Transcript ABF_Rearing_11-7

DIY Nematode Rearing for
Small Hive Beetle Control
Izzy Hill
Center for Urban Bee Research
Dr. Ashleigh Smythe
Virginia Military Institute
Today’s Topics
• Nematodes 101
• How to rear beneficial nematodes for SHB
control
• How to apply them
What is a Nematode
• Nematodes = roundworms
• Approx. 25,000 types of known nematodes,
maybe a million undescribed
• We’re interested in entomopathogenic
nematodes (EPNs)
– Two main types for SHB Control:
• Steinernema carpocapsae
• Heterorhabditis indica (we’re only discussing this one)
• 88-100% efficacy against pupae and larvae in the soil
(Ellis et al., 2010)
Source: McSorley (2009); Tofangsazi et. al (2012)
EPNS ARE NOT THE SAME
NEMATODES THAT FEED
ON ROOTS OR PEOPLE
OR DOGS
Entomopathogenic Nematodes (EPNs)
• Naturally occur in the soil
– H. indica’s biogeography includes N. America
• Type of
parasitoid
Source: Tofangsazi
et. al (2012)
Photo Credit:
James Kerrigan
Infective
Juvenile (IJ)
EPN Life Cycle
How the Bacteria Work
• H. indica have a mutualistic relationship with a symbiotic
bacteria (Photorhabdus luminescens)
• Bacteria live in intestine of H. indica IJ
• IJ enters host,
releases
bacteria in gut
and multiplies
Symbiotic Bacteria in
intestinal tract of IJ
bacteria
&
• Host dead in 24-48 hours
• Bacteria provides defense inside host by producing
antibiotics, nematicides, warning colors, and compounds
Photo Credit: Chich, 2007
to deter scavenging insects
Sources: Tofangsazi et. al , 2012; Chich, 2007; Ciche and Ensign, 2003
Photo Credit: D. Fenolino
The Host: Small Hive Beetle (SHB)
SHB Larvae
SHB Pupae
Pupating SHB
Photo Credits: Josephine
Ratikan (top); Lyle J. Buss
(bottom), UFL
Jon Zawislak
Nematodes as part of an IPM Plan
Action Thresholds
• No action thresholds set for SHB
Rearing your own nematodes
– Anyone can do it
– A great bee club program
– Another IPM tool in the toolbox
– Safe to handle, no risk to pets
– Exempt from EPA pesticide registration
– For a copy of these slides visit
bugonia.com/rearingguide
Rearing Heterorhabditis indica
• Step 1: Culture
• Step 2: Harvest
• Step 3: Storage
Rearing Heterorhabditis indica
• Step 1: Culture
• Step 2: Harvest
• Step 3: Storage
Materials
• Pipette (graduated in ml)
• Fresh, distilled water (chlorine-free)
• Culture flask or 1 L Tetra Pak juice carton
• Disposable gloves
• Wax worms
• Nematodes
• 2, four-inch Petri dishes and one 2-inch Petri dish
– Food-safe lids are a great substitute
• Coarse lab filter paper or unbleached coffee filter paper
• Scissors and pencil
Obtaining Nematodes
• Buy them from a trusted source.
– Southeastern Insectaries
– Bugs for Growers
• Be sure to request Heterorhabditis
indica.
Obtaining Wax Moth Larvae (Galleria)
• i.e., Greater Wax Moth Larvae
• Buy them at any pet store
or bait shop (ask for wax worms)
• You can try other hosts, but it
won’t be nearly as satisfying
• They MUST be alive; check
before purchasing
Photo Credit: Izzy Hill
• Store at 50°F in animal bedding to prolong life
• The Beekeeper’s Handbook by Sammataro and Avitable
has an appendix on how to rear wax moth larvae
Savor the Moment
Photo Credit:
Izzy Hill
Food Safe Lids = Petri Dish Substitute
• Four, 4-inch diameter
food container lids or
Petri dishes
• One, 2-inch food
container lid or Petri
dish
• Wash everything with
soap and rise 3x (nondistilled water ok)
Source: Gulco and Hazir, 2012; Thompson, 2012
Photo Credit: Izzy Hill
,
Unbleached Coffee Filters
• Typically coarse lab filter paper is used
– Non-toxic and weave is large enough to let
nematodes pass through
• Thompson (2012) shows
that unbleached coffee
filters are a nearly
identical product
• Simply cut filters to
needed size
Photo Credit: Izzy Hill
Culture Steps
1. Use pipette to collect
approximately 0.1 ml (or
less) of nematode
suspension
2. Add 1 ml of distilled water,
gently mix
3. Place in 4 inch circular,
doubled up coffee filters that
are placed in a 4 inch lid.
Photo Credit: Izzy Hill
Methodology adapted from Manual of Techniques
in Insect Pathology. Chapter by Kaya and Stock
(1997)
Culture Steps
4. Saturate filter with
nematode suspension
– Do not oversaturate filter
with nematodes:
•
•
Nematodes will compete
for resources
Puts nematodes at risk
from foreign bacteria
Photo Credit: Izzy Hill
Culture Steps
5. Place 10-15 wax worms
on the saturated filter
– Helps if they’re cold and
immobile
– Make sure they’re alive
• Cream colored
• Plump (not shriveled)
• Let them warm up just a
little to verify they are
alive
Photo Credits: Izzy Hill
Culture Steps
6. Cover loosely with another lid
– Let air in, don’t let wax worms out
– Put culture in a
partially unzipped
Ziploc bag
to maintain
humidity and
prevent filters
from drying out
Photo Credit: Izzy Hill
,
Step 1
Step 3
Step 2
Step 4
Overview of Culturing Steps
Culture Steps
7. Place culture at room
temperature in a dark
place
Not Infected
Infected
8. After 2-3 days,
infected cadavers
turns a rusty red color
w/ H. indica
9. Total culture time =
5-7 days
Photo Credit: Izzy Hill
Rearing Heterorhabditis indica
• Step 1: Culture
• Step 2: Harvest
• Step 3: Storage
Setting up the White Trap
A widely accepted
method for
capturing
nematodes
(Whitehead and
Hemming, 1965)
1. Set 2 inch petri dish/
food lid inverted
inside a four inch
food lid/ petri dish
Setting up the White Trap
2. Drape a new, doubled up
piece of coffee filter over the
2 inch food lid
– Filter will be around 3 inch
diameter
– Allow edges to hang over (coffee
filters better than lab paper for
this)
3. Fill the bottom lid with ~20 ml
of distilled water
– Let the water wick up the sides
Setting up the White Trap
5. Arrange infected wax
worms radially
– Only use infected wax
worms (and avoid
funky looking ones)
– Don’t pour water over
cadavers (nematodes
will die from anoxia)
Photo Credit: Izzy Hill
,
Setting up the White Trap
6. Put a loosefitting or
perforated lid
on it
7. Place in halfopen Ziploc
bag
8. Place in a cool,
dark place
Overview of Setting up the White Trap
Step 1
Step 2
Step 3
Step 4
Emergence
• Nematodes will emerge and
migrate to the water
• For H. indica, infective juveniles
will emerge after
14-15 days at
room temperature
Photo Credit: Bishwo Adhikari
Harvesting Steps
1.
Remove 2 inch lid with
cadavers
2.
Pour liquid from 4-inch lid
into a clean glass or beaker
3.
Replace 2 inch lid with
cadavers back onto 4 inch dish
4.
Add new, fresh, distilled water
to 4 inch dish, then repeat
Steps 1-3 three times to collect
as many nematodes as possible
5.
Nematodes will settle to the
bottom in 20 min.
–
You can see them!
Double Check Infectivity
1. Take a small amount of newly reared
nematodes.
2. Repeat the culturing process step but with
just a couple of wax worms.
3. Check in 2-3 days to see if wax worm turns a
rust color.
– If so, they’re infective.
DIY Rearing Heterorhabditis indica
• Step 1: Culture
• Step 2: Harvest
• Step 3: Storage
Storage
• Typically tissue culture
flasks are used for
storing nematodes
(polystyrene)
• Gulco and Hazir (2012)
found that Tetra Pak
containers (polyethylene
+ aluminum) work just as
well—actually better!
Storage (cont.)
• H. indica has a relatively short lifespan if not
stored properly
– Complicated to store long-term
• Can usually get two weeks out of them if
refrigerated.
– Note you can store them a bit longer if you
purchased them and they are stored in gel
Short-Term Storage
1. Use a 1 liter Tetra Pak container
2. Rinse container with soap, then rinse 4x
with water
3. Add 10-15 wax worms worth of nematode
suspension
4. Add no more than 1/3 inch of distilled
water
5. Store loosely covered in fridge (try your
butter shelf on the fridge door)
Application
Rate of Application
• 3 successfully infected wax worms = enough
for one hive when applying 1 ft out (3:1 ratio)
– 23,000 nematodes per 1ft2
• Rate matters much less than placing them in a
good environment
– If they all die, application rate is moot
– Using this method you’d just be guessing how many
were reared (approximately 50k per wax worm).
– Happy nematodes will thrive and reproduce in the
soil around your hive
Time of Application
• H. indica are most effective between 68-75°F soil
temperatures.
• SHB prefer 82-90°F air temperature.
• Both can do quite well outside of these ranges.
• If your personal SHB threshold is exceeded and soil
temperatures are above 60°F, apply H. indica.
• Not as effective if you apply them too early or too late.
• Use NRCS’s Soil Climate Analysis Network to determine
local soil temperatures in your area.
Applying Nematodes
• Mix a portion of
nematodes with 1/2 to
1 gallon of water
• Apply around
hive with a
watering can
• Tap water may
be ok
Applying Nematodes (cont.)
• Avoid overly sunny, dry, bare, and/or hot areas.
– Heat and ultraviolet light inactivate nematodes.
• Apply after sundown
– Avoids ultraviolet rays + keeps soil moist + SHB larvae
migrate to soil in evening (Ellis and Hepburn 2006).
• Some sort of ground cover (grass, weeds) may
help support a good, moist microclimate
– But remove deep thatch to apply directly to soil
• Best to apply when soil temps 68-75 ͦF (mid-June)
Checking for Overwintering
• Wait until after a rain or soak ground around hive.
• Take a wax worm and place it in moist (not soaking wet) soil
in a perforated lid.
• Cover lid with a moist coffee filter, rubber band it in place.
• Bury lid just below soil surface in front of your hive (be sure
to do this after watering around the hive).
• Check in 6 days to see if wax worm is red.
– If so, the nematodes overwintered successfully
• If done when temperatures are too cold, nematodes maybe
present but not mobile min. soil temps ~60°F.
Always Use Caution with Biological
Controls
• H. indica are great for controlling white grubs and Japanese
beetles.
• However, may inadvertently target non-target, beneficial
species (e.g., soldier beetles).
• Use caution if doing a broadcast spray for white grubs or
Japanese beetles.
• Losing beneficial insects could also result in pest outbreaks
in vegetable gardens. Avoid applying around hives located
directly in a vegetable garden. Keep notes to determine if
you see unusual pest (esp. aphid) outbreaks after a
nematode application.
• Earthworms and lady bugs, aren’t harmed by H. indica.
Next steps
• These slides are located at:
– www.bugonia.com/rearingguide
• Formal rearing guide to be released in the
next month
• Questions? Email me at [email protected]
• Consider starting a nematode rearing program
with your bee club
Great for many other garden pests
• Great for many other garden pests:
– Fall armyworm, Spodoptera frugiperda
– Fungus gnat, Bradysia difformis
– Indian meal moth, Plodia interpunctella
– Root weevil, Diaprepes abbreviatus
– Western flower thrips, Frankliniella occidentalis
– White grubs, Holotrichia serrata, Leucopholis
lepidophora, Hoplia philanthus, Popillia japonica
Source: Ansari et. al (2008); Ebssa et. al (2006), Jagdale, et. al (2007); Mbata and ShapiroIlan (2010); Negrisoli, et. al (2010a, b); Shapiro-Ilan, et. al (2010)