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The COVID-19 PandemicJohn Sudsbury2020-12-17T17:43:21-06:00
The Mercedes-Benz Superdome, known as one of the loudest stadiums in the bowl world, found itself at a much-lower volume level in 2020. The New Orleans Saints, whose deafening crowds have given them a decided home-field advantage, played their home games with several thousand fans, at most, in 2020. Photo by Derick E. Hingle.
The Allstate Sugar Bowl Has the Wherewithal to Weather Adversity
by Trey Iles for the Allstate Sugar Bowl, December 17, 2020
In its 87-year existence, the Allstate Sugar Bowl has seen its share of history and been through plenty of adversity. Born out of the depression, the Sugar Bowl has witnessed and endured World War II, the assassination of President John F. Kennedy, numerous economic downturns, the evolution of college football's postseason and, perhaps its biggest test, Hurricane Katrina in 2005-06.
But the current COVID-19 pandemic may top them all or, at least, equal the worst of times. When Sugar Bowl President Ralph Capitelli took the reins of the organization in February in 2020, he noted there would be challenges as there were every year. But not even he could have imagined what was to come.
When the effects of the pandemic cascaded in the middle of March of 2020, life all over the world was altered. It certainly has had a profound effect on the Sugar Bowl as well. It's upended the organization's budget, altered – several times over – how the event will take place and cancelled many of the satellite functions in which the Sugar Bowl takes as much pride in putting on as the game itself.
ASM Global, the management company for the Mercedes-Benz Superdome, developed the VenueShield plan, a best-in-class hygienic re-opening plan, to ensure fan-safety in the pandemic. VenueShield utilizes electrostatic cleaning, a state-of-the-art technology that creates a germ-free between people's hands and all surfaces they touch in the building.
"Every day a new dip or turn," Capitelli said. "It's been quite a challenge. I saw (former Sugar Bowl President) Mark Romig a while back. He was the president the year of Katrina when we had to move the game to Atlanta. Of course, everybody thought that his year was the most unusual year because of the storm's aftermath. He looked at me and said, 'You topped me.' "
As difficult as this year has been, Sugar Bowl Chief Executive Officer Jeff Hundley said facing the battles of the past have allowed the organization and its staff to work through the many challenges posed by the unrelenting pandemic. Heretofore, Katrina was the benchmark for the organization when it came to overcoming adversity. Yet despite having to move the game to Atlanta and navigate through the ruins, both economic and physical in the New Orleans area, the Sugar Bowl emerged as strong as it ever was.
"In speaking to our membership a couple of months ago, I drew back on times over the history where we've had to adapt and overcome," Hundley said. "An organization this old doesn't stay as relevant as it has for decades without being able to adapt to change and circumstances. That's a credit to the committee members and leadership here. They are willing and able to do so.
"There are bowl games around the country that have elected not to play. Fortunately, we're in the position of being able to push forward until the finish line or until the virus says we can't."
College Football Playoff Executive Director Bill Hancock said no bowl or organization is better suited to face the adversity presented by the pandemic than the Sugar Bowl because of its history.
"The Sugar Bowl staff and committee are terrific," Hancock said. "You can always count on them to calmly and thoroughly assess every situation. They have been adaptable and thoughtful throughout the pandemic — and always, for that matter.
"Everyone in college football learned from the example that the Sugar Bowl folks set in responding to Katrina. (Former Sugar Bowl CEO) Paul Hoolahan, Jeff Hundley and the committee quickly rose to the challenge then, teaching us all how to deal with the unthinkable. I particularly appreciate their good humor and spirit. Whatever cards are dealt to them, they play it quite well."
But it certainly has not been at all easy. And when the pandemic finally ends, it will leave scars on the game as it will every person and facet of life around the world.
"This has been a constantly shifting and evolving situation that has had us planning and re-planning," Hundley said. "Every time you think you have your arms around a certain topic or subject, something changes. And you've got to rethink it or plan again. From the time this started to where we are now, we've probably done almost 20 different budgets just trying to prepare and cut back."
Through seasoned leadership over the last 30 years, the Sugar Bowl has been an outstanding steward of its resources. That's a vital reason as to why it has remained one of college football's premiere bowl games and part of the College Football Playoff.
But the pandemic is having a profound effect on those resources. Last year, the Sugar Bowl was the primary host of the College Football Playoff National Championship in which LSU defeated Clemson to cap off a storybook season. But that came with a significant cost. The combination of putting on the regular Sugar Bowl game, which featured Baylor and Georgia, as well as the title contest cost the Sugar Bowl organization about $10 million.
"We were looking to get a small portion of that back this year," said Hundley, who noted this year's game is a CFP semifinal contest. "That's not likely to happen. Now the exercise is to try and break even. But even that is out of our control. Even into December, we don't know how many fans we'll be able to have in the stadium on game day [NOTE: that number is expected to be 3,000]. That all goes to the bottom line. So it's a little different deal that we're having to accept. It's not easy for an organization that has a tried-and-true template on how to do it."
Not being able to roll out the full Sugar Bowl experience for the teams, support personnel and fans has been equally disappointing, Capitelli and Hundley said.
"As it turns out now, we may have the game and not much else," Capitelli said. "That part is so disappointing. The people who make up our organization love college football. They love interacting with the schools and the coaches and the administrators and the fans that come. This year the team will be in a bubble and we won't be able to do any of that. It's hard. But everyone has pulled together and I'm proud of that."
Another disappointing part of the equation is not being able to hold the numerous satellite events. The Sugar Bowl puts on more than 50 events during the year and supports and honors thousands of student-athletes each year. The organization takes as much pride in those aspects as it does the actual game.
"That's been one of the hardest pills to swallow," Hundley said. "We understand the situation but it doesn't make the loss any less. The Sugar Bowl committee is a volunteer organization. The payoff for them is to produce and participate in all the events that go around the game and leading up to the game and having access to the teams and coaches which we won't have. It's unfortunate. But another fact life in this COVID time. We've all adapted and moved on. There is a sense of disappointment. But we'll rally and put a smile on and finish this thing off."
Among the duties for this year's game is preparing the teams, Sugar Bowl staff and all those who assist with the game with COVID preparation and protocols. Hundley said having Tulane's Dr. Gregory Stewart assist with that has been invaluable. Stewart was one of the leaders in helping the American Athletic Conference develop its COVID protocols for its athletes and support staff.
"None of us had any feeling for COVID protocol at the time when it started," Hundley said. "Luckily, we've gotten a lot of help from Dr. Stewart. He's been a tremendous resource.
"And even the development of those protocols has changed over time. We think of things that we didn't early on. Testing for teams, testing everybody involved with game management. Testing for Sugar Bowl staff during the month of December and leading up to the game. We can't afford to have an outbreak in the office and not be able to produce the game when the time comes. It has been interesting to say the least."
In addition, the Sugar Bowl has called upon the expertise of the College Football Playoff organization as well as the conferences that make up the Football Bowl Subdivision. Learning from them has helped the Sugar Bowl develop its plan to hold this year's contest.
"Bill Hancock and his staff have been another set of eyes for us," Hundley said. "That's because of their access to the conference commissioners, they have great access to the medical reports and experts that each conference uses. That has been helpful as we've gone through this process. They've had to be flexible as well. We have had weekly calls with (Hancock's) staff for several months now going back to the summer. We have been able to devise a plan that we believe will work."
While the College Football Playoff will be different this year, Hancock said it will be no less compelling than the previous editions of what has captivated the college football world.
"Of course, fewer people will be in the stadium," Hancock said. "The teams will be in town only two days as compared to five or six in the past. But the players and coaches have worked hard all season, with the semifinals as the goal. I like to emphasize the experience for the players; certainly they will all remember 2020, including the days they spend in New Orleans."
Capitelli said he's proud of how the entire organization has reacted to the pandemic. The effort has been above and beyond the call of duty, he said, and with many in the organization dealing with the effects of the pandemic on themselves and their families.
"I want to compliment Jeff and the entire staff for being so resilient and working through all these issues and be able to adjust on the fly every day," Capitelli said. "And our whole committee has been supportive. We've tackled other problems in the past and you can see that experience come through as we've gone through the pandemic.
"I believe that, despite all the tough headwinds we've faced, we'll still show the best face of New Orleans. We're resilient. And this may not be a year that we like but you'll see us back in the future. I think even in this limited capacity, we'll still put on as good an event as we possibly can."
A half-million dollar donation by the Sugar Bowl helped organizations like Second Harvest Food Bank provide aid to assist the people in Louisiana hardest hit by the pandemic. Photo by Jay Vise.
Allstate Sugar Bowl Aids Pandemic Relief Effort with $500,000 Contribution
The Sugar Bowl Committee, which has been a part of the fabric of New Orleans since the organization's inception in 1934, made a $500,000 donation to multiple local organizations working to alleviate the challenges of the current COVID-19 crisis.
The Allstate Sugar Bowl's donation went to assist the Second Harvest Food Bank, the Greater New Orleans Foundation's Service and Hospitality Family Assistance Fund and the Hospitality Cares Pandemic Response Fund (managed by United Way of Southeastern Louisiana).
"For decades, the Sugar Bowl has taken great pride as a good citizen of our local community," said Sugar Bowl Committee President Ralph Capitelli at the time of the donation. "This organization's primary mission is to generate tourism through amateur athletics. With the Covid-19 outbreak we're temporarily on hold, but that doesn't mean we can sit on the sidelines. With this donation, our aim is to help people in need, especially those in the hospitality industry whose livelihoods have been so negatively impacted by the Coronavirus."
Second Harvest Food Bank is the largest charitable anti-hunger network in South Louisiana. Its mission is to end hunger by providing food, access, advocacy, education and disaster response. The Service and Hospitality Family Assistance Fund of the Greater New Orleans Foundation is a program providing grants for service and hospitality workers whose families are experiencing financial crisis due to the Covid-19 outbreak. The Hospitality Cares Pandemic Relief Fund of the United Way of Southeastern Louisiana provides crisis grants to hospitality workers who are unable to afford basic financial needs during the Covid-19 outbreak.
"United Way of Southeast Louisiana is grateful for the Sugar Bowl Committee's contribution to our Hospitality Cares Pandemic Response Fund as it further demonstrates the committee's longstanding dedication to the betterment of our community," said Michael Williamson, President and CEO, United Way of Southeastern Louisiana. "The support will improve the well-being of 200 families across our region – for that, we are forever thankful."
"We are so excited that the Allstate Sugar Bowl, an annual supporter of our Foundation, was able to help increase the impact of the Hospitality Cares fund," said Jennifer Kelley, Executive Director, Louisiana Hospitality Foundation, which works in conjunction with the United Way of Southeastern Louisiana. "This gift shows their passion to support our hospitality workforce that has been displaced from their jobs due to the pandemic."
"This incredible gift of love will help us provide 1.2 million emergency meals to our neighbors in need," Natalie Jayroe, President and CEO of Second Harvest said. "During this response, we've had to purchase more food than ever before. Once again, this shows that sports is so much more than just a game to New Orleans and South Louisiana."


Elsa is starting a new cosmetic salon and there are clients coming from all the corners of the world. Today she gets a special visit from Ariel the mermaid. Help Elsa give Ariel a new trendy look with a cool face painting and makeup.


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Boone schools considering consolidation
October-17-2016
By Ryan Quinn, The Charleston Gazette-Mail
Boone County's cash-strapped public school system, which closed three elementary schools at the end of last school year, posted notices Friday for a public hearing on a proposed closure of Whitesville Elementary.
The hearing is planned for 6 p.m. Nov. 15 at the school, located on W.Va. 3/Boone Street in the town of Whitesville.
Whitesville's students would be...
PEIA takes another troubling proposal out to public hearings
PEIA board takes another troubling proposal out to public hearings
By WVEA Communications staff
Active state employees could face $25 million in benefit cuts and retirees face about $17 million in cuts to PEIA in the coming year.
The PEIA Finance Board voted Thursday to take a draft of a fiscal year 2018 plan out on public comment next month.
This is another unacceptable proposal that solely burdens employees, retirees and their families with the...
State board changes science testing grades, sets A-F cut scores
The West Virginia Board of Education voted Wednesday to change the grade levels in which public school students take end-of-year science standardized tests and set cut scores to determine the broader grades for entire schools in the upcoming A-F accountability system.
Board members approved both moves in voice votes with no nays heard. Of the nine...
State Board of Education makes changes to science test administration
The general science assessment that has been administered in grade 4 will be administered to fifth-grade students this spring, and the science assessment previously administered in grade 6 will be given to eighth graders in the spring. Students in 10th grade will continue to take the high school general science assessment in that grade.
The state Board of...
Democratic candidate for auditor wants biweekly pay survey
By Eric Eyre, The Charleston Gazette-Mail
If elected, the Democratic candidate for West Virginia auditor said Tuesday she would commission a survey of state employees about the state's ongoing switch to a new payroll system.
After a change to a 26-week pay-period system, most state workers receive their paychecks every two weeks instead of twice a month. Many employees have grumbled they're being...
WVEA Communications

redpajama_set_name: RedPajamaCommonCrawl


In and out of the classroom, life would be unthinkable without interacting with fellow humans. This book urges more cooperative and group activities in the English language classroom.
In and out of the classroom, life would be unthinkable without interacting with fellow humans. This book urges more cooperative and group activities in the English language classroom for all the advantages: students use the target language more, help each other with comprehension, receive attention from peers as well as the teacher, are motivated by group support, learn collaborative skills that transfer to life, and get to work with people different from themselves. The book also provides guidelines for problems that may arise when teachers do group activities, principles for interaction and bonding in the English language classroom , and some easy activities to implement in your classroom TODAY!


Heather Giustino Blog: We built a well!
I am over the moon, that we have fully funded a well! Now that we've funded one, the goal is to fund two! I really can't word things better than Julie, so I won't even try. Check out her post. There is still time to donate, and time to win some really amazing things!
To quote Maya Angelow "Giving liberates the soul of the giver"
shut up!!!! that is AMAZING heather!! i feel blessed you just having you in my life. you are so giving and have such a heart of gold. this is seriously amazing.


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360songwriting.com
Book Stuff
Experiences With The Book
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Ask the Prof
Here in the "Ask the Prof" area I'll post replies to questions about songwriting, tunewriting, collaboration, and teaching—with a focus on teaching songwriting and artistry, my particular bailiwick. You'll find specific responses or questions about my book, Songwriting Strategies: A 360o Approach, in the BOOK STUFF Tab, under "Experiences with the Book." But the book condenses a large body of techniques, exercises and tools I've developed or adapted from others over the years; I hope to explore this material in more depth, based on the interests of readers and fellow songwriters.
From Keppie Coutts
My first entry question comes from my friend and colleague Keppie Coutts. This question is typical of Keppie's far-reaching perspective and endless curiousity.
I met Keppie when she was a student at Berklee. We worked together on the Songs for Social Change contest that ran for many years, continuing that work as Keppie moved into a staff role with the Office of Cultural Diversity.
Later she taught in the Songwriting Department for a few semesters, then took her keen insights and passion for songwriting off to LA, and eventually back to her native Australia. She's now inaugurating a new songwriting course at the Australian Institute of Music in Sydney.
(It was Jimmy Barnes in Australia who started calling me "The Prof" by the way; and I adopt the moniker here as a fond nod to him.)
I'd like to have some different reference points for a discussion about different approaches to lyric writing. So I have a songwritey-teachy-type question for you:
What types of songs are there?
I'm not here referring to musical genre (although of course that will influence lyrical content), but more like:
Narrative songs
Songs centered around a metaphor for an emotion Abstract songs etc.
These are kind of random examples, and I know this is a highly subjective question, but I'm interested in how you would put contemporary songs into different 'types' or even 'approaches', if any... Warmth, Keppie
My kind of question! If Aristotle were a songwriter, he'd be all over this like a cheap suit! Since I know Keppie is focused for the present on lyric content, I'll answer her question not with a fixed set of categories but with some "polarities" I work with as both songwriter and teacher.
I do think narrative or "story songs" are a distinct genre that poses very specific challenges of form and skill for the songwriter. It's generally an important skill for a songwriter to be able to hear their song from the listener's point of view, asking what part of the story is being revealed with each line. But this skill is critical in writing a good story song, where you are counting on a clear unfolding narrative.
What is the complementary kind of song to a narrative song? There are songs that get called "situational" songs, describing a snapshot of a moment and an emotional situation, usually involving the Singer (in the world of the song) and someone else. Often that someone else is the Sung-To, and so situational songs work well in combination with first-person or "direct address" voice. For similar reasons, narrative songs can work well in third person, with the singer a narrator commenting from outside the world of the song.
One thing that makes a polarity more useful than fixed categories or a dichotomy is that you can acknowledge the seamless, organic connections to the principles involved. So, even a strict situational song tends to have at least a nod to "back story" which brings in some elements of the past to help motivate the current scene. (A second verse is often where this occurs.) Even when the back story doesn't show up in the song itself, the writer (and especially co-writers) may need to think or talk through that back story to write a clear song, with a consistent character's persona and point of view expressed. And similarly, a situational song may include a leap of expectation or worries toward the future (often in a bridge or last verse).
Just as a situational song has narrative components, most narrative songs proceed a bit like movies, as a procession of "scenes" or episodes each of which can have the immediacy of a situational encounter. Old traditional narrative ballads in particular work in that fashion, mixing dialogue (often highly patterned and formulaic) with fast-montage summaries of events, interspersed with evocative descriptions—of milk-white steeds, maidens, glinting swords and occasionally snuffling boars coming out of the woods. So narrative and situational songs can be seen as a continuum.
That's one polarity: call it NARRATIVE – SITUATIONAL. Another polarity involves the use of metaphor in the song. Here I distinguish songs that are primarily descriptions of outward events (which may use passing metaphors as a way of intensifying that description) from songs that are primarily reports of interior states of thought and feeling. Compare, for example, "Poncho and Lefty" by Townes Van Zandt (also a narrative song) and "I Will Always Love You" by Dolly Parton, so memorably covered by Whitney Houston. In between these end-points, I think, lies a beautiful world of songs built around a central metaphor, often found in the Great American Songbook canon: "Skylark" by Hoagie Carmichael and Johnny Mercer.
As with our other polarity, most songs move between these elements in a complex and delightful dance. Andrea Stolpe, in her book Popular Lyric Writing, first turned me on to pay attention to shifts between exterior and interior language in lyric structure. I augmented her framework by considering metaphorical lines as a third, intermediate texture. You can see these distinctions also at the level of the entire song. Then there are songs written as exterior narratives, but where the landscape as a whole is metaphorical, a dreamscape. Like Dylan's "All Along the Watchtower": somehow we know those two riders approaching have a lot to say, and that the howling wind ain't just the wind.
I could spin polarities endlessly, but I often find a small set of them are best to work with, especially when you begin to crisscross and tug at them. So the third polarity for "kinds of songs" (in lyric terms) I'll offer here is the polarity between songs that are intended to make referential "sense" with their lyrics, vs. songs that are about the sound of the lyrics. We're engaged in some discussions now in Berklee's Songwriting department about how to give appropriate honor to great dance songs, for example, with amazing melodies and progressions, but where the lyrics are not setting out to be metaphor-drenched, soul-baring threnodies. Sometimes we mis- speak in these discussions and say that in these latter types of songs, lyrics "don't matter." I prefer to say they matter a different way; and often they are generated via different processes as well.
Some beloved songs of mine have cryptic lyrics that still affect me deeply on many levels. Many songs of Neil Finn (the great New Zealand songwriter from Crowded House) strike me this way. I don't judge such songs as inferior because I can't "understand" all the lyrics in rational terms; nor do I believe my experience of those songs would be as rich with no lyrics present, or with any arbitrary lyrics.
We could compare this, perhaps, to the tension between representational and non- representational visual arts. With that comparison in mind, it is useful (though mysterious) to ask: how can we tell great non-representational lyrics from piss-poor first-cuff blather? My answer is: listen to the song, and pay careful attention to how every element of the song affects you as a listener. Then you'll know.
Now we have a starter set of three polarities to work with:
NARRATIVE — SITUATIONAL
DESCRIPTIVE/EXTERIOR — METAPHORICAL — ABSTRACT/INTERIOR
LYRICS BASED ON SENSE — LYRICS BASED ON SOUND
Now we can start to play with these dimensions or aspects, in light of specific examples and new writing challenges we can set ourselves. As you reflect on these and other polarities for lyric content, try to find aspects that, in your mind, are tightly locked together, and see if you can split them into new possibilities that stretch you as a writer.
For example, one of the clearest linkages is between narrative lyric content and a focus on outer, descriptive language. So, how about:
Can we write a narrative/abstract song? One that doesn't describe outer, but inner events, yet creates a clear narrative progression of events, a passing of time and consequences, in that emotional journey?
Can we write a narrative song based on a central metaphor? Many of the classic central-metaphor songs were situational in perspective. Or a narrative song that takes place in the metaphorical landscape, or a metaphorical song where changes to the metaphorical focus change over time?
Can we write a narrative song based on sound-driven lyrics? Where we are sure we are hearing a story, but don't have clear descriptive references for that sense of a story?
Write a situational song not as direct address but as a third-person narrative. Consider the beautiful traditional song "As I Roved Out" recorded by the iconic Irish band Planxty: an overheard conversation between two lovers. How does the presence of the listening narrator or observer change the meaning and emotion of this song? Can you write one of these for yourself?
Categories and distinctions are useful to artists when they liberate rather than confine, challenge and stretch rather than limit us. It's all in the way we use them. Aristotle, go back to bed!
KEPPIE'S REPLY:
I like the concept of thinking in polarities or on some kind of dimension. It helps for example, within a 'Narrative' framework, in which one might be tempted to pose a set of 'categories' that sub-divide it: Story songs (strict narrative, third person, clear sense of beginning-middle-end), Persona Songs (first person fictional), Temporal Specificity (years? weeks? days? 5 minutes?)...
I think 'temporal specificity' is where the concept of dimensionality is really useful - because it can be overlaid onto the other two subdivisions...and at the extreme end of that spectrum - as in a 'situation' that happens within 3 seconds - what we are really talking about might be that start of a new 'type': 'internal state'. Once we are in 'internal state', then we can overlay another dimension, 'descriptive - abstract'...
I think it is useful to consider how people approach these in terms of song function as well. For example, if we are talking about songs for film/TV, 'narrative' songs can actually be very ill-fitted to that particular market. And only certain types of metaphor (simple ones) are going to be appropriate.
In that setting, music supervisors are usually looking for a song (with both lyric and music being equally weighted I would argue) that perform 2 key functions:
1) Create an immediate and identifiable mood/emotion
2) Summarize a key emotion or relationship with a simple line or hook (that can be 'turned up' under the scene at the right moment)
And what they are looking to avoid are any details that tell a story that would in any way conflict or compete with the story being told by the visual. Which poses problems for 'narrative' song!
I think the question of what distinguishes the slop from the good stuff, with respect to 'sound based' or 'abstract' lyrical content is worthy of more investigation! Why do we still feel so much, and as you said, feel we have been taken through a story, even when we don't know exactly what has happened in a literal sense? What kinds of ambiguities are genuinely productive, and which are simply vague nothings? I think Dylan's 'Subterranean Homesick Blues' is another good example of that (unless there is some definitive 'translation' of that one that I don't know about...)
It would be wonderful to get feedback from readers about these ideas, specifically references to example songs that seem to lie in one zone or the other. Keppie's point about the function and context of songs in use is really well taken. My friend and co- writer Lisa Aschmann has had many songs placed in film and TV synch usages. She's observed a similar thing about the lyric requirements of songs that will be used as underscore for a visual medium and especially one with its own dramatic narrative. Songs that might appear vague or narratively weak taken on their own are sometimes exactly what the music supervisors are looking for: the scene already provides the "furniture" and the narrative. The song provides the emotional undercurrents, and if there is imagery in the song it's likely better if that imagery is metaphorical than literal and descriptive. So the discussion continues ...
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The latest Steam client beta includes a face-saving new feature; the ability to shovel rubbish games away into a "hidden" category. Joining the Steam beta is a pretty simple affair, you just have to find the option to do so under Settings and then Account.
To hide any games you might be ashamed of, head to your library, right-click on the offending article and choose the 'Set Categories' option. There'll be a little box in the bottom left hand corner for you to check if you wish to hide said game.
In other beta client news, Steam has been given a slightly new look. Or "neutralizing overall color palette" if you go by the update notes. In straightforward terms that means Steam has got a bit of a dark blue hue now, and seems to have a circular icon in the taskbar notification area which is, frankly, alarming. Change is frightening and confusing.


Following are a few documents that this Key Club uses often. Also, if there is an event that requires our members to miss school the permission slips will be found below.
Key Club keeps record of member activeness through attendance sheets, and if you find that our binder needs another sheet it can be downloaded here.
When Key Club spends money, you need to fill out this form and get your request approved by the Treasure, both Co-Presidents, and our Kiwanis Adviser.
This sheet is used at events such as club fair, and helps us to keep our email/ facebook listing up to date.
If you plan on attending the beach clean up on Oct. 18. 2014, you need to print and fill out this waiver.


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Authors: Marta Katarzyna Sawadro aff001; Agata Wanda Bednarek aff001; Agnieszka Ewa Molenda aff001; Agnieszka Izabela Babczyńska aff001
Authors place of work: Department of Animal Physiology and Ecotoxicology, University of Silesia in Katowice, Bankowa, Katowice, Poland aff001
Allatoregulatory neuropeptides are multifunctional proteins that take part in the synthesis and secretion of juvenile hormones. In insects, allatostatins are inhibitors of juvenile hormone biosynthesis in the corpora allata while allatotropins, act as stimulators. By quantitative real-time PCR, we analyzed the gene expression of allatostatin A (PtASTA), allatostatin B (PtASTB), allatostatin C (PtASTC), allatotropin (PtAT) and their receptors (PtASTA-R, PtASTB-R, PtASTC-R, PtAT-R) in various tissues in different age groups of female spiders. In the presented manuscript, the presence of allatostatin A, allatostatin C, and allatotropin are reported in females of the spider P. tepidariorum. The obtained results indicated substantial differences in gene expression levels for allatoregulatory neuropeptides and their receptors in the different tissues. Additionally, the gene expression levels also varied depending on the female age. Strong expression was observed coinciding with sexual maturation in the neuroendocrine and nervous system, and to a lower extent in the digestive tissues and ovaries. Reverse trends were observed for the expression of genes encoding the receptors of these neuropeptides. In conclusion, our study is the first hint that the site of synthesis and secretion is fulfilled by similar structures as observed in other arthropods. In addition, the results of the analysis of spider physiology give evidence that the general functions like regulation of the juvenile hormone synthesis, regulation of the digestive tract and ovaries action, control of vitellogenesis process by the neuropeptides seem to be conserved among arthropods and are the milestone to future functional studies.
Biology and life sciences – Biochemistry – Neurochemistry – Neurochemicals – Neuropeptides – Hormones – Peptide hormones – Neuroscience – Genetics – Gene expression – Anatomy – Nervous system – Reproductive system – Ovaries – Organisms – Eukaryota – Animals – Invertebrates – Arthropoda – Arachnida – Spiders – Insects – Medicine and health sciences – Research and analysis methods – Database and informatics methods – Bioinformatics – Sequence analysis – Sequence motif analysis
Neuropeptides represent the largest class of signal compounds, which also include steroids and sesquiterpenoids. Based on protein sequence similarity and main physiological functions, insect neuropeptides are grouped into families. One of the largest neuropeptide family comprise the allatoregulatory neuropeptides [1, 2, 3].
Allatoregulatory neuropeptides are multifunctional proteins with conserved protein sequences [4]. They are commonly found in insects from which they were isolated for the first time. They have also been identified in various groups of arthropods as well as in non-arthropod groups such as annelids and mollusks: Cancer borealis [5], Carcinus maenas [6], Triops newberryi [7], Penaeus monodon [8], Homarus americanus [9], Deroceras reticulatum [10, 11, 12]. Numerous manuscripts report demonstrating allatoregulatory reactivity in basal metazoans [13]. In addition to conservation of protein sequence, this further indicates, that allatoregulatory signaling might be conserved among metazoans, and thus it might be an ancestral mechanism. Insects are often regarded as a reference group for studies of allatoregulatory peptides in other groups of arthropods due to in insects allatoregulatory neuropeptides have been described in most detail so far. Based on these studies in insects, one of the primary roles of allatoregulatory neuropeptides is the regulation of the juvenile hormones (JHs) biosynthesis. JHs in insects (and methyl farnesoate–a standard product in JH synthesis in insects, serve different functions in arthropods [14, 15]) control crucial processes such as molting, metamorphosis, ovary development, vitellogenesis, mating behavior, and oviposition. Recent studies also confirmed the role of JHs in morphogenesis and caste determination in social insects [16]. Therefore, allatoregulatory neuropeptides indirectly affect these JH-dependent processes.
Allatostatins (ASTs) have an inhibitory effect, whereas allatotropins (ATs) stimulate biosynthesis and release of the JHs by the corpora allata (CA) [2, 17, 18, 19, 20]. ASTs are a superfamily of invertebrate neuropeptides that were initially defined by their action as inhibitors of JH biosynthesis in vivo. To date, more than 60 types of ASTs have been isolated and characterized from a variety of insect species. These peptides can be classified into three groups: the FGL-allatostatin (A type), the W(X)6W allatostatin (B type), and the lepidopteran (Manduca sexta) allatostatin (C type) [19] while allatotropins are not divided into subgroups. They were first isolated from M. sexta (Manse-AT), and their sequence (GFKNVEMMTARGF-NH2) was confirmed based on cDNA and was characterized in other insects like Spodoptera frugiperda as well as Aedes aegypti [11].
Among arachnids, ASTs have been identified in ticks. A-type ASTs were detected in the synganglion of Dermacentor variabilis [21], whereas AST C (Manse-AST) was described in the central nervous system of Ixodes scapularis [22]. Despite the detection of these peptides in ticks, their role has not been revealed. Furthermore, there are only a few publications that are based on the immunohistochemical data, AST A protein localization in the central nervous system of the model spider species Cupiennius salei [23]. However, the quoted data do not provide information on the role of ASTs and AT in spiders (compare with [11]).
In insects, in which allatoregulatory neuropeptides are relatively well-known in comparison with other arthropods, neuropeptides are produced and secreted in the central nervous system by neurosecretory cells and interneurons [2, 17, 18, 19, 20]. The major neurosecretory systems of insects are located behind the central nervous system and include corpora cardiaca (CC) and CA glands together with their connections. The CC, are neurohaemal organs and are among the most essential structures of the neuroendocrine system of insects. They stores and release the neuropeptides which are synthesized in the neuroendocrine cells of the brain. This importance is further highlighted by the ability of the CC glands to produce certain peptides from specific neurosecretory cells themselves. Apart from the CC, CA glands are also responsible for releasing the neuropeptides in the neuroendocrine system in insects, but their primary function is the synthesis of the juvenile hormones [2].
The neuroendocrine system of insects regulates most critical metabolic, behavioral, homeostatic, developmental, and reproductive processes [1, 24], which are widely studied in spiders because they are among the most abundant invertebrate predators in terrestrial ecosystems, agroecosystems, and woodlands [25, 26, 27]. Moreover, spiders are crucial in the development of efficient, sustainable, low-input agricultural systems [28]. However, how such important spider behaviors as web building, molting, sexual maturation, and complex behaviors like parental care are controlled on the neurohormonal level is still insufficiently studied. Unfortunately, knowledge about the function of the neuroendocrine and nervous systems of spiders is still rudimentary. Many of the quoted publications refer to research conducted in the 1960s [29, 30, 31, 32, 33].
It seems that the neuroendocrine and nervous systems of spiders may be the site of neuropeptide synthesis. Bonaric [32] described the neuroendocrine complex (Schneider I organs + neurohemal organs) as the CC homolog and Schneider II organs as the structure homologous to insect CA. Loesel et al. [23] also confirmed that in addition to the neuroendocrine system in C. salei, other nervous neurosecretory cells might be responsible for the secretion of AST. In addition, the neurohemal organ (tropfenkomplex) was identified as another secretory organ, which stores substances produced in the central nervous system and transported through the Schneider I organ [32]. Since neuropeptides are neurotransmitters, it can be assumed that they are secreted into the hemolymph by exocytosis and are transported to target cells where they bind to specific receptors [34]. Therefore, to analyze the function of allatoregulatory neuropeptides, it is thus first necessary to identify their tissue of synthesis as well as receptor location.
The main aims of this study, which is the first attempt to investigate spiders allatoregulatory neuropeptides, were to (i) detect the presence of these substances in the model spider species Parasteatoda tepidariorum C. L. Koch, 1841 (Araneae, Theridiidae) and to (ii) identify the tissues where the synthesis takes place and target sites of their action by the determination of expression of genes encoding allatostatin A (AST A), allatostatin B (AST B), allatostatin C (AST C) and allatotropin (AT) as well as their receptors in various tissues of female spiders. In addition, age-dependent expression rates were investigated.
Spider breeding
P. tepidariorum females were obtained, from laboratory-bred strains of the Department of Animal Physiology and Ecotoxicology, University of Silesia. The animals were bred at 25 ± 1°C at 70% relative humidity under L: 16 h, D: 8 h photoperiodic cycle. They were fed with laboratory cultured Drosophila melanogaster or D. hydei and were irrigated regularly.
For the needs of the presented study, the following ontogenetic stages of P. tepidariorum females were selected: the penultimate nymphal stage (35th day of life), the last nymphal instar (38th day of life), mature females (40th day of life), females after mating (43rd day of life) and females after oviposition (47th day of life). The spider development time was counted from leaving the cocoon, according to Miyashita [35] and behavioral observations.
Five different tissues and organs of P. tepidariorum–the midgut glands with midgut (MG), neuroendocrine and nervous system (NS), ovaries (OV), hindgut (HG), and integument (INT)–were used for the analysis. Moreover, the entire body of adult spiders (EB) was used as a biological material. The number of tissues/organs per sample was determined during a pilot study and varied depending on the size and age of spiders (Table 1). The preliminary results were based on the analysis of the weight of tissues needed to obtain the proper concentration of total RNA, which was used to prepare a cDNA template in the reverse transcription process. The required weight of tissues/organs is correlated with the number of dissected individuals. Due to the efficiency of reverse transcription (80%), a minimum of 700 ng/μl of total RNA had to be isolated. For example, in the case of females in the penultimate nymphal stage (day 35 of life), due to the small size of the ovaries, it was necessary to dissect the ovaries from 20 spiders to obtain 700 ng/μl of total RNA (Table 1).
The number of tissues/organs selected for detection of allatoregulatory neuropeptides in the spider P. tepidariorum depending on their stages of ontogenesis.
The tissues and organs were dissected on ice in sterile phosphate-buffered saline (PBS, 137 mM NaCl, 10 mM phosphate buffer (K2HPO4, KH2PO4), 2.7 mM KCl, pH 7.4). All tissues and organs were immediately frozen in liquid nitrogen and stored at -70°C until use. All samples were performed in six replicates.
In silico search
AST A, AST B, AST C, and AT and their receptor protein sequences of different insects (see S1 Table for species and accession number from the NCBI protein database [36]) were used to search homologous proteins in P. tepidariorum. The IAAA00000000.1 (TSA 25-JUL-2015) version of the current Transcriptome Shotgun Assembly database was searched with default parameters of tblastn [37] and the candidates with based on the sequence homology criterion that was proposed by Pearson [38] were identified.
The transcript sequences that were selected this way have been searched for the presence of ORFs and then transcribed into protein sequences using the Geneious (ver. 9.1.2) software. These sequences were used to confirm the identity of the putative neuropeptides and neuropeptide receptor genes in P. tepidariorum with gene sequences of other arthropods. For this purpose, the Geneious, Clustal Omega and InterProScan [39] software and algorithms were used. Primers for the further analysis of the section of putative genes encoding the neuropeptides and neuropeptide receptors in P. tepidariorum were designed using the Geneious software (Table 2).
Forward and reverse sequences for the PtASTA, PtASTA-R, PtASTB, PtASTB-R, PtASTC, PtASTC-R, PtAT, PtAT-R and PtRP49 primers.
Possible gene duplication was checked by performing multiple sequence alignment of chosen sequences in Clustal Omega and their pairwise comparison in BLAST software. The criterion of duplicated gene proposed by Ouedraogo et al. [40] was used. Phylogenetic tree was constructed with MEGA X [41] using the Neighbor-Joining method [42] with a bootstrap of 2000 replicates [43] The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree (S1–S13 Figs)).
The bioinformatics analysis of the P. tepidariorum genome has indicated that some of the tested neuropeptides and their receptors have paralogs. Therefore, the data about gene duplication have not been placed in the manuscript and have been only used in the primer designing.
RNA isolation and cDNA preparation
Total RNA was isolated from the tissues/organs using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the standard method [44]. Remaining genomic DNA contamination was removed using the TURBO DNA-free kit (Ambion, Austin, TX, USA) according to the manufacturer's protocol. Quality of RNA was assessed for using a NanoDrop 2000 spectrometer (Thermo Scientific, Wilmington, USA). Aliquots of 1 μg of total RNA were retrotranscripted using the Reverse Transcription System (Promega, Madison, WI, USA) and random primers according to the recommendations of the manufacturer. The reverse transcription products were then diluted with 80 μl of nuclear-free water. Prepared cDNA was used as the qPCR template.
Relative expression of selected genes was quantified by real-time PCR performed in the LightCycler 480 System (Roche, Basel, Switzerland) using SYBR Green Select Master Mix (2x) (Applied Biosystems, Foster City, CA, USA). Reactions for each of the six biological replicates in all age groups were run in triplicates in a 15 μl volume. The reaction master mixes were prepared using SYBR Green Master Mix 2x (7.5 μl), 20 mM forward and reverse primer (0.48 μl), cDNA template (3 μl), and nuclear-free water (4.02 μl) in 96-well white plates (Roche, Basel, Switzerland). All qPCR reactions were performed under the following conditions: 3 minutes for the pre-PCR denaturation and polymerase activation step at 95°C, followed by 40 cycles of 15 s denaturation at 95°C, a 20 s hybridization step at 57°C and a 45 s elongation step at 72°C. The quantification and calculation of the gene expression levels were performed according to the standard curve method [45]. Standard curves for every gene of five dilution series (from 1010 to 106 copies of DNA molecules) were constructed from purified cDNA (using the QIAquick PCR Purification Kit, following the manufacturer's protocol). Melting curve analyses were performed to confirm the amplification of a single PCR product. The thermal profile for the melting curve determination began with incubation of 1 min at 60°C with a gradual increase in temperature (1°C/15 s) to 95°C, during which time changes in fluorescence were monitored. No-template control tubes, containing water instead of template mRNA, were run under the same conditions for each gene. Real-time PCR efficiencies were calculated using the standard curve methods and normalized to the expression of the housekeeping gene ribosomal protein 49 (PtRP49) for each sample. Preliminary tests (not shown) of the most commonly used references genes (RP49, RP18, α-actin, β-actin, 3-phosphate dehydrogenase; [46]) revealed that only the PtRP49 gene was stably expressed in various tissues, regardless of the stage of spider ontogenesis.
The results are reported as mean values ± SD. Normality was checked using the Kolmogorov-Smirnov test. The data were tested for homogeneity of variance using Levene's test of equality of error variances. Whenever a significant effect was observed, the Tukey multiple comparison test was used for a post hoc one-way analysis of variance (ANOVA). Analysis of variance for the level of genes encoding allatoregulatory neuropeptides (PtASTA, PtASTB, PtASTC, PtAT) and their receptors (PtASTA-R, PtASTB-R, PtASTC-R, PtAT-R) at different stages of ontogenesis of P. tepidariorum females was performed using two-way ANOVA with the experimental groups (various tissues and bands) and the stages of life as the sources of differences. Results with p≤ 0.05 were considered to be significant. The data were analyzed using GraphPad Prism ver. 6.
Expression of genes encoding allatoregulatory neuropeptides and their receptors was visualized by a two-step real-time polymerase chain reaction (qPCR) method with cDNA as a template extracted from tissues/organs and from the entire body of P. tepidariorum females in various stages of ontogenesis. Proper products, corresponding to the expected fragments, were amplified for PtASTA, PtASTC, and PtAT (Fig 1C). Among all tissues/ organs of the P. tepidariorum females analyzed in the experiment, no amplification was observed for genes encoding AST B and its receptor. The correct sequence of the allatostatin B receptor in the transcriptome of the P. tepidariorum spider has not been demonstrated by in silico analysis. The nucleotide sequences most closely related to ASTB-R do not contain TM1-TM7 conserved transmembrane domains (S7 Fig). It seems that AST B does not belong to the physiological neurosecretions of P. tepidariorum, and it does not play a role in the regulation of their physiological processes. On the other hand, this may also indicate a gene mutation resulting in a significant change of the nucleotide sequence in the genome. So far, among all allatostatins, peptides belonging to allatostatin B have been characterized and isolated only in five species of insects: Locusta migratoria [47], M. sexta [48], Carausius morosus [49], Gryllus bimaculatus [50] and D. melanogaster [51]. In contrast, allatostatin A and allatostatin C are widely distributed in various groups of insects. This may indicate a limited occurrence of this peptide in arthropods. Moreover, the potency of the B type allatostatins is approximately 50% lower than that of the A type allatostatins in G. bimaculatus. Lorenz et al. [52], as well as Wang [51], proved that the activity of AST B is limited to insects from the family of crickets (Gryllidae). These peptides isolated from C. morosus inhibited CA activity only in crickets but did not affect the CA of the C. morosus itself. Therefore, it can be assumed that limited occurrence of allatostatin B in insects and its limited activity may explain the possible lack of allatostatin B in P. tepidariorum females.
The relative expression level of genes encoding allatoregulatory neuropeptides in Parasteatoda tepidariorum females.
Changes of the expression level of allatostatin A (PtASTA), allatostatin C (PtASTC), allatotropin (PtAT) (A) and their receptors' genes (PtASTA-R, PtASTC-R, PtAT-R) (B) during the ontogenesis stages in the entire body of P. tepidariorum females. Differential expression of genes encoding allatoregulatory neuropeptides and their receptors in female (C) NS–neuroendocrine and nervous system, OV–ovaries, MG–midgut glands with midgut, HG–hindgut, Vgs–vitellogenesis process, Ma–mating, Ovi–oviposition.
The obtained results confirmed the presence of allatoregulatory neuropeptides in P. tepidariorum. They revealed substantial differences in genes encoding allatoregulatory neuropeptides expression levels and their receptors in the different tissues, which may indicate the diverse production of neuropeptides and their subsequent action in the target tissues. The level of gene expression also varied depending on the age of individuals. However, for all transcripts excluding the entire body (Fig 1A and 1B), the highest expression level coincides with the beginning of sexual maturation at the 40th day of life.
It is clear that there might be more paralogs or that the genes observed are most likely homologs of the insect neuropeptides, but that a detailed phylogenetic analysis was not performed. It seems, that to confirm the presence of paralogs it would be necessary to perform phylogenetic analysis based on the newly available genome described by Posnien et al. [53] or Schwager et al. [54].
Allatoregulatory neuropeptide expression in various tissues of mature females
Among all tissues/organs of the female P. tepidariorum analyzed in the experiment, proper products of amplification for PtASTA, PtASTC, PtAT, and their receptors were observed. The highest level of expression of AST A, AST C, and AT genes was recorded in the neuroendocrine and nervous systems. Moreover, the same level of expression for the gene encoding AST C was observed in the hindgut (Fig 2), whereas the maximum expression level of receptors was found in the hindgut for PtASTA-R and PtAT-R as well as in the ovaries for PtASTC-R (Fig 2). In case of integument, no products of the amplification of all analyzed genes was obtained.
Tissue-specific expression of allatoregulatory neuropeptides.
Relative expression of allatostatin A, allatostatin C, allatotropin and their receptors of P. tepidariorum females at the 40th day of ontogenesis (the day of sexual maturity) [mean ± SD]. NS–neuroendocrine and nervous system, OV–ovaries, MG–midgut glands with midgut, INT–integument, HG–hindgut (HG). Different letters indicate statistically significant differences: a-d differences between levels of expression of genes encoding allatoregulatory neuropeptides (PtASTA, PtASTC, PtAT) in tested tissues, A-D differences between levels of expression of genes encoding allatoregulatory neuropeptide receptors (PtASTA-R, PtASTC-R, PtAT-R) in tested tissues. Asterisks indicate statistically significant differences between levels of expression of gene encoding allatoregulatory neuropeptide and its receptor in the same tissue. Tukey's multiple comparison test, p≤0.05.
For all allatoregulatory neuropeptides significant differences in gene expression levels were found compared to their receptor expression. In the neuroendocrine and nervous systems, the relative level of PtASTA was significantly higher than the PtASTA-R level (Fig 2). The same correlation was observed for the gene encoding AST C and its receptor (Fig 2). In the remaining tissues, the inversed tendency was observed, where the expression of PtASTA-R gene was significantly higher than the expression of the gene encoding PtASTA. The most significant difference in the expression of the neuropeptide and its receptor was observed in the hindgut, where the expression of PtASTA-R was 20-fold higher than that of PtASTA (Fig 2). Analysis of the expression of PtASTC and PtASTC-R genes revealed that the relative level of PtASTC expression was also higher than the level of PtASTC-R in the hindgut. Furthermore, comparison of expression of the gene encoding the receptor of AT was higher than the level of PtAT in all tissues/organs excluding neuroendocrine and nervous systems, where a significant difference between the level of both transcripts was not observed (Fig 2).
The highest expression of genes encoding PtASTA, PtASTC, and PtAT in the neuroendocrine and nervous systems in comparison to other tissues of P. tepidariorum confirms the presence of neurosecretory cells, constituting the main site of synthesis and secretion of allatoregulatory neuropeptides. Our results are the first evidence consistently indicating the neuroendocrine and nervous system as a place of synthesis of allatoregulatory neuropeptides in spiders and confirming the assumptions described before by Bonaric [32]. Moreover, these results coincide with the data published for C. salei, where the presence of allatostatin A in the arcuate body of the central nervous system by immunochemistry method has been demonstrated [23]. This clearly confirmed the presence of allatostatins in the nervous system of spiders.
In addition, to the neuroendocrine and nervous systems, high differences of the transcripts levels have been demonstrated in the different tissues. The expression of genes encoding AST A, AST C, and AT was primarily confirmed in the ovaries, midgut glands with midgut and in the hindgut. In a study conducted by Stay et al. [55] in Diploptera punctata as well as by Witek and Hoffmann [56] in Gryllus bimaculatus, it was demonstrated that allatoregulatory neuropeptides can also be secreted by the ovaries, which would confirm the high expression of genes encoding these neuropeptides obtained in the ovaries.
The results of the expression of genes encoding the allatoregulatory neuropeptide receptors also indicate considerable variation according to tissue and ontogenesis stage. The expression of genes encoding the receptors of allatoregulatory neuropeptides was also demonstrated in the neuroendocrine and nervous systems, but its level was lower than in the other tissues. As explained earlier, the tissue referred to as the neuroendocrine and nervous systems contained all the structures of the neuroendocrine system described by Bonaric [29, 30, 31, 32] as well as by Bonaric and Juberthie [33]. In the aforementioned manuscripts, the neuroendocrine and nervous systems were selected as the site of JH synthesis and/or its analog as well as other substances belonging to the neurosecretions of spiders. The presence of allatoregulatory neuropeptide receptors in this complex of structures indicates the allatoregulatory control of the functioning of the neuroendocrine and nervous systems. We can assume that these neuropeptides influence the secretion of other neurotransmitters through neurosecretory cells of the neuroendocrine and nervous systems [1, 20, 57, 58]. To date, the expression of genes encoding allatoregulatory neuropeptide receptors in the nervous system of insects has been reported in A. aegypti [59], D. melanogaster [60] or Apis mellifera [61]. Similar results obtained in insects and for P. tepidariorum suggest that the allatoregulatory neuropeptides in spiders may play the primary control function of JH secretion. Additionally, it has been previously shown that AST can affect visual information processing [62], learning and memory [61]. The multitude of nervous system functions makes it difficult to deduce, based only on the expression of the studied transcripts.
Analysis of the expression of PtASTA-R and PtAT-R genes confirmed their highest level in the hindgut, while the maximum expression level of the gene encoding the AST C receptor (PtASTC-R) was observed in the ovaries. The strong expression of analyzed genes in the hindgut may indicate the use of these neuropeptides for the regulation of gastrointestinal function as well as in intestinal peristaltic contractions, as in insects [58, 63, 64, 65, 66, 67]. Furthermore, it has also been shown that allatoregulatory neuropeptides control the secretion of digestive enzymes and stimulate their activity [68, 69, 70]. It cannot be excluded that similar mechanisms may occur in spiders. This conclusion is confirmed by the high expression of these transcripts in the midgut glands with midgut responsible for the production and secretion of digestive enzymes in spiders. The obtained results may also indicate the participation of allatoregulatory neuropeptides in other processes that take place in the midgut glands and midgut: energy metabolism processes, protein synthesis, and mobilization of spare substances. Protein synthesis in midgut glands and the midgut is also associated with the initiation of the vitellogenesis process. Hitherto, there is only one publication reporting the effect of AST A on reducing the production of vitellogenins in the fat body, as well as the inhibition of secretion of these proteins to hemolymph in B. germanica [71].
Proper products, corresponding to the expected fragments, amplified for genes encoding receptors of allatoregulatory neuropeptides in the ovaries suggest that they may regulate oocyte development, vitellogenesis, and choriogenesis, which has been confirmed by Woodhead et al. [72] in D. punctata, and the biosynthesis of ecdysone in the ovaries presented by Lorenz [73] in G. bimaculatus and Lorenz et al. [74] in Blaptica dubia.
Results from this study may confirm that in P. tepidariorum females the digestive tract and ovaries are the main tissues under allatoregulatory control. The process of vitellogenesis in P. tepidariorum is controlled on various levels (the gene expression, protein synthesis, protein deposition in ovaries) by allatoregulatory neuropeptides, indicated by allatotropin and allatostatin injections [75]. The allatoregulatory neuropeptides regulate the spider P. tepidariorum metabolism by inducing changes in the concentration of glycogen, lipids, and proteins in the midgut glands with midgut [76].
Age-dependent allatoregulatory neuropeptide expression
The expression profile of genes encoding allatoregulatory neuropeptides and their receptors was dependent on the stage of ontogenesis of P. tepidariorum females. Analysis of the entire body of P. tepidariorum females revealed that the expression of PtAT, PtASTA-R, and PtASTC-R was stable in most cases, throughout their life, except the 35th day of ontogenesis when the PtASTA-R expression was significantly higher than the level in the other stages (Fig 1A and 1B). In females at the last nymphal stage (38th day of life) and 3 days after copulation (43rd day of life), the level of PtASTA and PtASTC expression was significantly lower compared to the earlier stages of ontogenesis. Compared to other genes encoding allatoregulatory neuropeptides, a lower level of expression for the PtAT gene in the entire body of P. tepidariorum females was confirmed (Fig 1A).
Expression of all transcripts in selected tissues or organs was also age-dependent (Fig 1C). The same relation of changes in the expression level as in the entire body was confirmed. The maximum level of expression of every gene was observed on the day of sexual maturity (40th day of ontogenesis) in all tested tissues, except for the expression of PtASTA in the hindgut, where the highest expression level was observed in the penultimate nymphal stage. The highest expression of transcripts on sexual maturation day may indicate an increase in neuropeptide production. Such dependence may indicate the role of these substances in physiological processes that take place very intensively during that period. Among them, vitellogenesis is the essential process; it begins in spiders in the last nymphal stage [77] and intensifies significantly when sexual maturity is reached. Because vitellogenesis requires high energy expenditure, the high level of expression of transcripts in the midgut glands with midgut may also indicate their influence on the regulation of energy mobilization from the energy storage compounds.
Moreover, on the 38th and 43rd days of life, the relative expression levels of PtASTA, PtASTA-R, PtASTC, and PtASTC-R were lower than in the earlier stages of ontogenesis. In the neuroendocrine and nervous systems as well as in the midgut glands with midgut the expression levels of PtASTA, PtASTC and PtASTA-R transcripts on the 38th day were twice as low as on the 35th day, whereas in the ovaries the expression level of PtASTC was 11-fold lower. Inhibition of the expression of genes encoding ASTs at this stage of ontogenesis may indicate a reduction in the rate of physiological processes associated with the preparation of the body to achieve sexual maturity. The PtAT gene expression pattern was different from genes encoding ASTs; PtAT gene expression was not observed until the 40th day of individual development. The effect of AT on the secretion of JH and/or its analog, indispensable in development and larval growth, confirmed in insects may explain the lack of PtAT gene expression on days 35 and 38 of ontogenesis. These results may confirm that the synthesis of AT in P. tepidariorum females does not occur in the nymphal stages. In insects, it has been proved that larval-larval molting and metamorphosis can only occur as a result of the significant reduction of the JH level, which inhibits both of these processes [78, 79]. Based on the main AT function in insects, which is the stimulation of JH secretion, it may be relatively easy to explain the lack of its expression in the nymphal stages. A high level of JH due to the presence of allatotropin would prevent the molting and growing of spiders. This is also confirmed by the high level of allatotropin after the last molt (the day of sexual maturity) and the level of gene expression encoding enzyme of the juvenile hormone biosynthetic pathway (PtCYP15A1 enzyme responsible for catalyzing the epoxidation of methyl farnesoate to the juvenile hormone) in the same ontogenesis stages of P. tepidariorum. Bednarek et al. [80] confirmed the lack of PtCYP15A1 transcript in nymphal stages and high level of this product on the day of sexual maturity.
It was also observed that on day 43 of life the expression in the neuroendocrine and nervous systems was 7-fold lower in the case of genes encoding AST A and nearly 6-fold lower for the AST A receptor gene in relation to the day of sexual maturity. The changes in PtASTC and PtASTC-R expression levels were similar, but the differences were much more extensive. On the 43rd day of ontogenesis, a more significant difference in the obtained results was confirmed. In the midgut glands with midgut and hindgut, the expression of PtASTC was 14-fold lower with respect to the results from the 40th day of ontogenesis, whereas expression of the PtASTC-R gene in the midgut glands with midgut at the 43rd day of development was 20-fold lower compared to the previous stage of ontogenesis. Again, higher levels of expression of the PtASTA, PtASTA-R, PtASTC, and PtASTC-R transcripts were confirmed in the neuroendocrine and nervous systems and the ovaries in females after the formation of the first cocoons (47th day). In other tissues, neither transcript was expressed during the day of ontogenesis. The overlap of results obtained for genes encoding the allatoregulatory neuropeptides and results for genes encoding their receptors confirm the increased use of AST A and AST C during egg laying. The high expression of the PtASTA and PtASTC genes in the neuroendocrine and nervous system confirms their increased synthesis, while the highest expression of PtASTA-R and PtASTC-R genes in ovaries indicate their role in the regulation of the ovaries' action. Garside et al. [81] reported that in D. punctata the expression of AST A (Dipp-AST A) was deficient in the ovaries during the initiation of vitellogenesis (analogous results on the 38th day of spider ontogenesis) and increased until spermatophore deposition. Immediately after this process, the level of Dipp-AST A was lower, and after laying eggs, it increased rapidly. However, Woodhead et al. [72] reported that the level of AST in D. punctata ovaries was increasing from 3 days after pupation, while after spermatophore deposition, AST A content rapidly increased, with the maximum level on the day of egg laying. The authors noted that the rapid increase in the level of ASTs in the ovaries is correlated with the process of choriogenesis.
In contrast, the expression profiles for PtAT and PtAT-R genes were different. No expression in the nymphal stages (35th and 38th) was detected, although, after sexual maturity, expression rates were significantly higher than PtASTA and PtASTC (Fig 1C). In all tissues, PtAT gene expression decreased gradually from days 40 to 47 of spider development. The increased expression of both the PtASTA and PtASTA-R genes in the nymphal stages and the confirmed AST A activity in relation to the synthesis of JH indirectly confirms the occurrence of JH and/or its analog in P. tepidariorum. The secretion of AST A in the nymphal stages and the lack of AT secretion may indicate inhibition of JH synthesis and/or its analog, which enabled the proper course of molting. Such mechanisms of action of allatoregulatory neuropeptides have been confirmed in insects [58, 78, 79].
To conclude, the results demonstrated in this paper confirm the presence of allatoregulatory neuropeptides (AST A, AST C, and AT) in females of the spider P. tepidariorum, which complements the current knowledge of both the physiology of spiders and the occurrence of allatoregulatory neuropeptides. Moreover, the neuroendocrine and nervous systems have been reported to be the main sites of synthesis of these compounds, whereas the digestive tract and ovaries were primarily affected by the allatoregulatory control. The highest levels of all tested neuropeptides were observed on the 40th day of females' lives. This indicates increased production of these proteins and their increased use during the physiological processes that took place on that day, like vitellogenesis, oocyte development, or mating. These results are a starting point for future functional studies, which will advance our understanding of the role of neuropeptides in the regulation of spider physiology.
S1 Fig [a]
S6 Fig [tif]
Multiple alignment of allatostatin A receptor from and other arthropods.
Multiple alignment of allatostatin B receptor from and other arthropods.
Multiple alignment of allatostatin C receptor from and other arthropods.
Multiple alignment of allatotropin receptor from and other arthropods.
S10 Fig [tif]
Multiple alignment of allatostatin A precursors from and other arthropods.
Multiple alignment of allatostatin B precursors from and other arthropods.
Multiple alignment of allatostatin C precursors from and other arthropods.
Multiple alignment of allatotropin precursors from and other arthropods.
S1 File [pdf]
The real-time PCR results.
S1 Table [pdf]
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Assessment of glucose-6-phosphate dehydrogenase acti


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      onDataLoaded.subscribe(updatreGridOnDataLoaded);
    }

    function destroy() {
      onDataLoaded.unsubscribe(updatreGridOnDataLoaded);
      _grid.onSort.unsubscribe(onGridSort);
      _grid.onViewportChanged.unsubscribe(onGridViewportChanged);
    }

    function onGridSort(e, args) {
      var
        result = [], column,
        cols = _grid.getColumns(),
        sortCols = _grid.getSortColumns();

      $.each(sortCols, function(k, col) {
        column = cols[_grid.getColumnIndex(col.columnId)];

        var direction = col.sortAsc ? 1 : -1;
        if (column.order_fields) {
          $.each(column.order_fields, function(k,v) {
            result.push({
              field: v,
              dir: direction
            });
          });
        } else {
          result.push({
            field: column.field,
            dir: direction
          });
        }
      });
      _sortCols = result;
      refresh();
    }

    function updatreGridOnDataLoaded(e,args) {
      for (var i = args.from; i <= args.to; i++) {
        _grid.invalidateRow(i);
      }
      _grid.updateRowCount();
      _grid.render();
    }

    return {
      // properties
      "data": _data,
      "defaults": defaults,

      // methods
      "clear": clear,
      "init": init,
      "isDataLoaded": isDataLoaded,
      "refresh": refresh,
      "ensureData": ensureData,
      //"reloadData": reloadData,
      "getIdentities": getIdentities,

      "setQuery": setQuery,
      "addQuery": addQuery,
      "clearQuery": clearQuery,

      // events
      "onDataLoading": onDataLoading,
      "onDataLoaded": onDataLoaded,
      "onDataLoadError" : onDataLoadError,
      "onDataLoadAbort": onDataLoadAbort
    };
  }

  function ODataSortAdapter(argument) {
    // ...
  }

  // Slick.Data.RemoteModel
  RemoteModel.PagingAdapter = PagingAdapter;
  RemoteModel.ArrayAdapter = ArrayAdapter;

  $.extend(true, window, {
    Slick: {
      Data: {
        RemoteModel: RemoteModel,
        ODataSortAdapter: ODataSortAdapter
      }
    }
  });
})(jQuery);

redpajama_set_name: RedPajamaGithub


New Wave Friday will be at our bigger, better, NEW location right on Auahi Street! With a live performance by music legend Kapena as well as live art by Hawaii's own Boz Schurr, you're not going to want to miss this summer market!
We are bringing together the best of the best in the 3rd Annual Best of Honolulu Festival presented by American Savings Bank. A free, family-friendly event featuring 'ono food, keiki zone, entertainment and a marketplace.
FREE PARKING Parking Instruction for the Zone 20 Parking Structure: All cars must enter through the Dole Street entrance. Please inform the gate attendants that you will be attending the Moiliili Summer Fest. Drivers will receive a parking ticket to display on their cars upon entry. Parking is complimentary. Shuttles will be provided; however, it is also a short 10 minute walk to the festival.
Honolulu Night Market is back for our summer event and it's going to be bigger and better than ever! That means more vendors, more entertainment, more music and more fun than ever before.
Look out for some new additions to our event, plus all the local vendors and food you've grown to love!