DIY Nematode Rearing for Small Hive Beetle Control

Izzy HillCenter 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

Infective Juvenile (IJ)

Source: Tofangsazi et. al (2012) Photo Credit: James Kerrigan

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 bacteria in gut & and multiplies

• Host dead in 24-48 hours

• Bacteria provides defense inside host by producing antibiotics, nematicides, warning colors, and compounds to deter scavenging insects

Photo Credit: Chich, 2007Sources: Tofangsazi et. al , 2012; Chich, 2007; Ciche and Ensign, 2003

Symbiotic Bacteria in intestinal tract of IJ

Photo Credit: D. Fenolino

The Host: Small Hive Beetle (SHB)

Photo Credits: Josephine Ratikan (top); Lyle J. Buss (bottom), UFL

Pupating SHB

SHB Larvae

SHB Pupae

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

• 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

Photo Credit: Izzy Hill ,

Savor the Moment

Photo Credit: Izzy Hill

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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 (non-distilled 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

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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.

Methodology adapted from Manual of Techniques in Insect Pathology. Chapter by Kaya and Stock

(1997)

Photo Credit: Izzy Hill ,

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

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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 2

Step 3 Step 4

Overview of Culturing Steps

Culture Steps

7. Place culture at room temperature in a dark place

8. After 2-3 days, infected cadavers turns a rusty red colorw/ H. indica

9. Total culture time = 5-7 days

Not Infected Infected

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 loose-fitting or perforated lid on it

7. Place in half-open 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 container2. 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 WF (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 izzy@izzyhill.com

• 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 Shapiro-Ilan (2010); Negrisoli, et. al (2010a, b); Shapiro-Ilan, et. al (2010)