Validity of morphological characters used to distinguish Culex restuans and Culex pipiens

Transcription

1 Validity of morphological characters used to distinguish Culex restuans and Culex pipiens Tyler McKinnish 1,2, Bruce Harrison 1, Kevin Caillouet 3, Michael Hutchinson 4, and Brian Byrd 1 1 Environmental Health Sciences Program, Western Carolina University 2 Biology Department, Western Carolina University 3 Biology Department, Virginia Commonwealth University 4 Dept. of Environmental Protection, Vector Management, Pennsylvania

2 Background Culex pipiens and Culex restuans are the primary vectors 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

3 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

4 Research Purpose To determine the validity of the three morphological characters for the differentiation of Cx. pipiens and Cx. restuans

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 (Unpublished)

6 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 morphological characters

7 Culex species ID PCR (Crabtree et al, 1995) 18S 5.8S 28S PQ10-CP Culex pipiens (698 base pairs) Conserved Primer: 28s (CP16) R6-CR Culex restuans (506 base pairs) Cx. pipiens Cx. restuans

8 Preliminary Results 141 mosquitoes (PA and VA) 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

9 Sensitivity and Specificity Sensitivity tells us the proportion of a particular species that will be correctly identified by the character CP-Primers Sensitivity: TP / (TP+FN) X 100

10 Sensitivity and Specificity 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

11 Scutum with Pale Scale Spots Scutum with two small white spots: Cx. restuans Scutum without two small spots: Cx. pipiens

12 Scutum with Pale Scale Spots Culex restuans (PCR ID) Character Present (Pale Scale Spots) Character Missing (No Pale Scale Spots) 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), were correctly 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.

13 Abdominal Band Characters Abdominal segments II and III with continuous white basal bands with straight or nearly straight margins: Culex restuans Abdominal segments II and III usually with enlarged cream or light yellow central spot that is narrowly attached (or not attached) to white lateral basal spots: Culex pipiens Culex restuans Culex pipiens

14 Abdominal Band Characters Culex restuans (PCR ID) Character Present (straight bands) Character Missing (centrally enlarged bands) 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), were correctly identified based on the abdominal characteristic. Furthermore, 84% of the Culex pipiens (PCR confirmed) were correctly identified with the presence of centrally enlarged bands.

15 Pale Head Scale Characters Top of head with long black erect forked scales on median and 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

16 Pale Erect Scales (Head) Characteristics Culex pipiens (PCR ID) Character Present (Pale erect forked scales) Character Missing (Dark erect forked scales) 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 correctly identified 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.

17 Missing Character States? Visible scutal scales were missing* 16.3% (95% CI: ±6.1) of the time n=141 Abdominal bands were missing * 11.3% (95% CI: ±5.23) of the time Erect scales on the occiput were missing* 39.7% (95% CI: ±8.08) of the time *missing/indistinguishable

18 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 ID d 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%)

19 Summary - 2 Abdominal Banding: 91% of Cx. restuans were correctly identified, and only 14% of Cx. 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

20 Summary - 3 Pale Erect Forked Scales on the Occiput: 92% of Cx. 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 is 88%. (True Negative; 95% CI: 60%-98%)

21 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 occiput characters (pale vs black scales) is a good character. 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%). Both of these characters have a positive predictive value of 95% for Culex pipiens The occiput character does a little (~10%) better job at identifying Cx. restuans than the abdominal character does. Using the combination of characters will increase the likelihood of correctly distinguishing the two species.

22 Limitations These results are preliminary Need to increase PCR numbers Need to ID and PCR NC specimens (Forsyth County) 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)

23 Selected References Crabtree, M.B., Savage, H.M. and B.R. Miller Development of a Species-diagnostic Polymerase Chain Reaction Assay for the Identification of Culex Vectors of St. Louis Encephalitis Virus Based on Interspecies Sequence Variation in Ribosomal DNA Spacers. American Journal of Tropical Medicine and Hygiene. 53(1): Harrington, L.C., and R.L. Poulson 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 Host-Feeding Habits of Culex and Other Mosquitoes (Diptera: Culicidae) in the Borough of Queens in New York City, with Characters and Techniques for Identification of Culex Mosquitoes. Journal of Medical Entomology. 39(5): 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 Host Feeding Patterns of Established and Potential Vectors of West Nile Virus in the Eastern United States. Vector-Borne and Zoonotic Diseases. 4(1):

24 Acknowledgements Funding: WCU College of Health and Human Sciences WCU Honors College Virginia Commonwealth University

25 Questions?