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Articles posted by caroline

2014-12-15 Update

Posted on December 16, 2014 by caroline in Caroline

PhD Program of Study Progress

Scheduling Quals for the Spring
To Do – email committee with update and to schedule date

Chapter 2: Invasion history of X. crassiusculus

Library containing the following individuals sequenced.
To Do – Analyze data!

Brunei	1
Cameroon	8
China	20
Ghana	12
Ankasa	9
Kakum	3
Honduras	3
Indonesia	12
Japan	20
Aichi	6
Iriomote Is.	2
Okinawa	12
Madagascar	8
Malaysia	2
New Guinea	7
Taiwan	7
Thailand	4
USA	111
AR	5
FL	24
GA	23
Hawaii	7
MD	8
NC	12
SC	12
VA	20
Grand Total	215

Chapter 3: What is a species? Molecular and morphological delineation of the X. ferrugineus/bispinatus species complex

Begin with measurements of simple characters of a few practice specimens. For example, width, length, elytra, and color
To Do – practice mounting elytra
To Do – Finalize specimen list
To Do – Plan collecting trip for the Spring?

Chapter 4: Invasion history of an outbreeding invader D. valens

Follow up on acquisition of specimens
Finalize specimen numbers
Begin dissections

Collecting Beetles – Red Turpentine Beetles

Posted on May 7, 2014 by caroline in Beetles

For a printer friendly version of these instructions please download the PDF here.

The red turpentine beetle (RTB), Dendroctonus valens, (Figure 1) is a common bark beetle in the U.S. and has recently become a threat to China’s pine forests as an exotic pest. In its native range these beetles are found under the bark of dying or injured pine trees.

Figure 1. The red turpentine beetle.

Known distribution in the U.S.

The RTB is found throughout pine forests in most of the US except for the several states in the southeast (see Figure 2).

Figure 2. Number of recorded RTB collections in the U.S. from T. Atkinson’s curated bark and ambrosia beetle database; http://www.barkbeetles.info/.

Known hosts:

The RTB is ubiquitous wherever pines grow, in any forest, shade, and park trees 8 inches or larger in diameter throughout most of North America (except the South East). Look for resin exudates on freshly cut stumps, the bases of trees that are dying, and trees disturbed by fire, logging, land clearing, or construction.

Signs of an RTB attack

From a distance, pines with needles that have lost their color and turned red may indicate a potential RTB attack. “The best indicators of red turpentine beetle attack on pines are: large pink-white pitch tubes on the lower bole (Figure 3); accumulations of reddish brown sawdust at the base of the tree and in bark crevices (Figure 4); and accumulations of cream to pink colored crystallized resin granules at the tree base.”

Figure 3. Pitch tubes and resin at the base of a pine from an RTB attack.

Figure 4. Sawdust and frass buildup at the base of an RTB attacked pine.

Extracting the RTB from wood

RTB “egg galleries under the bark are fairly wide and linear to irregular in shape. Galleries extend downward from the entry hole 7 cm to 1 m and may even extend into large roots.” Unlike some other bark beetles larval feeding occurs in a gregarious fashion within the gallery.The easiest way to extract RTB from their galleries is to use a wide chisel or the blade of a hatchet to gently lever off the bark surrounding the pitch tube and entry hole. Once part of the gallery is exposed (Figure 5), beetles can be collected by hand, forceps, or aspirators and deposited in a labeled collection vial. Immediately after collecting, specimens should be stored in high quality ethanol (95%).

Figure 5. RTB adults and feeding larvae inside their gallery under the bark of an RTB infested pine.

Trapping the RTB

Funnel traps baited with a frontalin+turpentine+ethanol lures are the most successful method for trapping the RTB. If needed we can send you a specialized RTB lure from Synergy Semiochemicals. Alternatively, freshly cut pine is a perfect lure as well! If trapping beetles, traps should be checked every day/every-other-day to ensure that fresh, live specimens are being collected for preservation. Immediately after collecting, specimens should be stored in high quality ethanol (95%).

Storing collected beetles

Beetles collected from the same location can stored in the same vial. The vial should could contain 95% ethanol and a label containing collection info. Please store the vial in a freezer until they can be shipped or delivered by another collaborator.

Pictures and quoted text are from the U.S. Forest service Management Guide for Red Turpentine Beetle; http://www.fs.usda.gov/Internet/FSE_DOCUMENTS/stelprdb5191791.pdf.

2014-02-28 Updates

Posted on February 28, 2014 by caroline in Caroline

Follow op on program of study petition
– Jiri will check petition status
– Have acquired all committee signatures approving change in program of study requirements

Ch. 1: Lit. Review
– Should only be ~4 pages and structured to provide introductory material for each chapter

Ch. 2: Global population structure of X. crassiussculus
– Anticipated timeline is to have DNA extracted from abdominal muscle tissue by mid-May for Adam Payton to prepare sequencing library.
– May need more Chinese specimens for Ch. 4
– Specimens & Locations
Ghana – 26
Honduras – 3
Japan – 26
Malaysia – 3
New Guinea – 26
Taiwan – 6
Thailand – 24
Hawaii – 10
China – 5 (Need more. Will be going through Craig’s specimens)
Cameroon – 6
SE US – 56

Ch. 3: Continuous morophological variation in X. ferrugineus/bispinatus
– Need Jiri to contact Tom Atkinson about future collaboration on this project.
– In the process of acquiring an assistant to began sorting specimens and assessing morphological variation.
– Need to finalize number of specimens and locations.
– Specimens & Locations
X. bispinatus
North Carolina – 6
Georgia – 45
Florida – 165
Honduras – 2
X. ferrugineus
North Carolina – 8
Georgia – 12
Florida – 171
Costa Rica – 5
Honduras – 49
Panama – 7

Ch. 4: Pop. Structure and diversity of inbreeding and outbreeding Scolytines in their native and exotic ranges
– Comparing X. crassiusculus to Dendroctonus valens
– Need lot more D. valens specimens. Jiri will contact some folks about this.
– Specimens & Locations
X. crassiusculus
Same as Ch.2
Dendroctonus valens (list of all available specimens)
Michigan – 10
Wisconsin – 2
California – 3

Other: Wallacellus spp. phylogenetic revision
– Adding new X. eurygraphus sequences to test multiple hypothesis for phylogenetic relationships between similis, piceus, and Euwallace
– Also adding X. horrius and Cyclorhipidion spp. sequences when building new phylogeny, which is for another project

Field Collecting Equipment List

Posted on May 20, 2013 by caroline in Miscellaneous

This page is currently a work in progress intended to help aid in packing for international and domestic collecting trips

Collecting tools

The selection of tools partly depends on which types of scolytine beetles you are mostly interested in. For example, for twig bark beetles you will not need any of the heavy duty hardware. On the other hand, trying to pry ambrosia beetles out of a branch with a knife routinely leads to squashed specimens (for xylophages we recommend sawing out a wood “cookie” with the gallery in it, splitting it out with a chisel, and peeling pieces off with clippers, until you get at the beetle). Our recommendations of brands are based on years of experience, not on any relationship with the vendors.

If you have everything shown in the photo- you should be set for both wood and trap collecting.

Essential – extracting beetles alive or into ethanol

Tool/gear bag with strap
Hatchet
Anvil-style clippers (we recommend Bahco)
Folding saw
Chisel
Knife/box cutter (heavy duty), extra blades
Collection tubes prepped with ethanol, in collection box
Soft forceps (for grabbing beetles), with spare and loop for neck
Hard forceps (for careful bark dissections)
Headlamp (useful even in daytime)
Pre-cut collection labels
Labeling pens (we recommend .5 Pigma MICRON archival pen)
Collection bags, various sizes (for carrying sticks to dissect later)
Any tubes or other container with holes poked (for live specimens)
Kimwipes/tissues (for live specimens)

Funnel/bottle trapping

Lindgren traps or bottle traps (2L bottles or materials to make them (see below, also: http://ambrosiasymbiosis.org/wp-content/uploads/2013/03/bbtrap.pdf)
Knife for constructing bottle traps, cutting rope
Rope and/or twine
Twist-ties or cable ties
Tape (electrical)
Lures, or >95% un-denatured etoh (seal in drinking alcohol bottle for checked luggage to pass TSA)
Bags for lures/ethanol
Squirt-bottle for re-filling etoh bags and tubes
Plastic pasteur pipette
Collection bags, various sizes (for carrying sticks to dissect later)
Any tubes or other container with holes poked (for live specimens)
Kimwipes/tissues (for live specimens)
Labeling equipment (Pigma pens, labels)

Light-trapping

Light source of choice, batteries, etc.
1. We typically use at least two white (UltraFire Sk98 Cree Xml-t6) and two black (UltraFire SK98 UV) flashlights per sheet
Voltage converter if necessary
glasses with UV protection
White sheet (Queen size is best, usually can borrow from a hotel)
Rope/twine to hang sheet
Clips/clothespins
Bowl/tray for ethanol

Not essential but useful

Ethanol-resistant labeling markers
Tarp for cutting-up wood indoors (easy clean-up)
Multi-tool
Collecting notebook/log
Aspirator (pooter)
Electric or regular chain saw (remove oil if packing, also pack oil and allen wrench)
Scalpel (for very small galleries)
Pin/teasing needle (small galleries, can also just use hard forceps)
Watch glasses for IDing in the lab, under a scope.

Generally useful in travel

Power adapter
…

If culturing fungi from wood/beetles:

Dry media

Plates

Scalpel and blades

Spreaders

Rubber gloves (1 box, transferred to zip-loc bag)

Minuten pins

100 uL Pipette

100 uL pipette tips – autoclaved (figure 20 tips/beetle, e.g. 30 beetle isolations = 600 tips)

Parafilm

Lighter

Pellet pestles (~20)

1.5 mL tubes

1.5 mL tubes prepped with PBS for serial dilutions (15/beetle, 30 beetle isolations =450 tubes)

Tube of Tween

Tube rack

Cryotube slants (pre-made, in cardboard vial boxes, generally 125+)

Extra empty sterile cryotubes

Extra empty sterile 1.5 mL tubes

Mite paper – 3’ folded and stored in zip-loc bag to extend effective life

Gallon zip-loc bags to put plates in (mite protection)

Permits and labels for shipping fungi back

Padded envelopes/small boxes for shipping slants back

Packing tape

Arrange for in advance

Permits for shipping fungi arranged

Travel authorization submitted to Cindy Love

Completed online travel registration checklist found here: http://ufic.ufl.edu/travelregistration.html

Team Assist insurance set up, card printed to take on trip

Vaccinations if required/recommended for destination

International calling setup on cell phone

International driver’s license (if renting car)

Pcard/personal credit/debit card travel authorizations

Accommodation reservations

Things to make sure are available at destination/hosting lab in advance (if needed)

Plates plus potentially other lab tools, consumables listed above (allow time for hosting lab to order to ensure arrival in time)

Autoclave

Glassware for autoclaving media

Sterile hood

Bulk ethanol (lots necessary if trapping – illegal to fly with large amounts of ethanol)

Microscopes

Microscope camera

Printing capabilities

WiFi and printing capability

Incubators

traps to borrow

Vials for -80C Freezer

Posted on April 24, 2013 by caroline in Brian

Hi Brian,

This is not urgent, but sometime in the near future…

The box in the lab freezer labeled “Ready for -80” has both new vials that do not have a box in the -80C basement freezer and old vials that need to get put back in their correct boxes.

Also for any new vials (4000’s and up) could you please leave space in the boxes for any missing vial numbers. For example if you have 4011,2013, & 4014 please leave a space between 4011 and 4013.

Feel free to ask me questions.

Thanks, Caroline

Project Meeting 2013-04-15

Posted on April 18, 2013 by caroline in Caroline

Investigating a collecting expedition to either Taiwan or Okinawa and Iriomote in Japan. Contacting Drs. Roger Beaver and Phill Ward to ask about taxonomic richness at these locations.
Picking samples of Xylosandrus crassiusculus for RAD trial run with Stuart McDaniel’s lab
Currently troubleshooting DNA extraction methods
Optimizing qPCR methods with Sedonia

Making 1X TAE

Posted on April 16, 2013 by caroline in Brian

Also could you make 6Liters of 1x TAE to refill the 2 Gallon plastic carboy sitting on the left corner of the sink please?

To make:

Measure out 6L of DI water (from other sink tap, let tap run a few seconds first) into the carboy 1L at a time using the 1L graduated cylinder.

Then measure out 120 mL of 50X TAE buffer (on the shelf with chemicals) using a 100mL graduated cylinder and add to carboy.

Mix the contents of the carboy as best as you can via swirling

Thanks!

Autoclaving

Posted on April 16, 2013 by caroline in Brian

Hi Brian,

When you are in could you clean and autoclave the blue plastic pestesls in a flask covered with foil please.

Also could you please autoclave 2 1L Flasks full of 1.5ml microcentrifuge tubes. If you run out of tubes in the drawer there are more above the sink cabinets.

Thanks!

Collection Tube prep

Posted on March 22, 2013 by caroline in Brian

Autoclave collection tubes and stuff with a kimwipe

Things to do

Posted on March 22, 2013 by caroline in Brian

Make plates – 1 L PDA Hard
Add yellow caps to cryo tubes
Fill 1.5 mL microcentrifuge tubes with 1 mL of PBS