Hybridisation in UK mosquitoes Dallimore, T.,¹ Strode, C.,¹ Beatty, G.,² Fernández-Martínez, L.T.,¹ Provan, J.² ¹ Department of Biology, Edge Hill University, England. ² IBERS, Aberystwyth University, Wales. Contact:
[email protected]
Introduction Hybridisation events among Culex mosquitoes has been recorded, but it is not known if these events occur in other groups of mosquitoes. A system of screening for potential hybridisation events are important as these events have been recorded to lead to; • Changes in feeding habits/behaviour • A bridge for zoonotic transmission • Extreme founder effects
A cost effective method of screening for hybridisation events using genetic markers A species specific SNP based approach has been developed from novel anonymous nuclear markers using the ISSR cloning methods outlined in Provan and Wilson (1). This method provides a fast and easy PCR method of screening populations for hybridisation events.
Methods
Nuclear internal transcribed spacer 2 (ITS2) has also been tested as an additional tool for verification.
Hybridisation potential and a targeted species approach. A candidate group of
mosquitoes including Culiseta morsitans, Cs. fumipennis and Cs. litorea, were selected based on current knowledge of; 1.Genitalia ‘lock and key’ effect (l & K) 2.Gamete compatibility (gam) 3.Sympatric occurrence (sym) 4.Vector potential (vec)
Mitochondrial COI primers were designed and tested to verify maternal species lineage.
Sample collection A dataset of 2500 mosquitoes were collected in 2014/16 from 75 locations across the UK, using sweepnets, larval dipping and baited traps. 289 specimens of Cs. morsitans, Cs. litorea or Cs. fumipennis have been identified ready for screening.
Table 1. Hybridisation potential for UK mosquito groups Candidate mosquito groups
l&k
gam
sym
vec
An. maculipennis
+++
-
++
+++
++
-
+++
+
Cx. pipiens s.l. /torrentium
++
++
+++
+++
Ae. cinereus/geminus
+++
-
-
+++
Oc. annulipes/cantans
+
-
+++
+
Oc. caspius/dorsalis
++
-
+
+++
Oc.punctor/punctodes*
+++
-
-
++
Oc. sticticus/nigrinus
++
-
+
+
++/+++
-
++/+++
+++
++/+
-
+
++
An. claviger/plumbeus/algeriensis
Cs.litora/morsitans/fumipennis Cs. alaskaensis/annulata/subochrea
*Oc.punctodes not yet recorded in the UK, but common across Europe. +++ = high, ++ = medium, + = low, - = no data available (2, 3, 4).
A
B
C
Map 1. Blue = collection sites, no candidate species. Orange = collection sites, candidates collected.
Results
Image 1. Selected candidates for development of genetic markers. ‘Lock and key’: a notable similarity in male genitalia can be suggestive of hybrid potential. A. Cs. fumipennis, B. Cs. litorea, C. Cs. morsitans .
Image 2. Scanning electron micrograph of Cs. morstitans head and mouthparts.
• 84 anonymous ISSR sequences have been isolated randomly from across the nuclear genome using Cs. morsitans, 10 of which have been selected to develop species specific SNP’s and screened across all 3 Cs. species. • Initial analyses of COI regions shows very little interspecific variation < 1%, and may not be suitable for parental lineage analysis. • Initial analysis of ITS2 suggests this gene is suitable for hybridisation analysis between Cs. species with 2 x 8 bp indels, and 12 > species specific SNP’s.
References 1. Provan, J. & Wilson, P.J. (2007) Molecular Ecology Notes, 7, 254–256. 2. Medlock, J.M. & Vaux, A.G.C. (2015) Parasites and Vectors, 8, 142. 3. Byrne, K. & Nichols, R, A. (1999) Heredity, 82, 7-15. 4. Ciota,A.T. et al. (2013) Parasites and Vectors, 6, 305.
