SMolecular identification of swede midge

SMolecular identification of swede midge










Scouting for Swede Midge


Identification of Swede Midge Insect Specimens


The swede midge (Contarinia nasturtii Kieffer) (Diptera: Cecidomyiidae) is a Eurasian pest of crucifers. It has been reported in Austria, Belgium, Czechoslovakia, Denmark, France, Germany, Great Britain, Ireland, Italy, the Netherlands and other countries (Ellis 2005). Swede midge is a relatively new insect pest in North America and poses a serious threat to cruciferous vegetables and field crops (Kikkert et al., 2009).There are approximately 400 Contarinia spp. worldwide. Approximately 1,200 species in the family Cecidomyiidae reside in North America, with at least 59 Contarinia spp. described. Because many cecidomyiids are not yet described, this number is probably an underestimate (Ellis 2005).

Accurate identification of the pest is critical to evaluate its potential risk, develop survey strategies, and determine the level and manner of control. Like many other midges, it is difficult to accurately identify swede midge eggs, larvae and pupae based on morphology information. The adult stage has more morphological features that could be used for identification. However, molecular tools make it possible to accurately identify swede midge regardless of life stage. To identify swede midge, it is important, first of all, to collect and process midges associated with known host plants since swede midge reportedly feeds on cruciferous plants only.

Currently, we identify swede midge using two methods, 1) morphological features (good for swede midge adults and 2) swede midge mitochondrial cytochrome c oxidase subunit 1 (COI) gene sequence (good for all life stages).



Morphological identification
Although it is difficult to morphologically identify swede midge eggs, larvae and pupae, the eggs, larvae and pupae have some characteristics that could be used for identification if they are found in conjunction with cruciferous plant material. Listed below are these characteristics (Ellis 2005).

Eggs
Use the following characters to identify eggs:
• Color is white
• Size is 0.27 mm by 0.08 mm with a 0.06 pedicel
• Batch size is 2-50 eggs


Larvae
Use the following characters to identify first and second instars:
• Legs are absent
• Color is translucent
• Length is between 0.3 mm to 4 mm
Use the following characters to identify third (last) instars:
• Color is pale lemon
• Body length is 3-4 mm at maturity
• During the last instar, larvae possess a sclerite—the sternal spatula—on the ventral side of the prothorax
• Third instars are capable of jumping

Pupae
Pupae are spherical or oval cocoons formed primarily in the top 5 cm of soil near the host plant (Hornig 1953; Readshaw 1966; Chen and Shelton 2007).

Adults
Swede midge adult stage is the only life stage that has unique characteristics for an accurate morphological identification. The following diagram (Fig.1) is a swede midge identification decision tree developed by James D. Heal at University of Guelph and provides key characteristics of swede midge adults for morphological identification.

Figure 1
Swede midge adult identification decision tree (JD Heal, University of Guelph)

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Swede midge adult identification decision tree




Wing venation (Fig. 2) and antenna shapes are the most useful diagnostic tools for swede midge adult identification. Swede midge wing venation is reduced (cross veins are absent, radial vein is straight or nearly so, and cubital fork is present in middle third of wing). Swede midge antennae are long, filiform. Female antennal segments are cylindrical (Fig. 3A). Male antennae with 12 flagellomeres; each is divided into two separate nodes surrounded by a threadlike looped sensillum (Fig. 3B) (Ellis 2005).

Figure 2
Swede midge wing venation characteristics (Ellis 2005)
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 Swede midge wing venation characteristics

Figure 3
Swede midge female (A) (Ellis 2005) and male (B) (www.contarinia.faw.ch) antennae
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Swede midge female (A) (Ellis 2005) and male (B) (www.contarinia.faw.ch) antennae

Figure 4
Molecular identification of swede midge adults emerged from cruciferous weed species collected from Niagara County, New York State in 2006 (Chen et al., 2009)
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Molecular identification of swede midge adults

Swede midge adults were from 1 laboratory colony (positive control), 2 Lepidium campestre, 3 Sinapis arvensis, 4 Erysimum cheiranthoides, 5 Capsella bursa-pastoris and 6 Rorippa islandica. M: DNA molecular weight markers.




Molecular Identification
For molecular identification, the mitochondrial cytochrome c oxidase subunit I gene (COI) sequence of swede midge (Genbank accession No. EU812560) is commonly used as the diagnostic molecular marker (Frey et al., 2004; Kikkert et al., 2006; Chen et al., 2009). Swede midge molecular identification procedure includes:

1. Extracting genomic DNA from 1 swede midge suspect sample (whole larva or adult or body parts).
2. 1st PCR: polymerase chain reaction (PCR) amplification of a COI fragment (488 bp) using a pair of universal primers (5’-GGATCACCTGATATAGCATTCCC-3’ and 5’-CCCGGTAAAATTAAAATATAAACTTC-3’).
3. 2nd PCR: a swede midge diagnostic PCR using the 488 bp PCR fragment as the temperate with a pair of diagnostic primers (5’-CAATTATTGGAGATACTCGAAGATGA-3’ and 5’-ATTCGAACTCCTGCTCCTATTCGATCTAGG-3’).
4. Positive amplification of a 286 bp specific swede midge PCR fragment indicates the positive identification of a C. nasturtii specimen.
5. If a swede midge suspect sample was collected from a place/site where no swede midge occurrence had been reported previously, the COI gene fragment gene of the suspect sample needs to been sequenced and compared with the known swede midge COI gene.

For example, in 2004 we found swede midge larvae in plants in Niagara County, NY and they were first identified using PCR and then confirmed by comparing its sequence to the known swede midge COI gene. Subsequent samples of swede midge collected in the same area from cruciferous crops and weeds were identified usually using only 2 PCR tests (Fig. 4).



References cited

Chen, M, A. M. Shelton, P. Wang1, C. A. Hoepting, W. C. Kain, and D. C. Brainard. 2009. Occurrence of the new invasive insect, Contarinia nasturtii (Kieffer) (Diptera: Cecidomyiidae), on cruciferous weeds. J. Econ. Entomol. 102: 115-120.
Chen, M., J. Z. Zhao, and A. M. Shelton. 2007. Control of Contarinia nasturtii (Diptera: Cecidomyiidae) by foliar sprays of acetamiprid on cauliflower transplants. Crop Prot. 26: 1574-1578.
Ellis, S. E. 2005. New pest response guidelines: swede midge. USDA APHIS PPQ PDMP. http://www.aphis.usda.gov/ppq/manuals/
Frey, J. E., B. Frey, and R. Baur. 2004. Molecular identification of the swede midge (Diptera: Cecidomyiidae). Can. Entomol. 136: 771-780.
Hornig, H. 1953. The significance of C. nasturtii for swede cultivation with special reference to conditions in Schleswig Holstein. Z. Angew. Entomol. 35: 271-318.
Kikkert, J. R., C. A. Hoepting, A. M. Shelton, and M Chen. 2009. Swede midge. Cornell University Cooperative Extension Fact Sheet no. 102VCFS751.3. http://www.nysipm.cornell.edu/factsheets/vegetables/cruc/sm.pdf
Kikkert, J. R., C. A. Hoepting, Q. J. Wu, P. Wang, R. Baur, and A. M. Shelton. 2006. Detection of Contarinia nasturtii (Diptera; Cecidomyiidae) in New York, a new pest of cruciferous plants in the United States. J. Econ. Entomol. 99: 1310-1315.
Readshaw, J. L. 1966. The ecology of the swede midge, Contarinia nasturtii (Kieffer) (Diptera: Cecidomyiidae). I. Life-history and influence of temperature and moisture on development. Bull. Entomol. Res. 56: 685-700.
 

 



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