Eradication Programs
Florida GALS Eradication Program Methods Development
Objectives • Chemical Control Techniques – Molluscicide bait efficacy testing – Effective soil drench development
• Trap Development – Most effective trap design – Most effective attractants
• Nematode Detection – Is A. cantonensis present in Florida
• Biological Studies – – – – –
Time to reproductive maturity (defined by first egg laying event) Growth rate over time and “natural” survivability Clutch size and frequency of egg laying and effect of burial depth on egg hatch Effect of population density on the animals Source of chirality in A. fulica using traditional crosses and molecular techniques.
Molluscicide Evaluation
Molluscicide Evaluation Product
Mortality (%) after 15days (Mean ± S.E.)*
Ortho® Bug-Geta® Plus
Choice Test 70.3 ± 4.3 a
No-Choice Test 72.9 ± 4.2 a
Niban®
45.3 ± 4.7 bc
75.5 ± 4.0 a
Sluggo®
48.9 ± 4.7 bc
59.7±4.6 ab
Ferroxx®
34.7 ± 4.5 c
50.7 ± 4.7 b
Control
2.2 ± 1.3 d
2.2 ± 1.3 c
Molluscicide Evaluation: No-choice Test Sluggo®
Ortho® Bug-Geta® Plus
80%
80%
70% Mean % Mortality
Mean % Mortality
70% 60% 50% 40% 30% 20%
60% 50% 40% 30% 20% 10%
10% 0%
0% 0
5
10
15
0
5
Time (days)
15
10
15
Time (days)
Ferroxx®
Niban®
80%
80%
70%
70% Mean % Mortality
Mean % Mortality
10
60% 50% 40% 30% 20%
60% 50% 40% 30% 20% 10%
10%
0%
0% 0
5
10 Time (days)
15
0
5 Time (days)
Molluscicide Evaluation: Choice Test Ortho® Bug-Geta® Plus
Sluggo®
80% Mean % Mortality
Mean % Mortality
70% 60% 50% 40% 30% 20% 10%
80% 70% 60% 50% 40% 30% 20% 10% 0% 0
0% 0
5
10
5
10
15
Time (days)
15
Time (days)
Niban®
Ferroxx®
80%
80%
70% Mean % Mortality
Mean % Mortality
70% 60% 50% 40% 30% 20%
60% 50% 40% 30% 20% 10%
10% 0%
0% 0
5
10 Time (days)
15
0
5
10 Time (days)
15
Snail Collection – Iron Phosphate Logarithmic Specimen Live/Dead/Totals Since Program Inception 10000
35000
* 30000 1000
25000 20000
100 15000 10000
10
5000 Totals
Live/Dead Axis 1
0Axis
*Note: 31,000 snails added in March '12 retroactively after the snails from the first few weeks were counted.
Live
Dead
Total
April 4 – Metaldehyde Snapshot • April 4, 2013 – 46 properties surveyed – 23 props with only dead snails – 402 snails (63 alive; 339 dead) = 84% mortality rate.
Soil Drenches • Natural Oils: clove oil, neem oil, lucky nut (Thevetia peruviana), oleander (Nerium spp., etc.). • Fungicides (high copper content) • Insecticides • Molluscicides (formulated for drench)
Trap Efficacy Testing
Chemical Barrier Testing
Attractants • Environmentally safe, non-toxic ingredients are being evaluated as baits: wheat, alfalfa, soybean meal, dehydrated apples, calcium citrate, Lglutamine, yeast, sugar, etc. • GALS attraction is evaluated for each developmental stage.
Rat Lungworm Testing • Currently testing for: – Angiostrongylus cantonensis
• Could possibly include the following species in the future: – – – –
A. costaricensis A. dujardini A. malaysiensis A. vasorum
http://dpd.cdc.gov/dpdx/html/ImageLibrary/A-F/Angiostrongyliasis/body_Angiostrongyliasis_il5.htm
• Quantitative PCR • Traditional identification using light microscopy
Quantitative-PCR Testing
Remove tissue from snail and add reagents to digest and extract the DNA
Prepare super-mix for testing samples Test samples using the real-time PCR machine
Results
• A positive sample is used in each batch of samples tested. • A sample is positive if the curve goes above the 30 relative fluorescence units (RFU)
Traditional Nematode Extraction
Rat lungworm Testing Results: To Date • Over 500 snails tested so far • 30 specimens tested a week • Samples tested from 17 of the 20 Cores • RLW detected in Cores 4, 7 and 12.
Biology Studies • Growth rate over time • Egg Burial depth study • Reproductive Studies • Self-fertilization? • Chirality Breeding Study (dominant / recessive)
Time of First Egg Laying
All treatments (58 Cages)
Average:
(2 snails per cage) Minimum:
Parental Size
Days after Hatch
Months after Hatch
Height (mm)
Width Whorls Weight (mm)
193.3
6.2
85.1
44.3
6.99
69.32
149.0
5.0
69.5
34.2
6.25
36.36
Synthetic Diet - GM
Average:
187.3
5.9
84.4
44.1
6.96
67.90
(51 cages)
Minimum:
101.0
5.0
69.5
34.2
6.25
36.36
Lettuce
Average:
237.1
8.0
90.4
45.7
7.18
79.64
(7 cages)
Minimum:
235.0
8.0
80.5
38.8
6.75
51.01
Significant Life Table Data • Egg to fully mature adult (2 snails per 4L cage) – Average = 193.3 days (6.2 months) – Minimum = 149 days (5 months).
• Size at first egg laying (both parents were measured) – Average height = 85.1 mm (3.4 inches). – Note: the smallest parent was 69.5 mm (2.7 in.) in height, but it is impossible to say whether that individual had a fully mature female reproductive system or was only the sperm donor (which develops first).
• Greatest number of eggs produced by one cage – 2,207 eggs (7 clutches) within 76 days. – Isolated snails, (1 snail per 4L cage), have not produced any eggs.
Egg Incubation Depth
• Eggs buried at a range of depths up to 17” • Emerged up to 15” so far
Reproductive Potential- Field Studies • Smallest reproductively mature snails • Smallest snails with eggs – Viability?
• Do snails retain eggs longer during winter?
Mating Configuration • Reciprocal
• Non-reciprocal
Chirality • Pair desired crosses prior to reproductive maturity • Observe pairs for egg production • Hatchlings evaluated for chirality of shell • Tissue samples saved for genetic evaluation
THANK YOU for your time and attention
Objectives • Chemical Control Techniques – Molluscicide bait efficacy testing – Effective soil drench development
• Trap Development – Most effective trap design – Most effective attractants
• Nematode Detection – Is A. cantonensis present in Florida
• Biological Studies – – – – –
Time to reproductive maturity (defined by first egg laying event) Growth rate over time and “natural” survivability Clutch size and frequency of egg laying and effect of burial depth on egg hatch Effect of population density on the animals Source of chirality in A. fulica using traditional crosses and molecular techniques.
Molluscicide Evaluation
Molluscicide Evaluation Product
Mortality (%) after 15days (Mean ± S.E.)*
Ortho® Bug-Geta® Plus
Choice Test 70.3 ± 4.3 a
No-Choice Test 72.9 ± 4.2 a
Niban®
45.3 ± 4.7 bc
75.5 ± 4.0 a
Sluggo®
48.9 ± 4.7 bc
59.7±4.6 ab
Ferroxx®
34.7 ± 4.5 c
50.7 ± 4.7 b
Control
2.2 ± 1.3 d
2.2 ± 1.3 c
Molluscicide Evaluation: No-choice Test Sluggo®
Ortho® Bug-Geta® Plus
80%
80%
70% Mean % Mortality
Mean % Mortality
70% 60% 50% 40% 30% 20%
60% 50% 40% 30% 20% 10%
10% 0%
0% 0
5
10
15
0
5
Time (days)
15
10
15
Time (days)
Ferroxx®
Niban®
80%
80%
70%
70% Mean % Mortality
Mean % Mortality
10
60% 50% 40% 30% 20%
60% 50% 40% 30% 20% 10%
10%
0%
0% 0
5
10 Time (days)
15
0
5 Time (days)
Molluscicide Evaluation: Choice Test Ortho® Bug-Geta® Plus
Sluggo®
80% Mean % Mortality
Mean % Mortality
70% 60% 50% 40% 30% 20% 10%
80% 70% 60% 50% 40% 30% 20% 10% 0% 0
0% 0
5
10
5
10
15
Time (days)
15
Time (days)
Niban®
Ferroxx®
80%
80%
70% Mean % Mortality
Mean % Mortality
70% 60% 50% 40% 30% 20%
60% 50% 40% 30% 20% 10%
10% 0%
0% 0
5
10 Time (days)
15
0
5
10 Time (days)
15
Snail Collection – Iron Phosphate Logarithmic Specimen Live/Dead/Totals Since Program Inception 10000
35000
* 30000 1000
25000 20000
100 15000 10000
10
5000 Totals
Live/Dead Axis 1
0Axis
*Note: 31,000 snails added in March '12 retroactively after the snails from the first few weeks were counted.
Live
Dead
Total
April 4 – Metaldehyde Snapshot • April 4, 2013 – 46 properties surveyed – 23 props with only dead snails – 402 snails (63 alive; 339 dead) = 84% mortality rate.
Soil Drenches • Natural Oils: clove oil, neem oil, lucky nut (Thevetia peruviana), oleander (Nerium spp., etc.). • Fungicides (high copper content) • Insecticides • Molluscicides (formulated for drench)
Trap Efficacy Testing
Chemical Barrier Testing
Attractants • Environmentally safe, non-toxic ingredients are being evaluated as baits: wheat, alfalfa, soybean meal, dehydrated apples, calcium citrate, Lglutamine, yeast, sugar, etc. • GALS attraction is evaluated for each developmental stage.
Rat Lungworm Testing • Currently testing for: – Angiostrongylus cantonensis
• Could possibly include the following species in the future: – – – –
A. costaricensis A. dujardini A. malaysiensis A. vasorum
http://dpd.cdc.gov/dpdx/html/ImageLibrary/A-F/Angiostrongyliasis/body_Angiostrongyliasis_il5.htm
• Quantitative PCR • Traditional identification using light microscopy
Quantitative-PCR Testing
Remove tissue from snail and add reagents to digest and extract the DNA
Prepare super-mix for testing samples Test samples using the real-time PCR machine
Results
• A positive sample is used in each batch of samples tested. • A sample is positive if the curve goes above the 30 relative fluorescence units (RFU)
Traditional Nematode Extraction
Rat lungworm Testing Results: To Date • Over 500 snails tested so far • 30 specimens tested a week • Samples tested from 17 of the 20 Cores • RLW detected in Cores 4, 7 and 12.
Biology Studies • Growth rate over time • Egg Burial depth study • Reproductive Studies • Self-fertilization? • Chirality Breeding Study (dominant / recessive)
Time of First Egg Laying
All treatments (58 Cages)
Average:
(2 snails per cage) Minimum:
Parental Size
Days after Hatch
Months after Hatch
Height (mm)
Width Whorls Weight (mm)
193.3
6.2
85.1
44.3
6.99
69.32
149.0
5.0
69.5
34.2
6.25
36.36
Synthetic Diet - GM
Average:
187.3
5.9
84.4
44.1
6.96
67.90
(51 cages)
Minimum:
101.0
5.0
69.5
34.2
6.25
36.36
Lettuce
Average:
237.1
8.0
90.4
45.7
7.18
79.64
(7 cages)
Minimum:
235.0
8.0
80.5
38.8
6.75
51.01
Significant Life Table Data • Egg to fully mature adult (2 snails per 4L cage) – Average = 193.3 days (6.2 months) – Minimum = 149 days (5 months).
• Size at first egg laying (both parents were measured) – Average height = 85.1 mm (3.4 inches). – Note: the smallest parent was 69.5 mm (2.7 in.) in height, but it is impossible to say whether that individual had a fully mature female reproductive system or was only the sperm donor (which develops first).
• Greatest number of eggs produced by one cage – 2,207 eggs (7 clutches) within 76 days. – Isolated snails, (1 snail per 4L cage), have not produced any eggs.
Egg Incubation Depth
• Eggs buried at a range of depths up to 17” • Emerged up to 15” so far
Reproductive Potential- Field Studies • Smallest reproductively mature snails • Smallest snails with eggs – Viability?
• Do snails retain eggs longer during winter?
Mating Configuration • Reciprocal
• Non-reciprocal
Chirality • Pair desired crosses prior to reproductive maturity • Observe pairs for egg production • Hatchlings evaluated for chirality of shell • Tissue samples saved for genetic evaluation
THANK YOU for your time and attention
