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Validity of morphological characters used to

distinguish Culex restuans and Culex pipiens

1Environmental Health Sciences Program, Western Carolina University

2Biology Department, Western Carolina University

3Biology Department, Virginia Commonwealth University4Dept. of Environmental Protection, Vector Management, Pennsylvania

Tyler McKinnish1,2, Bruce Harrison1, Kevin Caillouet3,

Michael Hutchinson4, and Brian Byrd1

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Background

Culex pipiens and Culex restuans are the primaryvectors of West Nile virus in the eastern US

Enzootic (avifaunal) transmission and magnification

Primary vectors in epidemic transmission of WNV in many

regions, but roles in transmission are complex and variable

(Apperson 2002; Apperson 2004)

Cx. pipiens is a more important vector than Cx. restuans

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Background

Difficult to differentiate morphologically

(NY) Harrington and Poulson 2008

Molecular ID by PCR more accurate

Requires molecular techniques

Resource intensive

Only takes one leg to contaminate

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Research Purpose

To determine the validity of the three morphological

characters for the differentiation ofCx. pipiens and

Cx. restuans

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Keys to the Mosquitoes of the Mid-Atlantic Region, Harrison et al. (Unpublished)

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Methods

Cx. pipiens and Cx. restuans mosquitoes collected from gravid traps

(PA, VA, NC)

Three morphological characters for all specimens assessed by one

reviewer (BB)

DNA extracted for PCR using DNAzol procedure (MRC, Inc)

PCR analyzed using conserved reverse primer (28s rDNA sequence)

and species-specific forward primers (Crabtree et al 1995)

Data analyzed to determine validity of the morphologicalcharacters

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Culexspecies ID PCR(Crabtree et al, 1995)

18S 5.8S 28S

PQ10-CP

R6-CR

Culex pipiens (698 base pairs)

Culex restuans (506 base pairs)

Conserved Primer:

28s (CP16)

Cx. pipiens Cx. restuans

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Preliminary Results

236 mosquitoes reviewed (to date)

Character Presence/Absence Determined

DNA extracted 56 mosquitoes with paired PCR results

36 Culex pipiens

20 Culex restuans Preliminary Validity Calculations

Sensitivity and Specificity

Positive and Negative Predictive Values

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Sensitivity tells us the proportion of a particular species

that will be correctly identified by the characterCP-Primers

Sensitivity: TP / (TP+FN) X 100

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Specificity tell us the proportion of the individuals

who are NOT members of a particular species that

will be correctly identified as NOT that species

Specifiity: TN / (TN+FP) X 100

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Scutum with Pale Scale Spots

Scutum with two small white spots: Cx. restuans

Scutum without two small spots: Cx. pipiens

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Scutum with Pale Scale SpotsCulex restuans (PCR ID)

+ -

Character Present

(Pale Scale Spots)6 0

Character Missing

(No Pale Scale Spots) 13 29

Sensitivity: 31.6% (95% CI: 13.6%- 56.5%)

Specificity: 100% (95% CI: 85.4%- 100%)

In other words, only 32% of the actual Culex restuans (PCR confirmed), werecorrectly identified by the presence of two small white spots on the scutum.

Furthermore, 100% of the Culex pipiens (PCR confirmed) were correctly

identified as NOT having two small white spots on the scutum.

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Abdominal Band Characters

Abdominal segments II and III with continuous white basalbands with straight or nearly straight margins: Culex restuans

Abdominal segments II and III usually with enlarged cream orlight yellow central spot that is narrowly attached (or not

attached) to white lateral basal spots: Culex pipiens

Culex restuans Culex pipiens

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Abdominal Band Characters

Culex restuans (PCR ID)

+ -

Character Present

(straight bands)13 6

Character Missing(centrally enlarged bands)

2 32

Sensitivity: 86.7% (95% CI: 58.3%-97.6%)

Specificity: 84.2% (95% CI: 68.1%- 93.4%)

In other words, 87% of the actual Culex restuans (PCR confirmed), werecorrectly identified based on the abdominal characteristic.

Furthermore, 84% of the Culex pipiens (PCR confirmed) were correctly identified

with the presence of centrally enlarged bands.

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Pale Head Scale Characters

Top of head with long black erect forked scales on medianand lateral areas of occiput and vertex: Culex restuans

Top of head with one or more light tan or pale, long, and

erect forked scales on midline of vertex and/or occiput,

with brown erect scales laterally: Culex pipiens

Culex restuans Culex pipiens

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Pale Erect Scales (Head) Characteristics

Culex pipiens (PCR ID)

+ -

Character Present

(Pale erect forked scales)23 1

Character Missing(Dark erect forked scales)

2 14

Sensitivity: 92.0% (95% CI: 72.4%-98.6%)

Specificity: 93.3% (95% CI: 66.0%- 99.7%)

In other words, 92% of the actual Culex pipiens (PCR confirmed), were correctlyidentified based on the pale head scale character.

Furthermore, 93% of the Culex restuans (PCR confirmed) were correctly

identified with the presence of dark erect forked scales.

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Character States: Presence?

*missing/indistinguishable

Erect scales on the occiput were missing* 39.7% (95% CI: 8.08) of the time

Visible scutal scales were missing* 16.3% (95% CI: 6.1) of the time

Abdominal bands were missing * 11.3% (95% CI: 5.23) of the time

n=141

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Summary - 1

White Spots on Scutum: Though all of the Cx.pipiens lacked

the character, too many Cx. restuans also lack the character-

69% would have been IDd as Cx.pipiens!

Culex restuans will be identified 100% of the time if the spots

are present. (True Positive; 95% CI: 52%-100%)

However, for any particular specimen without spots, the

probability that it is Culex pipiens is only 69%.(True Negative; 95% CI: 53%-82%)

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Summary - 2

Abdominal Banding: 91% ofCx. restuans were correctly

identified, and only 14% ofCx. pipiens were misidentified as

Cx. restuans (bands appear continuous)

For any particular specimen with straight white bands on the

abdomen, the probability that it is Culex restuans is 68%.

(True Positive; 95% CI: 43%-86%)

For any particular specimen with centrally enlarged bands,the probability that it is Culex pipiens is 94%.

(True Negative; 95% CI: 79%-99%)

Cx. pipiens

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Summary - 3

Pale Erect Forked Scales on the Occiput: 92% ofCx. pipiens

were correctly identified as Cx. pipiens, AND less than 7% of

Cx. restuans were misidentified as Cx. pipiens

For any particular specimen with pale erect scales on

the occiput, the probability that it is Culex pipiens is

96%. (True Positive; 95% CI: 77%-99%)

For any particular specimen with dark erect scales on

the occiput, the probability that it is Culex restuans is88%. (True Negative; 95% CI: 60%-98%)

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Take Home Messages

The presence of scutal spots is great for identifying Cx. restuans.

(However, present < 36% of the time in Cx. restuans)

The absence of scutal spots is not diagnostic.

The abdominal band character is also a good character, and is

present at a high rate in both species.

The abdominal characters are present more often than the occiput

character (89% vs 60%).

Using the combination of characters will increase the likelihood of

correctly distinguishing the two species.

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Take Home Messages

The occiput characters (pale vs black scales) is a good

character.

The occiput character does a little (~10%) better job at

identifying Cx. restuans than the abdominal character does.

The occiput character does a little (~10%) better job at

identifying Cx. restuans than the abdominal character does.

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Limitations

These results are preliminary

Need to increase PCR numbers (In progress)

Consider adding Cx. quinquefasciatus

Current results are only from PA and VA specimens

Limited number (56) of specimens with confirmed PCR identities

However, confidence intervals suggest significant trends

All specimens collected from gravid traps

Other traps may increase/decrease character presence rates

Single morphological reviewer for individual character states (BB)

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Selected References

Crabtree, M.B., Savage, H.M. and B.R. Miller. 1995. Development of a Species-diagnostic Polymerase

Chain Reaction Assay for the Identification ofCulexVectors of St. Louis Encephalitis Virus Based on

Interspecies Sequence Variation in Ribosomal DNA Spacers.American Journal of Tropical Medicine

and Hygiene. 53(1): 105-109.

Harrington, L.C., and R.L. Poulson. 2008. Considerations for Accurate Identification of Adult Culex restuans

(Diptera: Culicidae) in Field Studies.Journal of Medical Entomology. 45(1): 1-8.

Apperson, C.S., Harrison, B.A., Unnasch, T.R., Hassan, H.K., Irby, W.S., Savage, H.M., Aspen, S.E., Watson,

D.W., Rueda, L.M., Engber, B.R., and Nasci, R.S. 2002. Host-Feeding Habits ofCulexand Other

Mosquitoes (Diptera: Culicidae) in the Borough of Queens in New York City, with Characters and

Techniques for Identification ofCulexMosquitoes.Journal of Medical Entomology. 39(5): 777-785.

Apperson, C.S., Hassan, H.K., Harrison, B.A., Savage, H.M., Aspen, S.E., Farajollahi, A., Cranston, W.,

Daniels, T.J., Falco, R.C., Benedict, M., Anderson, M., McMillen, L., and Unnasch, T.R. 2004. Host

Feeding Patterns of Established and Potential Vectors of West Nile Virus in the Eastern United States.Vector-Borne and Zoonotic Diseases. 4(1): 71-82.

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Acknowledgements

Funding:

WCU College of Health and Human Sciences

WCU Honors CollegeVirginia Commonwealth University

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Questions?

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