Information relating to Monarch butterflies and Bt corn

Bt

"Bt" is the common term for a series of proteins that were discovered in the soil-living bacterium Bacillus thuringiensis. These Bt proteins are used as insecticides because they kill a variety of insect pests. They are believed to be toxic to insects but harmless to mammals. Because there are so many different proteins with different properties, they are assigned numbers and letters so we can keep them straight. The name for a Bt protein consists of Cry (for crystal protein), a number indicating what major group it belongs to, and one or more letters indicating what subcategory it belongs to. So the full name can look like this: CryIa or Cry1a. The older practice of using Roman numerals for the numbers is being replaced by a new style using Arabic numerals. You will see Bt names written both ways. Each protein targets a particular group of insects. The Cry1 group kills insects in the order Lepidoptera, the butterflies and moths. The Cry3 group kills insects in the order Coleoptera, the beetles. Bt has been available to gardeners and farmers for several decades in the form of a powder that is applied to plants to protect them from insect damage. In recent years, the bacterium's DNA sequence that directs production of the toxic protein has been engineered into crop plants so that applications of the powder to fields are not required. More information on Bt is available at http://helios.bto.ed.ac.uk/bto/microbes/bt.htm.

Bt corn

Bt corn refers to corn varieties that have been genetically engineered with a Bt gene. A Bt corn plant will produce Bt toxin within its cells. Insects that eat the tissues of the corn plant, for example a European corn borer feasting on leaves or a corn rootworm munching on roots, will eat the toxin and will die. Different varieties of Bt corn have different properties because they contain different Cry proteins. Some varieties have been engineered to manufacture the toxic Cry protein in all cells of the plant all season long. At least one variety did not make the Cry protein in the corn kernels; in this variety the amount that was made in other cells of the plant gradually decreased toward the end of the summer.

Event Promoters, enhancers Coding region Selectable marker Method used
Bt 176*

1) PEP carboxylase
(green tissues)
2) corn CDPK
(pollen-specific)

Modified Cry1A(b) bar gene + CaMV35S (herbicide tolerance) gene gun
MON810 CaMV35S + intron from corn hsp70 gene Modified Cry1A(b) CP4 EPSPS and gox genes (herbicide tolerance), each with CTP;
nptII (antibiotic resistance; not expressed)
gene gun
Bt 11 CaMV 35S Modified Cry1A(b) bar gene + CaMV35S (herbicide tolerance) PEG-mediated protoplast transformation
CB 351* (StarLink) CaMV 35S + petunia cab22L leader sequence Modified Cry9C bar gene + CaMV35S (herbicide tolerance);
bla gene (antibiotic resistance; not expressed)
gene gun

* No longer available in US corn hybrids.
bar: also called pat, phosphinothricin acetyltransferase
bla: beta-lactamase
CaMV35S: cauliflower mosaic virus 35S gene
CKPK: calcium-dependent calmodulin-independent protein kinase
CP4: Agrobacterium strain CP4
CTP: chloroplast transit peptide
EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase
gox: glyphosate oxidoreductase
hsp: heat shock protein
nptII: neomycin phosphotransferase (kanamycin resistance)
PEP carboxylase: phosphoenolpyruvate carboxylase

More information on Bt corn varieties is available at http://www.ianr.unl.edu/pubs/fieldcrops/nf409.htm.

 

Corn pollen

Corn pollen is the vehicle by which gametes from male flowers are carried to female flowers on the corn plant. Pollen is released from the anthers at the top of the corn plant during a two-week period in the summer. Pollen grains usually travel a short distance to a neighboring corn plant, where they fall on the ears and eventually pollinate the female flowers to produce corn kernels on the ears. The distance that pollen will travel depends on wind speed and direction. Actual distances will vary from year to year. The first table below shows data reported by several researchers for the distances that pollen traveled and the number of pollen grains that were found per square inch or per square centimeter at those distances. The second table shows some representative dates when corn pollen is shed in different geographic areas.

Concentrations of pollen deposited at various distances from corn field.

distance pollen grains per square centimeter pollen grains per square inch source
in the field 0 to 506 0 to 3,264 Hansen-Jesse and Obrycki, 2000
0 to 1,600 0 to 10,320 Pleasants et al., 2001
65 to 425 419 to 2,741 Sears et al., 2001
at the edge of the field 0 to 1,100 0 to 7,095 Pleasants et al., 2001
158 to 266 1,019 to 1,716 Sears and Stanley-Horn, 2000)
0.2 meters away 0 to 427 0 to 2,754 Hansen-Jesse and Obrycki, 2000
0.5 meters away 260 1,677 Zangerl et al., 2001
1 meter away 0 to 222 0 to 1,432 Hansen-Jesse and Obrycki, 2000
0 to 1,300 0 to 8,385 Pleasants et al., 2001
170 1,097 Zangerl et al., 2001
2 meters away 0 to 400 0 to 361 Pleasants et al., 2001
192 1,238 Zangerl et al., 2001
3 meters away 0 to 56 0 to 361 Hansen-Jesse and Obrycki, 2000
5 meters away 0 to 11 0 to 71 Hansen-Jesse and Obrycki, 2000
0 to 200 0 to 1,290 Pleasants et al., 2001
34 to 175 219 to 1,129 Sears and Stanley-Horn, 2000)
10 meters away 0 to 4 0 to 25 Hansen-Jesse and Obrycki, 2000

The paper by Pleasants et al. is available at http://www.pnas.org/cgi/reprint/211287498v1.pdf.
The paper by Sears et al. is available at http://www.pnas.org/cgi/reprint/211329998v1.pdf.
The paper by Sears and Stanley-Horn is available at http://www.ag.usask.ca/isbr/Symposium/Proceedings/Section7.htm#120.
The paper by Zangerl et al. is available at http://www.pnas.org/cgi/reprint/171315698v1.pdf.

Period of pollen shed in corn fields. (Oberhauser et al., 2001)

Region Site name Dates of pollen release
Minnesota/Wisconsin Farm July 31-August 7
Amery July 27-August 3
AG July 25-August 1
Rosemount July 21-28
Cedar Creek July 10-26
Iowa 595/280 July 7-13
Shipley S July 7-13
LincolnWay July 11-17
Coopers July 5-19
Ankeny July 6-12
Maryland Wye Farm July 19-31
Wye Island July 6-17
Gannon July 7-19
Airport July 27-August 9
Beaverdam July 6-18
Ontario, Canada Fergus August 7-14
New Hamburg August 1-15
Drayton August 10-17
Linwood August 9-16
Palmerston August 4-18

 

Bt corn pollen

The amount of Bt protein in the pollen differs from one Bt corn variety to another. The table below shows the amount of protein measured in the pollen from several varieties of Bt corn.

Bt event promoter element(s)

amount of Bt protein in pollen*

Bt 176 1) A promoter derived from a pollen-specific protein kinase in maize, linked to the Bt CryIA(b) gene. This promoter causes the Bt protein to be produced in the pollen.
2) A promoter derived from phosphoenolpyruvate carboxylase in maize, linked to the Bt CryIA(b) gene. This promoter causes the Bt protein to be produced in green tissues such as stems and leaves. There is little or no Bt protein produced in the kernels.
1.1 to 7.1 micrograms per gram of pollen
Bt 11 The promoter from the cauliflower mosaic virus 35S gene, linked to the Bt CryIA(b) gene. This promoter causes the Bt protein to be produced in all tissues of the plant. 0.09 micrograms per gram of pollen
MON810 The promoter from the cauliflower mosaic virus 35S gene, linked to the Bt CryIA(b) gene. This promoter causes the Bt protein to be produced in all tissues of the plant. 0.09 micrograms per gram of pollen

 

Monarch butterflies

Several web sites have information on the Monarch butterfly. Here are some links.
http://www.MonarchWatch.org/biology/cycle1.htm (life cycle, egg stage)
http://www.MonarchWatch.org/biology/cycle2.htm (life cycle, larva stage)
http://www.MonarchWatch.org/biology/cycle3.htm (life cycle, pupa stage)
http://www.MonarchWatch.org/biology/cycle4.htm (life cycle, adult stage)
http://www.MonarchWatch.org/biology/index.htm (anatomy, egg stage)
http://www.MonarchWatch.org/biology/anat2.htm (anatomy, larva stage)
http://www.MonarchWatch.org/biology/anat3.htm (anatomy, pupa stage)
http://www.MonarchWatch.org/biology/anat4.htm (anatomy, adult stage)
http://www.MonarchWatch.org/tagmig/index.htm (fall migration, click on the map to enlarge)
http://www.MonarchWatch.org/tagmig/spring.htm (spring migration, click on the map to enlarge)
http://www.MonarchWatch.org/tagmig/winter.htm (overwintering sites)
http://www.pgmonarchs.org/foml.html A page with links to mating, life cycle, and migration

 

corn varieties
http://filebox.vt.edu/cals/cses/chagedor/98corn.html (Bt varieties available in 1996)

 

Monarch butterfly life cycle

Several web sites have information on the life cycle of the Monarch butterfly. Here are some links.
http://www.MonarchWatch.org/biology/cycle1.htm (egg stage)
http://www.MonarchWatch.org/biology/cycle2.htm (larva stage)
http://www.MonarchWatch.org/biology/cycle3.htm (pupa stage)
http://www.MonarchWatch.org/biology/cycle4.htm (adult stage)
http://www.MonarchWatch.org/tagmig/index.htm (fall migration, click on the map to enlarge)
http://www.MonarchWatch.org/tagmig/spring.htm (spring migration, click on the map to enlarge)
http://www.MonarchWatch.org/tagmig/winter.htm (overwintering sites)
http://www.pgmonarchs.org/fomc.html (migration of the Western U.S. population)
http://www.pgmonarchs.org/foma.html (life cycle)
http://www.sbceo.k12.ca.us/~crane/monarch/life.html (life cycle)
http://www.sbceo.k12.ca.us/~crane/monarch/migr.html (migration)

Monarch adults lay their eggs on milkweed plants. The table below shows the number of eggs found on milkweeds in corn fields versus nonagricultural areas and the contribution to the Monarch population of corn fields versus nonagricultural areas. (Oberhauser et al., 2001) The paper by Oberhauser et al. is available at http://www.pnas.org/cgi/reprint/211234298v1.pdf.

Regions and habitats eggs/milkweed plant proportion of breeding habitat contribution of each habitat to the total Monarch population
Minnesota/Wisconsin
    corn
    nonagricultural
 
0.0612 0.124 73
0.0134 0.0011 1
Ontario
    corn
    nonagricultural
 
0.0125 0.302 0.055
0.0133 0.045 1
Iowa
    corn
    nonagricultural
 
0.190 0.436 45
0.114 0.0023 1

An article on ABCNews.com reports that scientists can determine the geographic birthplace of a Monarch butterfly by examining the chemicals in its wings. Fifty percent of the butterflies that were analyzed originated in a region corresponding roughly to the U.S. Corn Belt. http://more.abcnews.go.com/sections/science/dailynews/monarch981221.html The research on this topic was reported in an article by Leonard I. Wassenaar and Keith A. Hobson in 1998 in the Proceedings of the National Academy of Science, volume 95, pages15436-15439.

 

Monarch butterfly larval stage

http://www.MonarchWatch.org/biology/cycle2.htm
http://www.MonarchWatch.org/biology/anat2.htm

 

Bt pollen toxicity

The table below gives the amounts of pollen from various corn sources that were tested on butterfly larvae. Pollen amounts are expressed as number of grains per square centimeter of leaf, with the number of grains per square inch of leaf given in parentheses underneath. The LD50, short for "lethal dose for 50 percent," is the amount of pollen that killed half of the caterpillars tested. A low LD50 indicates that the pollen is very toxic and not much pollen is needed to kill half of the caterpillars. A high LD50 indicates that the pollen is less toxic and a large amount is needed to kill half of the caterpillars.

 
Bt transgene butterfly species LD50* reduced feeding* no effect* source
Bt 176 Monarch 389
(2,509)
133
(8,580)
  Sears and Stanley-Horn, 2000
Bt 176 Monarch 67
(432)
23
(148)
  Sears et al., 2001
Bt 176 Monarch   5 to 10
(32 to 65)
  Hellmich et al., 2001
Bt 176 Black swallowtail 613
(3,954)
    Zangerl et al., 2001
Bt 11 Monarch     more than 1,600
(more than 10,320)
Hellmich et al., 2001
MON810 Monarch     more than 1,600
(more than 10,320)
Hellmich et al., 2001
DBT418 (no longer available) Monarch     more than 1,600
(more than 10,320)
Hellmich et al., 2001
CBH351 (StarLink, no longer available) Monarch     1,001 to 1,600
(6,456 to 10,320)
Hellmich et al., 2001
TC1507 (Herculex, available soon) Monarch     1,001 to 1,600
(6,456 to 10,320)
Hellmich et al., 2001

* Pollen amounts are expressed as number of grains per square centimeter of leaf, with the number of grains per square inch of leaf given in parentheses underneath.

The paper by Hellmich et al. is available at http://www.pnas.org/cgi/reprint/211297698v1.pdf.
The paper by Sears et al. is available at http://www.pnas.org/cgi/reprint/211329998v1.pdf.
The paper by Sears and Stanley-Horn is available at http://www.ag.usask.ca/isbr/Symposium/Proceedings/Section7.htm#120.
The paper by Zangerl et al. is available at http://www.pnas.org/cgi/reprint/171315698v1.pdf.

More information on Bt corn pollen is available at http://www.checkbiotech.org/root/index.cfm?fuseaction=newsletter&topic_id=1&subtopic_id=8&doc_id=2694

 

The Losey experiment

John Losey and his colleagues, Linda Rayor and Maureen Carter, report that they fed Monarch larvae three different diets. For one diet, they collected pollen from N4640-Bt corn, a variety that incorporates the Bt 11 transgene. This pollen was sprinkled over milkweed leaves in an attempt to match the amount of pollen observed on milkweed leaves in corn fields. For a second diet, they collected pollen from a conventional corn variety and sprinkled it over milkweed leaves. The third diet consisted of milkweed leaves with no corn pollen sprinkled over it. Monarch larvae were placed on the leaves, which are their sole diet in nature. Five leaves of each diet were prepared and five larvae were placed on each leaf.

Losey and his colleagues observed the larvae for several days. They kept records of deaths, body weights, and amount of leaves consumed by the larvae. They recorded that larvae on the leaves sprinkled with Bt pollen ate less leaf area and gained less weight than larvae on the leaves sprinkled with conventional pollen or not sprinkled with pollen. Forty-four percent of the larvae eating Bt-sprinkled leaves died, while no larvae died on leaves sprinkled with conventional pollen or with no pollen.

The scientists prepared a report saying that their results had "potentially profound implications" for Monarch butterflies. They suggested that more research be done to determine whether Monarchs were at risk when farmers planted Bt corn in their fields and how the risk from Bt corn compared to the risk from pesticides normally used in corn fields. Losey and his colleagues first circulated their report to fellow scientists for review and suggestions, as is normal in the scientific community. Then they submitted the report to the American weekly journal Science, which declined to publish it. Later they submitted the report to the British weekly journal Nature, which published it.

The full text of the report, minus the two graphs, is available at http://www.biotech-info.net/transpollen.html.

 

John Losey

John Losey is an assistant professor of entomology at Cornell University. For more information about him, visit his homepage at http://www.entomology.cornell.edu/Faculty_Staff/Losey/Losey.html.

 

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