Autumn Mushrooms: Recent finds by EWU students and answers to some FAQs

by Dr. Jessica Allen and Amy Gray

IMG_8027 This year’s feverish collecting of fall mushrooms has just passed as truly cold weather sets in. Fall 2019 will be remembered as a season of exceptional mushroom hunting. The EWU Mycology class (mycology is the study of fungi) took full advantage of this year of great mushrooms, spending multiple days on field trips throughout the area, including to Fourth of July Pass, Mount Spokane, the Roosevelt Grove of Ancient Cedars, and Priest Lake.

Some of their notable finds included club fungi:

Club fungi and mushroom knife for perspective (Photo credit: Michelle Keller Pearson)

The elusive chanterelle:

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Chanterelles peaking through the duff (Photo: Amy Gray)

Some Lycogala (not a fungus – a plasmodial slime mold – but neat, and sometimes studied in mycology courses). The hand below is checking to see if they squirt if squished. Yes, they do.

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Pink slime mold on a dead conifer (Photo: Amy Gray)

Coral fungi:

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Photo: Amy Gray

Some “dyers polypore” (Phaeolus schweinitzii )

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Fresh velvet polypore (Photo: Amy Gray)

and an abundance of (normally) harder-to-find cup fungi.

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Photo: Amy Gray

These wild and often alien-looking organisms boggle the mind, gross people out, and spark curiosity. A number of mushrooms questions may have come to mind for you as you read this blog post. Here we have answered a list of frequently asked mushroom questions.

 

What are mushrooms?

Mushrooms are the parts of a fungus that reproduce. They are attached to large networks of hyphae, string-like strands of cells, that grow throughout wood or soil. When you see a mushroom, you are only seeing the ‘tip of the iceberg,’ as most of the fungus is hidden from view with the naked eye. The mushroom is where reproduction happens, and spores are produced that will fly off and start a new fungus.

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That’s not smoke (Photo: Andreas Fina)

 

Wait, mushrooms are related to the mold on my bread and the grout creepiness in my bathtub? I really don’t think I like fungi. Why should I care about them?

While some fungi are nuisances, cause disease, or initiate allergic reactions, most fungi are beneficial to humans and the environment.

  • Fungi are essential for healthy functioning of natural systems. Fungi break down fallen trees, leaves, and other dead matter, which makes them critical in nutrient recycling.
  • Almost every single plant that you see has mycorrhizal fungi attached to its roots. The mycorrhizae help the plant access and acquire more water and nutrients in the soil, and the fungus is given sugars by the plant in return. Without these root attached fungi plants cannot grow as well.
  • Many animals form mutualistic relationships with fungi. For instance, leaf-cutter ants don’t digest leaves at all, they simply bring them back to their colony, chew them up, and feed them to fungi that they farm. The fungi break down the leaves, then produce tiny mushrooms for the ants to eat.
  • Mushrooms are an essential food source for some mammals, including northern flying squirrels and red-back voles.
  • Fungi that form symbiotic relationships with algae are called lichens. Lichens hold soil together in arid systems, act as natural fertilizers in forest system, provide winter food for large mammals like caribou, and nesting material for birds.
  • Fungi produce thousands of chemical compounds that no other organisms can make. One fungal compound that we use frequently is penicillin, which is made by the fungus Penicillium. Many fungal metabolites are under investigation for potential use in cancer treatment, though no (exclusively) fungal-derived compound for that purpose has yet been identified.
  • Many fermentation processes rely on fungi. Brewer’s yeast, Saccharomyces cerevisiae, is essential for the production of beer, wine, and other beverages –S. cerevisiae is also used to make bread. Miso and soy sauce are produced through the fermentation of Aspergillus fungi. Brie, camembert, and “bleu” cheeses all require fungi to become their delicious final products.
  • A strong contender for the largest organism on the planet is a fungus – it occupies around 3.5 square miles of Oregon’s Malheur National Forest. The fungus is pathogenic; and thus, no friend to the Douglas firs and hemlock trees that grow there. It produces delicious mushrooms when in “fruiting” season, when honey-colored mushrooms emerge from infected trees and the ground below them every fall.

 

Are they all poisonous?

Some mushrooms are deadly, and others will make a person very sick. However, many mushrooms growing in the woods are not poisonous. That being said, just because a mushroom is “not poisonous” does not mean it is going to be a tasty treat. Choice edible mushrooms, like chanterelles and morels, are highly sought after for a reason! Many other mushrooms are technically edible, but have unpleasant textures and a blandness that can’t hold a candle to your average store-bought mushroom.

 

How can I tell which ones are edible and which ones are poisonous?

The only way to tell if a mushroom is edible or poisonous is to identify it to species using an appropriate reference, like Mushrooms Demystified by David Aurora. If you are ever unsure about the identify of the mushroom you find, don’t eat it. As Teri Pratchett memorably wrote, “All mushrooms are edible, some only once.”

 

I saw one of these, and I heard they can kill you. I also heard that they are hallucinogenic. Which is correct?

amanita muscaria

Amanita muscaria (Photo: Michael Maggs. https://creativecommons.org/licenses/by-sa/2.5/)

Aha! You’ve met the iconic Amanita muscaria! The Alice-in-Wonderland mushroom. This fungus can be found in treed regions over much of the planet, and it has a fascinating history imbedded in many cultures. Let’s answer the second question first – are they hallucinogenic? Well, they are considered toxic, as they produce a number of interesting compounds… at least two of which mimic neurotransmitters. Depending upon the metabolism of the consumer (and weight, and what the fungus is growing with, and a number of other factors), consumption of this raw mushroom can lead to nausea, vomiting, euphoria, seizures, slow heart rate, increased heart rate, and both visual and auditory disturbances, and “coma-like symptoms.”

So… can they hurt you?

Well, “coma-like symptoms” are not ideal.

More importantly, should any combination of those symptoms put you in the hospital, and you are incapacitated or reticent to explain what it is that you’ve eaten (as consumers of Schedule 1 Federally classified substances often are), then the treatment might kill you. There have been incidents where a drug was administered to counteract the cardiac symptoms upon a patient’s admission to the hospital (symptoms that vary depending upon the stage of metabolism of the toxins). This means that a medicine meant to bring a patient’s heart rate back up from far too slow may cause a heart attack when their heart speeds to racing after some more toxin metabolizing.

Take home message: if you’re seeking a bit of euphoria, these authors would recommend going for a run or trying yoga instead.

 

So, are any of the fungi, or fungi-like critters pictured up above edible, sought after, and delicious?

Yes, indeed.

 

Which ones can you eat? Where do you find them?

Absolutely not telling. You’d want to take a class or three on that, for sure.

 

Can I take classes on fungi and plants at EWU?

Yes! The EWU biology department offers a variety of classes on plants and fungi, including field mycology, lichenology, botany, plant physiology, and field botany. Sign up, fill your pockets with snacks, bring good walking shoes, and come join us!

EWU Graduate Students Present Research

The EWU Biology Department has many opportunities this spring to support our Biology Graduate Students and learn about their research! These presentations are open to all. Here is the current schedule:

Thursday, May 23rd:

Jade Clinkenbeard Prospectus: “An evaluation of aquatic plant and invertebrate communities in local wetland restoration projects,” at 3pm in SCI 247

 

Wednesday, May 29th:

Ethan Bean Prospectus: “Pseudoregneria Spicata: Selecting seed provenance for prairie restoration” at 12pm in SCI 244

 

Thursday, May 30th:

Abigail Keever Thesis Defense: “Sex-specific expressions and its effect on osteoclast differentiation” at 10am in SCI 247

Anwar Bushnaq Thesis Defense: “How Do Fine Sediments and Hangman Creek Discharge Affect Benthic Macroinvertabrates in the Spokane River?” at 3-5PM in SCI 243

 

Tuesday, June 4th:

Coty Jasper Thesis Defense: “Analyzing the Diet Composition of Lake Trout in Upper Priest Lake Idaho,” at 11am in SCI 247

 

Wednesday, June 5th:

Jared Lamm Thesis Defense: “Targeted Short-term Nutrient Reduction to Manage an Invasive Annual Grass: Soil and Plant Responses,” at 11am in PUB 321/323

Katie Johnson Prospectus: “Evolution of virulence after a host shift and serial passage of Drosophila C virus in Drosophila hosts,” at 2pm in SCI 243

 

Thursday, June 6th:

Marissa Medina Thesis Defense: “The effects of warming on carbon and microbial community dynamics in wetlands at Turnbull National Wildlife Refuge, Washington” at 1pm in SCI 247

Justin Donahue Thesis Defense: “High Resolution Modeling of Tick Density and Detection of Ricjettsia spp. in Dermacentor Ticks at Turnbull National Wildlife Refugeat 2:30 pm in SCI 280

 

Friday, June 7th:

Alexa Whipple Thesis Defense: “Riparian Resilience in the Face of Interacting Disturbances: Complex Interactions Between Wildfire, Erosion and Beaver (Castor canadensis) in Grazed Dryland Riparian Systems of Low Order Streams, North Central, WA” at 2pm in SCI 137

Lily Crytser Prospectus: “Measuring Variation in Body Morphology and Life History in Brook Stickleback in Eastern Washington,” 12pm in SCI 280.

 

Monday, June 10th:

Erik Peterson Prospectus: “Remnant Assessment and Soil Inoculation to Inform Large-scale Restoration at Eastern Washington University,” at 10am in SCI 244

Joseph Weirich Prospectus: “Beaver Moderated Fire Resilience in the North Cascades,” at 12pm in SCI 244.

Shelby Fettig Prospectus: “Effect of Nutrition on Honey Bee Gut Microbiome, Disease Occurrence, and Hive Growth,” at 3pm in SCI 247.

 

Tuesday, June 11th:

Philip Campos Prospectus: “Impact of Chytrid Fungus on the Skin Microbiome of Amphibians in Northern Idaho and Turnbull National Wildlife Refuge, WA,” at 10am in SCI 247

 

Thursday, June 13th:

Veronica Albrecht Prospectus: “Characterizing the role of sRNA-cagI in Helicobacter pylori gene regulation” at 1pm in SCI 247

Sarah Hill Prospectus: “Advancing the Field of Prairie Restoration in the Inland NM: A Community Needs Assessment and Forb Establishment,” at 4pm in SCI 246.

Darren Ginder Thesis Defense: “Improving Dopamine Monitoring with NCAM and the effects of Intranasal Oxytocin on Dopamine Signaling in the Rat Brain,” at 2PM in SCI 247

 

Friday, June 14th:

Brandon Flatgard Prospectus “Analysis of Helicobacter pylori
sRNA-cagII and sRNA-cagIII and Identification of Genes it Regulates” at 1 pm in SCI 247

 

*DISCLAIMER: This Schedule is subject to change, so please check it regularly if you plan on attending a presentation.*

EWU Grad Students: on the March Toward Science Outreach

Amy Gray

March for Science 1

As Bloomsday runners were busy at packet pickup on May 4th, the March for Science was in full swing. Grad student Veronica Albrecht (Dr. Castillo lab) and Christina Ramelow (Dr. Daberkow lab) kindly gave up the better part of their Saturday to show off microbes and explain to excited children how one can build an EWU Eagle on a Petri plate with pigment-producing bacteria. Their table was busy with kids and adults curious about skin microbes, endosymbionts in termites, and hypercolor media. Our students also found themselves unexpectedly – but enthusiastically – explaining hemolysis (where a microbe plated on a Petri dish with media containing animal blood produces hemolysins that actually break open the blood cells, leaving a zone that looks clear around the bacteria)

March for Science 2

… and Kirby-Bauer disk diffusion antibiotic tests (where paper disks impregnated with different antibiotics are placed on a Petri dish covered with bacteria, and a clear zone of bacterial death right around the paper tells you whether the microbe is sensitive to the drug)

March for Science 3

first to EWU and Gonzaga faculty, and then on… live radio.

Three cheers for Veronica and Christina’s enthusiasm about science outreach! You did us proud.

Drone Research at EWU Track Sediment Movement in Latah Creek

Dr. Jessica Allen

Sedimentation is one of the most common types of pollution in freshwater bodies, including rivers, streams and lakes. The negative impacts of high levels of sediment to organisms in the water are numerous, including destroying habitat for small animals, preventing larger animals from seeing their prey, suffocating fish, and blocking sunlight from reaching plants. Treating water with high concentrations of sediments is expensive, and water can still be left with an off flavor.

You don’t have to travel far to see the negative impacts of sedimentation. The Spokane River has been seriously impacted by many different types of pollution over the past century, including sedimentation. One major source of sediments for the Spokane River is Latah Creek, where upstream agricultural practices are washing large quantities of silt into the waterway. While it is clear that Latah Creek is a pollutions source for the Spokane River, what is less clear is just how much sediment is moving into the Spokane River and how it is moving in that body of water once it gets there.

How exactly is it possible to see how sediment moves through such a large river system? The short answer is drones.

Drones, unmanned aerial vehicles, aren’t only for hobbyists. They are frequently used in scientific research. Drones allow easy access to a bird’s-eye-view on landscapes, where they can be used for projects like tracking forest pests and predicting flooding. Research applications in wildlife management are also extensive, as drones provide some of the most accurate counts of animal populations.

Two EWU biology professors, Dr. Camille McNeely and Dr. Paul Spruell, along with graduate student Lily Cryster, are using a drone to track sediment flow from Latah Creek into the Spokane River. The aerial images they capture are used to monitor how much sediment flows out of Latah Creek, and the path it takes as it moves downriver. They will combine these data with water quality measurements to better understand how this sediment source affects water quality in the Spokane River. The project is funded by Rose Foundation’s Mike Chappell Fund for the Spokane River. By monitoring how sediments flow from Latah Creek into the Spokane River, EWU scientists hope to find solutions for cleaner water in our local river.

Below are images of sediment flowing from Latah Creek into the Spokane River take in March, 2019. Images were captured with the DJI Phantom 4 from 200 feet altitude.

 

Marcos Monteiro is EWU’s first Barry Goldwater Scholar

Andrea Castillo

Marcos Monteiro is Eastern Washington University’s first Barry Goldwater Scholar.

Marcos Monteiro

The Barry Goldwater Scholarship is one of the most competitive and prestigious STEM scholarships in the country. Marcos is among eight undergraduate students in Washington and 496 nation-wide to be awarded this scholarship!

What does it take to be a Barry Goldwater Scholar?

You must have impressive academic and research experience and go through a rigorous application process, including a nomination from your academic institution!

Marcos invests significant time and effort in his academic studies; after having taken multiple upper division biology courses and part of the organic chemistry series, his cumulative GPA is a 4.0.

Marcos’ research experience journey began last spring with an independent study in the lab of Dr. Andrea Castillo. Here, he worked with another undergraduate student, Laurisa Ankley, to study Manuka Honey antimicrobial mechanisms against the bacterial pathogens, including Pseudomonas aeruginosa. Because of his interest in research and pursuing a PhD in Microbiology/Molecular Biology, Marcos applied to the Ronald E. McNair Scholar’s and S-STEM Programs. The Ronald E. McNair Scholar’s Program, directed by Christina Torrez-García at EWU, provides scholarships and graduate school preparation for students interested in applying to PhD Programs. The S-STEM Scholarship Program, lead by Dr. Joanna Matos, provides financial support to undergraduate students so they can work less and invest time enriching their education through activities like independent studies. Marcos was successful with both applications and has benefited from the financial support and mentoring provided with these programs.

Last fall and winter, Marcos presented his work at multiple conferences. Notably, he gave oral presentations at the Murdock Undergraduate Research Conference and the West Coast Bacterial Physiologists Annual Asilomar Meeting. The data generated from Marcos’ experiments will contribute to a manuscript we are working to publish, “Manuka honey chelates iron and impacts iron regulation in key bacterial pathogens.”

After completing his Manuka Honey studies, Marcos started a new independent study project with Drs. Andrea Castillo and Javier Ochoa-Repáraz. He is working to engineer a probiotic that expresses high levels of the inhibitory neurotransmitter g-aminobutyric acid. This probiotic will be tested as a therapeutic to mitigate the negative symptoms of Multiple Sclerosis.

This summer Marcos will take a break from the probiotic project and travel to Seattle for an internship in the Fred Hutchinson Cancer Research Center Summer Undergraduate Research Program!

In this whirlwind of research opportunity and success, Marcos still needed to complete his B.S. in Biology. While considering scholarships to support another year at EWU, Marcos came across the Barry Goldwater Scholarship. He contacted the EWU Barry Goldwater Scholarship Campus-Representative, Associate Dean, Leslie Cornick and worked with Leslie and his mentors to submit the application.

This is what it takes to compete and be successful as a Barry Goldwater Scholar!

Congratulations Marcos!

The fate and future of prairie restoration at EWU

Sarah Hill, Graduate Student, Department of Biology

“OH NO! WE’VE GOT APHIDS!!!” That was the text I sent to my project partner, Alison
Last Friday. Another surprise bestowed on us by the fates of the greenhouse. For the last few months, Alison and I have been raising plants that will go into the prairie reconstruction project at EWU next fall, and every few days we are greeted with new opportunities and challenges. Some days the fates are kind – these are the days when you find a new species has germinated and is ready for transplant, or when you notice the first few supple cotyledons poking out of the soil. Sometimes the fates surprise you by revealing that the Wooly Sunflower (Eryophyllum lanatum) seeds that you were cold stratifying in the fridge are starting to germinate a month earlier than expected, and they need to be planted ASAP! Other days you have aphids, grow lights that are too low, or venting fans that accidently got turned off, leading to some very hot little seedlings. The ancient Greeks believed that the Fates did not pre-determine the destiny of a person, but rather would intervene at critical points with decisions that would either be helpful or harmful to the person’s future. In a similar way, the greenhouse Fates are not determining the outcomes of our plant propagation – but they sure are providing us with opportunities to make critical decisions that will affect the survival of our seedlings!

The surrounding community is also getting a chance to shape the future of the prairie at
Eastern. The same day that we discovered the aphids, Alison and I had been at Cheney Middle School, starting a germination experiment with the 7th grade science students in Mrs. Hansen’s class. This day built on a session we had with the students in the fall where these students planted In total almost 500 Arrowleaved Balsamroot (Balsamhoriza sagitatta) and Fernleaved lomatium (Lomatium dissectum) plugs for the restoration. Students that day were excited to take part in this historic project; when learning about the significance of EWU’s restoration and one student exclaimed “WAIT! You mean Cheney is special and important?!” When we revisited the students, among grumblings about the classroom smelling like dirt, we overheard students hypothesizing what was happening to the seeds they planted in the fall. Underneath their nonchalant attitudes, they were keenly interested in the fate of their seedlings. Other partnerships with Cheney Parks and Rec, the West Valley School District, Tekoa School district, and the Spokane Salish school are also underway to build community wide prairie appreciation, and to raise plants for the restoration here at Eastern. In the fall community members will be invited to lend a hand in the first round of prairie planting and laying the foundations for the work yet to come. As the plants grow up alongside the community members, they will be able to take future generations out for a stroll among the hills, and recall their roles in building biodiversity in Cheney.

Raising prairie plants is an exercise in patience and humility. Plants do not necessary operate on timescales amenable to contemporary human schedules, and we are still learning the best way to put back the pieces of the prairie in this region. Having a large scale restoration project on campus at Eastern will be a boon to prairie restoration throughout the region. The research possibilities are endless, as is the amount of information this project can contribute to our understanding of prairie ecology and restoration techniques. My time at Eastern will have ended long before much of the restoration project has been completed, but I hold onto the vision of a functional prairie with a diverse array of grasses, wildflowers, and wildlife. Appreciating the slow march into the future makes the first unfurling of a true leaf thrilling, gives me the patience to squish thousands of aphids, and encourages me to embrace all the learning opportunities the fates send my way.

A Deep Dive into a Graduate Student’s Research Experience

Josh Chastek

 

josh in the lab

My name is Josh Chastek. I am a native Spokanite, science enthusiast, and outdoor adventurer. I enjoy backpacking, photography, orchids, and airplanes. I am also interested in genetics, microscopy, and cell culture. I completed my undergraduate degree at EWU and continued at EWU because I was not (and am not) done learning about biology.

My initial focus in biology was with cryobiology, the study of the effects freezing temperatures on living tissues. That interest brought me into contact with Dr. Charlie Herr, with whom I now work. The Herr lab focuses on the development of technologies for endangered species preservation. The kinds of research I’ve been able to participate in have transformed my interest in biology into a love of cell culture systems and technology development. During my time as a graduate student, I have worked on freezing fish sperm and insect embryos. I have also worked on culture system development for growth of reproductive organs in fish, cats, dogs, Drosophila (fruit flies), and silkworms.

One of the coolest aspects of working at a university is getting undergraduates involved in real world research. Some of our research includes:

  • The development of reproductive cell culture technologies.

We have worked on optimizing cell culture systems to grow tissues of plants, vertebrates, and invertebrates. Our insect research has included using honey bees, Drosophila species, and silkworms as models. Prior to my arrival, Dr. Herr had developed technology for the long-term storage of honey bee semen. This technology (combined with the ability to artificially inseminate honey bees) gives researchers the ability to bring in bee semen from around the world and produce hives with greater genetic diversity.

  • The development of cell culture systems for the storage of insect gametes and reproductive tissues.

This is an important issue because at this time there are no genetic storage techniques for many species of insects. Drosophila have been an important research model for the last century (Jennings 2011). Current and future drosophila research depends on the maintenance of many, some 40,000 plus, different fly strains. No reliable cryogenic methods have been developed to store gametes or embryos and all of these stocks must be maintained alive. There are large resource and time costs associated with maintaining these stocks. Our lab has been using what was learned from freezing honey bee sperm to develop methods for freezing Drosophila gametes and embryos.

josh blog image 3

Live/dead cell stain of drosophila embryo

  • The optimization of cell culture systems for growing insect reproductive organs to use for in vitro fertilization experiments.

silmoth portrait

Another insect model we work with in our lab is the silkworm, Bombyx mori. Silkworms are important for the luxury resource they provide as well as their role as research model. They are specialized feeders who only eat the leaves of mulberry trees and can no longer be found in the wild. Due to their extended time in domestication (over 5000 years) silkworms are extremely susceptible to pathogens and environmental irregularities. Technologies to store their genetics would be beneficial to the world’s silk producers and researchers. We have reason to believe that because of their unique mating style, silkworms may be a good insect model to attempt in vitro fertilization with. In order to successfully produce progeny, in vitro, mature eggs and sperm must be collected and mixed together in media that will support them. Silkworms mate directly after emerging as moths, if they can find a mate, suggesting that the male and female gametes are mature and ready for fertilizing after emerging from their cocoon. Mating moths will connect for 12-24 hours and during this time the male will use a peristaltic pumping action to mix his sperm with all of the females eggs. The moths will separate after the sperm transfer which is followed by the female depositing all of the fertilized eggs. This is different than other insects like Drosophila who use individual sperm to fertilize eggs as they are produced and matured over the insects lifespan. Using information gained from research using honey bees and drosophila, we have been working to develop a cell culture system that allows for the survival of silkworm gametes and reproductive tissues. Optimizing a media to mix the sperm and eggs in has been the main focus of our silkworm research.

In the fall of 2018 we established a silkworm colony here at EWU. The goals of building the colony were to learn how maintain a population of silkworms, and also to be able to provide silkworms at all stages of their life cycle to do research with. We were successful in raising and mating silkworms. Undergraduate researcher Rachael Doty helped establish and maintain the colony (thanks Rachael)!

We were not able to attempt any in vitro fertilization trials last year, but we were able to keep silkworm testicular tissue alive in one of two of our selected cell culture media. We are starting another colony this spring and will use what we learned from the previous experiments to attempt in vitro fertilization this quarter. Once we have optimized a system to allow for fertilization we would like to begin working on freezing the sperm, eggs, and fertilized embryos.

If you, like me, have a passion for research that hasn’t been sated by an undergraduate degree, you should consider continuing your education. Acquiring an MS will not only give you new tools and some teaching experience, but will allow you to dive deep into a research experience.

Works cited:

1).B. H. Jennings, Drosophila – a versatile model in biology & medicine, Mater. Today, 2011, 14, 190–195

https://www.sciencedirect.com/science/article/pii/S1369702111701134

2).Hopkins, Brandon and Herr, Charles.(2010). Factors affecting the successful cryopreservation of honey bee spermatozoa. Apidologie 41: 548-556.

https://www.apidologie.org/articles/apido/full_html/2010/05/m09054/m09054.html

3).Hopkins, Brandon & Cobey, Susan & Herr, Charles & Sheppard, Walter. (2016). Gel-coated tubes extend above-freezing storage of honey bee (Apis mellifera) semen to 439 days with production of fertilised offspring. Reproduction, fertility, and development.

https://www.ncbi.nlm.nih.gov/pubmed/27977389

4).Stucky M., Hopkins BK, Mr. C. (2008) Cryopreservation of honey bee spermatozoa,     Reprod. Fert. Dev. 20, 127-128.

https://doi.org/10.1071/RDv20n1Ab94

5). Hopkins Brandon K., Herr Charles, Sheppard Walter S. (2012) Sequential generations of honey bee (Apis mellifera) queens produced using cryopreserved semen. Reproduction, Fertility and Development 24, 1079-1083.

http://www.publish.csiro.au/RD/RD11088