Complex Genome Evolution in A. coluzzii Associated with Increased Insecticide Usage in Mali

This talk was presented to the Center for Population Biology group at UC Davis on October 6, 2015. I highlight previous work on speciation involving the sister mosquito species A. gambiae and A. coluzzii, most of which was done by the Vector Genetics Lab (led by Greg Lanzaro) and Drosophila folks from Davis. I also share some more preliminary work on complex insecticide resistance that appears to be increasing rapidly in these mosquitoes.

Transcript

1. Complex  Genome  Evolu.on  in  A.  coluzzii   Associated  with  Increased  Insec.cide  Usage     in  Mali   Bradley  J.  Main   UC  Davis   Vector  Gene.cs  Laboratory  
2. Outline   •  Background:   – Cryp0c  species     – Gene0c  differences     – Adap0ve  Introgression   – Selec0on  on  standing  varia0on   •  Bioassay  data   •  Expression  data   •  Preliminary  SEM  data    
3. sister species (M) Anopheles coluzzii (S) Anopheles gambiae COETZEE, M., HUNT, R. H., WILKERSON, R. C., DELLA TORRE, A., COULIBALY, M. B. & BESANSKY, N. J. 2013. Anopheles coluzzii and Anopheles amharicus, new members of the Anopheles gambiae complex. Zootaxa, 3619, 246-274.
4. (M) Anopheles coluzzii (S) Anopheles gambiae -­‐  Savanna  Form   -­‐  Bamako  Form   sister species
5. A.  gambiae  -­‐  Savanna  Form:   temporary  puddles   A.  gambiae  -­‐  Bamako  Form:   riverine  rock  pools   A.  coluzzii:  permanent  rice   fields   Segregation by Larval Habitats
6. Hybrid  progeny  have  normal:       -­‐Egg  batch  size   -­‐Hatching  rate   -­‐Larval  development  success     -­‐sex  ra0o.       (Di  Deco  et  al.  1980,  Diabate  et  al.  2007,  Hahn  et  al.  2012)   No Intrinsic Incompatibilities have been detected
7. Assortative mating also occurs in mixed male swarms, so it is not simply due to spatial or temporal segregation. (Debire et al. 2013) Strong but imperfect Assortative Mating in Mali (Tripet  et  al.  2001)  
8. Species Distribution Across Mali (Tripet  et  al.  2007)  
9. Outline   •  Background:   – Cryp0c  species     – Gene.c  differences   – Adap0ve  Introgression   – Selec0on  on  standing  varia0on   •  Bioassay  data   •  Expression  data   •  Preliminary  SEM  data    
10. Whole Genome vs. Genic View of Speciation •  Variable  divergence  between  chromosomes.   – Genome-­‐wide  microsatellite  data     •  “Genomic  Islands  of  Specia.on”  –  X  and  2L   – Genome-­‐wide  microarray       (Wu  2001)   (Lanzaro  et  al.  1998)   (Turner  et  al.  2005)  
11. “Instrumental or Incidental to Speciation” 3L   (White et al. 2010) Whole Genome vs. Genic View of Speciation
12. islands  of  genomic  divergence  (IGD)   pericentric  heterochroma0n  (HET)   The Islands Are Contained Within Heterochromatin (Sharakova et al. 2010, Lawniczak  et  al.  2010)   - Low gene density, repeat rich, and low recombination.
13. (Cruickshank  and  Hahn  2014)   Islands of Relative Divergence Whole Genome vs. Genic View of Speciation
14. Contemporary Gene Flow -­‐  Insec.cide  resistance  related  in  Mali  and  Ghana.   -­‐  Loss  of  2L  island.   -­‐  Common  in  West  Africa  –  e.g.  Guinnea-­‐Bissau.   -­‐  Loss  of  2L  and  3L.         (Clarkson  et  al.  2014,  Norris  et  al.  2015)   (Pennisi  2014)   More:   (Marsden  et  al.  2011,  Nwakanma  et  al.  2013)   So,  the  whole  genome  view  is  wrong  or  this  is  a   breakdown  in  the  specia.on  process  ~through  secondary   contact.  
15. Outline   •  Background:   – Cryp0c  species     – Gene0c  differences   – Adap.ve  Introgression   – Selec0on  on  standing  varia0on   •  Bioassay  data   •  Expression  data   •  Preliminary  SEM  data    
16. 1991       2002     2004               1996   2010   2011   2012   2006   (Lee  et  al  2013)   15  fixed  SNPs  between  the  3  islands  
17. Prevalence of the insecticide resistance gene, kdr in four villages in Mali. 1993 - 2004 Tripet et al., (2007) Am. J. Trop. Med. Hyg. 76: 81-87 0.00 0.25 0.50 0.75 1.00 1993 2002 2004 M-form rr rs ss 0.00 0.25 0.50 0.75 1.00 1993 2002 2004 S-form BANAMBANI 0.00 0.25 0.50 0.75 1.00 1996 2002 M-form rr rs ss 0.00 0.25 0.50 0.75 1.00 1996 2002 S-form PIMPERENA Frequency Frequency Frequency Frequency ss ss 0.00 0.25 0.50 0.75 1.00 1993 2002 2004 M-form rr rs ss 0.00 0.25 0.50 0.75 1.00 1993 2002 2004 S-form SELENKENYI 0.00 0.25 0.50 0.75 1.00 1996 2002 M-form 0.00 0.25 0.50 0.75 1.00 1996 2002 S-form SOULOUBA Frequency Frequency Frequency Frequency 2004 2003 ss rr rs ss ss Anopheles coluzzii Anopheles gambiae Anopheles coluzzii Anopheles coluzzii Anopheles coluzzii Anopheles gambiae Anopheles gambiae Anopheles gambiae Banambani Selenkenyi Pimperena Soulouba
18. X 2L 3L post-2006 2006 pre-2006 Bam (7) A.gam (43) F1+n (99) A.col (22) Bam (12) A.gam (9) F1+n (22) F1 (10) A.col (36) Bam (43) A.gam (12) F1+n (7) A.col (136) 1073-213 1061-057 1039-358 4679-157 4691-107 4707-118 kdr 0313-052 0315-679 0317-546 post-2006 Bam (7) A.gam (43) F1+n (99) A.col (22) Bam (12) 01073-213 01061-057 01039-358 04679-157 04691-107 04707-118 kdr 10313-052 10315-679 10317-546 DIS genotypes A. coluzzii heterozygote A. gambiae kdr genotypes wt / wt kdr / wt kdr / kdr Introgression of the 2L island with kdr (Main  et  al.  2015)   kdr  >  Mixed  on  2L  
19. X 2L 3L post-2006 2006 pre-2006 Bam (7) A.gam (43) F1+n (99) A.col (22) Bam (12) A.gam (9) F1+n (22) F1 (10) A.col (36) Bam (43) A.gam (12) F1+n (7) A.col (136) 1073-213 1061-057 1039-358 4679-157 4691-107 4707-118 kdr 0313-052 0315-679 0317-546 post-2006 Bam (7) A.gam (43) F1+n (99) A.col (22) Bam (12) 01073-213 01061-057 01039-358 04679-157 04691-107 04707-118 kdr 10313-052 10315-679 10317-546 DIS genotypes A. coluzzii heterozygote A. gambiae kdr genotypes wt / wt kdr / wt kdr / kdr Introgression of the 2L island with kdr Main  et  al.  2015   Why didn’t this happen after 2002? kdr  >  Mixed  on  2L  
20. Insec.cide  Treated  Bed  Net  (ITN)  Usage   Altered  the  Fitness  Landscape  Post-­‐2006   0 0.2 0.4 0.6 0.8 1 Proportion A. coluzzii A. gambiae - Savanna form A. gambiae - Bamako form 1980 1985 1990 1995 2000 2005 2010 2015 Year 25 50 75 ITN %
21. Outline   •  Background:   – Cryp0c  species     – Gene0c  differences   – Adap0ve  Introgression   – Selec.on  on  standing  varia.on   •  Bioassay  data   •  Expression  data   •  Preliminary  SEM  data    
22. 2004   2006   3L   2L   X   2010   PURE  coluzzii   BACKCROSS  coluzzii   Did  addi.onal  beneficial  alleles  introgress   from  A.  gambaie  into  A.  coluzzii  aaer  2006?  
23. Two  Differen.ated  Regions   -40 -20 0 20 40 60 Tajima’s D u c j b -40 -20 0 20 40 60 Tajima’s D a -40 -20 0 20 40 60 Tajima’s D 0 5 10 15 20 0 0.1 0.2 0.3 0.4 0.5 FST CYP9K1 X 0 10 20 30 40 50 0 0.1 0.2 0.3 0.4 0.5 FST 2R 0 10 20 30 40 kdr 2L 0 10 20 30 40 50 position [Mb] 0 0.1 0.2 0.3 0.4 0.5 FST 3R 0 5 10 15 20 25 30 35 40 position [Mb] 3L (Post-­‐2006  D)  –  (Pre-­‐2006  D)   CPR125  
24. Selec.on  on  Standing  Varia.on     Intronic reg-1 reg-2 2002 2004 2010 2014 2002 2004 2010 2014 T A T 4% 23% 85% 99% 0% 0% 0% 9% A G A 46% 31% 4% <1% 0% 0% 0% 0% A G T 48% 39% 12% 0 100% 97% 100% 87% 2% 7% 0% <1% 0% 3% 0% 4% 26 35 26 158 13 18 21 21 Frequency of CYP9K1 haplotypes Anopheles coluzzii Anopheles gambiae Haplotype 3' UTR Intronic reg-1 reg-2 2002 2004 2010 2014 2002 cyp-l G T A T 4% 23% 85% 99% 0% cyp-ll A A G A 46% 31% 4% <1% 0% cyp-lll A A G T 48% 39% 12% 0 100% cyp-other 2% 7% 0% <1% 0% N 26 35 26 158 13 Frequency of CYP9K1 Anopheles coluzzii An Haplotype 3' UTR Intronic reg-1 reg-2 2002 2004 2010 2014 2002 cyp-l G T A T 4% 23% 85% 99% 0% cyp-ll A A G A 46% 31% 4% <1% 0% cyp-lll A A G T 48% 39% 12% 0 100% cyp-other 2% 7% 0% <1% 0% N 26 35 26 158 13 Frequency of CYP9K1 h Anopheles coluzzii Ano CYP9K1   CYP9K1  
25. Es.mate  Metabolic  Resistance  by   Inhibi.ng  P450’s  with  PBO   +  =  Acetone  Control     pbo  =  4%  PBO  Exposure  (PBO  inhibits  P450s)     P  =  Permethrin  Exposure     Mortality  24hours  Post  Treatment   Treatment  1  (1  hour)   +  =  Acetone  Control     Treatment  2  (1  hour)   SEL2014  M-­‐form   cyp-­‐1:kdr   Pimperena  S-­‐form   cyp-­‐3:kdr   PBO_P   PBO_+   +_P   +_+   PBO_P   PBO_+   +_P   +_+  
26. MOPTI(cyp-­‐2:wt)  x  SEL2014(cyp-­‐1:kdr)     F1’s  Allowed  to  Mate   F2  Genotype  Permethrin  Bioassay   N=120   N=120   cyp:        2        1        2        2   wt/wt   kdr/wt   kdr/kdr    1/2    2/2    1/2    2/2   1/2   wt/wt   kdr/wt   kdr/kdr        1   2/2   Propor0on  KD  @  60min  
27. Cis-­‐regulatory  Varia.on  at  CYP9K1   and  CPR125   r=.974  from  iPLEX  on  DNA  Dilu0on     Log2(cyp1/cyp2)   ASE  Es0mates  in  cyp-­‐1/2  F1  hybrids   CYP9K1   RNAseq   CYP9K1   iPLEX     (corrected;  N=18)   (N=4)   CPR125   RNAseq   (N=4)  
28. Cu0cle  thickness  
29. Future:  Cu0cle  Thickness  Es0mates      Normalized  by  Wing  Size  
30. Anton     Cornel   Catelyn   Neiman   Gregory  Lanzaro   Abou   Fofana   Cheick   Traore   Malaria  Research  &   Training  Center,  Mali   Laura   Norris   Travis   Collier   Yoosook   Lee   Amanda     Everiq       “Complex  and  evolving  mechanisms  of  insec.cide  resistance  in  malaria  vectors”   Youki     Yamasaki   T32  Training  Grant   R21   YOU?