolution images were captured using this grid as a reference to enable the observation of fixed fields through time using an inverted microscope and camera (N = 5 images per sample, 2.479 mm x 1.859 mm).
After 24 d post sporulation, gametophytes were counted from microscope images (N = 300 images from 60 samples). To test for differences in gametophyte counts, generalized linear mixed effects models were employed with Poisson distribution using the function glmmTMB() in package glmmTMB41, and pairwise comparisons were conducted with emtrends() from package emmeans42in R. Our results illustrate that the response of gametophytes to thermal variability was different between K1 and K4 populations (t = 2.7, p = 0.007), where temperature did not have an effect for the warmer K4 population (estimate = -0.01, standard error [SE] = 0.01, confidence interval [CI] = [-0.03, 0.01]), but did have an effect for the cooler K1 population (estimate = -0.06, SE = 0.02, CI = [-0.10, -0.03]) (Figure 6A), suggesting a possible adaptive divergence in thermal tolerance traits. Kelp gametophytes are often depicted as a resistance stage43, meaning they produce an all-purpose phenotype that is stress tolerant and relatively insensitive to environmental variability. However, these results indicate that thermal variability imposes a significant pressure at this early stage.
After 32 d post sporulation, visible sporophytes with lengths greater than approximately 1 mm were counted on the entirety of each 2.5 cm by 7.5 cm glass slide (N = 72 total samples). To test for differences in visible sporophyte counts, generalized linear mixed effects models were employed with Poisson distribution using the function glmmTMB() in package glmmTMB and pairwise comparisons were conducted with emtrends() from package emmeans in R. Our results illustrate that the response of sporophytes to thermal variability is similar between K1 and K4 differentiated populations (z = 0.92, p = 0.36), where the temperature had an effect for the warmer K4 population (estimate = -0.66, SE = 0.04, CI = [-0.74, - 0.57]), as well as the cooler K1 population (estimate = -0.85, SE = 0.13, CI = [-1.10, -0.60]) (Figure 6B). Samples reared at 20 &#176;C grew few visible sporophytes (mean &#177; SE = 0.4 &#177; 0.2) compared to those reared at 12 &#176;C (mean &#177; SE = 82.4 &#177; 9.8). This result suggests that sporophyte production is more sensitive to temperature than the gametophyte stage, and that site-specific culturing temperatures must not exceed 15 &#176;C to achieve sporophyte development as outlined in the protocol.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66092/66092fig01.jpg" /> 
Figure 1: Diagram of &#39;green gravel&#39; incubator system. (A) Red light source for vegetatively bulking gametophyte cultures. (B) Access port for electrical wires and tubing, leading to an external outlet. (C) Structure to block full-spectrum light out of the red-light section. (D) A &#39;green gravel&#39; culturing section. (E) full-spectrum light sources. (F) Tubing lines connected to an external filtered air source. (G) Check valves to reduce airborne contamination. (H) Individual culture containers that minimize contamination. <a href="https://www.jove.com/files/ftp_upload/66092/66092fig01large.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/66092/66092fig02.jpg" /> 
Figure 2: Diagram of &#39;green gravel&#39; water bath system. (A) Chiller with submerged pump (in I). (B) 20 gallon tub for water bath. (C) Drain to recirculate water bath. (D) Valve for recirculation of water bath. (E) Light source. (F) 2.5 L &#39;green gravel&#39; container with transparent lid and aeration opening. (G) Aeration source. (H) Pipes that recirculate water with use of submerged pumps. (I) Water bath receiver from/to chiller from/to tubs with submersible pumps. (J) Acrylic cover to minimize water bath evaporation. (K) Mesh shade to adjust light intensity. <a href="https://www.jove.com/files/ftp_upload/66092/66092fig02large.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/66092/66092fig03v2.jpg" /> 
Figure 3: Macrocystis pyrifera development. Developmental life history stages of Macrocystis pyrifera from laboratory growth trials. <a href="https://www.jove.com/files/ftp_upload/66092/66092fig03large.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/66092/66092fig04.jpg" /> 
Figure 4: &#39;Green gravel&#39; seeded with Macrocystis pyrifera. &#39;Green gravel&#39; seeded with Macrocystis pyrifera is cultured in the laboratory until sporophytes reach 1-2 cm. &#39;Green gravel&#39; is then deployed and continues to grow in the field. <a href="https://www.jove.com/files/ftp_upload/66092/66092fig04large.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/66092/66092fig05.jpg" /> 
Figure 5: Experimental time series. Example images from a time series following the experimental growth and development of Macrocystis pyrifera gametophytes and sporophytes originating from two populations collected in California (USA) and cultured at two different temperatures. K1 = Santa Cruz, K4 = San Diego. <a href="https://www.jove.com/files/ftp_upload/66092/66092fig05large.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/66092/66092fig06.jpg" /> 
Figure 6: Representative Results. Macrocystis pyrifera life stages observed for K1 Santa Cruz San Diego K4 Santa Cruz populations of origin cultured at constant thermal conditions of 12 and 20 &#176;C. Error bars, mean &#177; 1 SE. Asterisk (*) denotes statistically significant differences (p &#60; 0.05). (A) Gametophytes at day 24 (N = 300 total images from 60 samples). (B) Visible sporophytes at day 32 (N = 72 samples, within a standardized 2.5 cm by 7.5 cm area). <a href="https://www.jove.com/files/ftp_upload/66092/66092fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1. <a href="https://www.jove.com/files/ftp_upload/66092/Supplemental File_Calendar R2.zip" target="_blank">Please click here to download this File.</a>

This protocol describes the field collection and regular laboratory maintenance of substrates seeded with canopy-forming giant kelp for use in restoration trials to address the success and limitations of the 'green gravel' technique in field settings.

Field Collection and Laboratory Maintenance of Canopy-Forming Giant Kelp to Facilitate Restoration

Canopy-forming kelps are essential foundation species, supporting biodiversity and providing ecosystem services valued at more than USD$500 billion ...

field-collection-laboratory-maintenance-canopy-forming-giant-kelp-to

Research

JoVE Journal

Environment

Helminth Collection and Identification from Wildlife

Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are &#34;roundworms&#34; (nematodes), &#34;thorny-headed worms&#34; (acanthocephalans), &#34;flukes&#34; (trematodes), and &#34;tapeworms&#34; (cestodes). The optimum method for collecting helminths is to examine a host that has been dead less than 4-6 hr since most helminths will still be alive. A thorough necropsy should be conducted and all major organs examined. Organs are washed over a 106 &#956;m sieve under running water and contents examined under a stereo microscope. All helminths are counted and a representative number are fixed (either in 70% ethanol, 10% buffered formalin, or alcohol-formalin-acetic acid). For species identification, helminths are either cleared in lactophenol (nematodes and small acanthocephalans) or stained (trematodes, cestodes, and large acanthocephalans) using Harris&#39; hematoxylin or Semichon&#39;s carmine. Helminths are keyed to species by examining different structures (e.g. male spicules in nematodes or the rostellum in cestodes). The protocols outlined here can be applied to any vertebrate animal. They require some expertise on recognizing the different organs and being able to differentiate helminths from other tissue debris or gut contents. Collection, preservation, and staining are straightforward techniques that require minimal equipment and reagents. Taxonomic identification, especially to species, can be very time consuming and might require the submission of specimens to an expert or DNA analysis.

Wild animals are commonly parasitized by a wide range of helminths.&#160; The four major types of helminths are &#8220;roundworms&#8221; (nematodes), &#8220;thorny-headed worms&#8221; (acanthocephalans), &#8220;flukes&#8221; (trematodes), and &#8220;tapeworms&#8221; (cestodes).&#160; Here we describe how helminths are collected from a vertebrate animal and how they are preserved and taxonomically identified.&#160;

Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are &#34;roundworms&#34; (nematodes), ...

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is described herein. During a 135-day laboratory experiment, we fed bloater (Coregonus hoyi) that had been caught in Lake Michigan to lake trout (Salvelinus namaycush) kept in eight laboratory tanks. Bloater is a natural prey for lake trout. In four of the tanks, a relatively high flow rate was used to ensure relatively high activity by the lake trout, whereas a low flow rate was used in the other four tanks, allowing for low lake trout activity. On a tank-by-tank basis, the amount of food eaten by the lake trout on each day of the experiment was recorded. Each lake trout was weighed at the start and end of the experiment. Four to nine lake trout from each of the eight tanks were sacrificed at the start of the experiment, and all 10 lake trout remaining in each of the tanks were euthanized at the end of the experiment. We determined concentrations of 75 PCB congeners in the lake trout at the start of the experiment, in the lake trout at the end of the experiment, and in bloaters fed to the lake trout during the experiment. Based on these measurements, &#947; was calculated for each of 75 PCB congeners in each of the eight tanks. Mean &#947; was calculated for each of the 75 PCB congeners for both active and inactive lake trout. Because the experiment was replicated in eight tanks, the standard error about mean &#947; could be estimated. Results from this type of experiment are useful in risk assessment models to predict future risk to humans and wildlife eating contaminated fish under various scenarios of environmental contamination.

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout Salvelinus namaycush from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is presented. To maximize applicability of the laboratory results to the field, the piscivorous fish should be fed prey fish that are typically eaten in the field.

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and screening for the selection of a fast-acting, highly virulent entomopathogenic fungus are important steps. Entomopathogenic fungi, such as Metarhizium, Beauveria, and Nomurea, which act by contact, are better suited than Bacillus thuringiensis or nucleopolyhedrosis virus (NPV), which must be ingested by the insect pest. In the present work, we isolated 68 Metarhizium strains from infected insects using a soil dilution and bait method. The isolates were identified by the amplification and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA region. The most virulent strain of Metarhizium anisopliae was selected based on the median lethal concentration (LC50) and time (LT50) obtained in insect bioassays against III-instar larvae of Helicoverpa armigera. The mass production of spores by the selected strain was carried out with solid-state fermentation (SSF) using rice as a substrate for 14 days. Spores were extracted from the sporulated biomass using 0.1% tween-80, and different formulations of the spores were prepared. Field trials of the formulations for the control of an H. armigera infestation in pigeon peas were carried out by randomized block design. The infestation control levels obtained with oil and aqueous formulations (78.0% and 70.9%, respectively) were better than the 63.4% obtained with chemical pesticide.

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Here, we report the different stages involved in the knowledge-based development of an effective mycoinsecticide, including the isolation, identification, screening, and selection of the "best-fit" entomopathogenic fungus, Metarhizium anisopliae, for the control of insect pests in agriculture.

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation 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 ...

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

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 ...

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Wastewater Irrigation Impacts on Soil Hydraulic Conductivity: Coupled Field Sampling and Laboratory Determination of Saturated Hydraulic Conductivity

Since the early 1960s, an alternative wastewater discharge practice at The Pennsylvania State University has been researched and its impacts monitored. Rather than discharging treated wastewater to a stream, and thereby directly impacting the stream quality, the effluent is applied to forested and cropped land managed by the University. Concerns related to reductions in soil hydraulic conductivity occur when considering wastewater reuse. The methodology described in this manuscript, matching soil sample size with the size of the laboratory-based hydraulic conductivity measurement apparatus, provides the benefits of a relatively rapid collection of samples with the benefits of controlled laboratory boundary conditions. The results suggest that there may have been some impact of wastewater reuse on the soil&#39;s ability to transmit water at deeper depths in the depressional areas of the site. Most of the reductions in the soil hydraulic conductivity in the depressions appear to be related to the depth from which the sample was collected, and by inference, associated with the soil structural and textural differences.

Here we present a methodology which matches a soil sample size and a hydraulic conductivity measurement device to prevent the so-called wall flow along the inside of the soil container from being erroneously included in water flow measurements. Its use is demonstrated with samples collected from a wastewater irrigation site.

Since the early 1960s, an alternative wastewater discharge practice at The Pennsylvania State University has been researched and its impacts monitored. ...