Widespread Chemical Detoxification of Alkaloid Venom by Formicine Ants

1 Widespread chemical detoxification of alkaloid venom by formicine ants

3 Supplementary Materials

5 Supplemental Table S1: Venom gland volumes and venom solution concentrations used for all 6 acidopore grooming assays.

Concentration Volume of Average of defensive Defensive Defensive defensive defensive compound gland compound Test species compound Source Species gland N solution equivalen source size category solution volume2 applied (ul ts per gland applied (mm3) gland volume / dose4 (dose) (ul) ul carrier)3 Solenopsis Small 0.2 0.12 0.09 Venom 0.24±0.07 13 invicta1 Large 0.6 0.36 0.66 Monomorium Small 0.2 0.02 0.49 Venom 0.007±0.001 3 minimum Large 0.6 0.05 4.28 Pogonomyrme Small 0.2 0.12 0.09 Venom 0.25±0.06 5 x barbatus Large 0.6 0.36 0.66 Solenopsis Small 0.2 0.12 0.12 Venom 0.19±0.05 6 geminata1 Large 0.6 0.36 0.87 Crematogaster Dufours 0.44±0.18 3 Small 0.2 0.12 0.05 laeviuscula1 Linepithema Pydgidial 0.02±0.01 10 Small 0.2 0.12 0.97 humile Tetramorium Venom 0.02±0.003 6 Small 0.2 0.12 1.0 bicarinatum 7 1 Only large workers used for venom extraction. Mean±SD.

8 2 The volume of defensive compound in a defense gland was estimated by measuring the volume of the 9 turgid glands. Under 100x magnification three gland axes were measured and the formula for 10 calculating the volume of an ellipsoid used to calculate gland volume. The largest worker size class was 11 used for polymorphic species.

12 3 With the necessary exception of M. minimum, solutions were formulated to match the concentration 13 of S. invicta venom solution.

14 4The number of defensive glands (venom, Dufours, or pydgidial) that would contain the equivalent 15 amount of defensive compounds present in the solution. Defensive gland equivalency concentrations 16 were calculated as: G/D where G = the number of glands placed in solution and D = the number of doses 17 in total solution volume. D was calculated as: (carrier volume + total volume of venom glands added) / 18 dose volume.

20 Supplemental Table S2: Results of sealed acidopore assay. Ants had their acidopore sealed or were 21 sham treated with nail polish and then their heads immersed in venom or carrier solution. Survivorship 22 was monitored for 7 hours.

Median % Survival Species Treatment1 Surviving N P2 Time 7 Hours (hrs) Acidopore sealed - carrier 90 7+ 10 - Camponotus Sham sealed – Si venom 90 7+ 10 sansabeanus <0.005 Acidopore sealed - Si venom 21 3.5 14 Acidopore sealed - carrier 70 7+ 10 - Myrmecocystus Sham sealed – Si venom 80 7+ 10 placodops <0.02 Acidopore sealed - Si venom 30 3.5 10 23 1Acidopore sealed - Ants had the opening to the acidopore sealed with nail polish. Sham sealed - Ants 24 had nail polish applied to the top of their first gastral tergite. Si venom – Ants were treated with pure 25 Solenopsis invicta venom. Carrier – distilled water with 0.002% v/v Triton X-100™.

26 2 Results of Kaplan-Meier survival time analysis contrasting survivorship of acidopore sealed versus sham 27 sealed ants treated with S. invicta venom.

29 Supplemental Table S3: Comparison of the relative resistance of formicines and other ants to 30 displacement by Solenopsis invicta. Four studies have published tables of species occurrence in 31 matched invaded and uninvaded habitats during the course of an S. invicta invasion (Camilo and Phillips 32 1990; Porter and Savignano 1990; Morris and Steigman 1993; Jusino-Atresino et al. 1994). We used the 33 pitfall trap data published in these studies as a rough assessment of the relative resistance of formicine 34 and non-formicine species.

Total Total Resistant Resistant Study Species1 Formicines Species2 Formicines Porter and 12 1 2 1 Savignano 1990 Jusino-Atresino 13 2 8 2 and Phillips 1994 Morris and 6 2 0 0 Steigman 1993 Camilo and 14 2 5 1 Philips 19903 36 1 Total number of common species trapped: those for which at least 10 workers were caught. See 37 footnote 3 for an exception.

38 2 A species was scored as resistant to S. invicta invasion if its abundance in highly invaded habitat was at 39 least 50% of its abundance in uninvaded habitat. 3

40 3 This study examined multiple habitat types. For this study, we contrasted species that occurred in the 41 disturbed habitat lacking S. invicta (Assemblage Two) with those in the disturbed habitat containing high 42 densities of S. invicta (Assemblage Four). To isolate the impact of S. invicta from disturbance, we 43 ignored species present only in undisturbed habitat (Assemblage One) or in the lightly-invaded, 44 disturbed habitat (Assemblage Three). This study published only presence absence data, so all species 45 were included in count. Resistant species were those that were present in both habitats.

50 Supplemental Figure 1: Testing effectiveness of acidopore grooming in detoxifying Crematogaster 51 laeviuscula venoms. Symbols report mean and 95% confidence intervals for fraction of ants surviving 7 52 hours. Ants were treated with venom or carrier solution while anesthetized (thus unable to perform 53 acidopore grooming) or awake. Kaplan-Meier survival time analyses contrast 7-hour survivorship of 54 venom treated, awake ants with venom treated, anesthetized ants. Numbers in parentheses report 55 sample size. Asterisks report significance levels. All species treated with pure C. laeviuscula venom: 56 Nylanderia fulva, (χ2=0.8, P=0.38), Nylanderia vividula, (χ2=6.5, P<0.02); Paratrechina longicornis, (χ2=4.2, 57 P<0.04). 5

59 Supplemental Video 1: A sequence of edited video clips showing Camponotus rufipes performing 60 acidopore grooming while competing with Solenopsis saevissima for tuna baits. Video shot at Serra do 61 Japi, Brazil in April of 1995.

63 Supplemental Video 2: A sequence of edited video clips of all 8 formicine species performing acidopore 64 grooming after exposure to Solenopsis invicta venom.

66 Camilo GR, Phillips SA, Jr. (1990) Evolution of ant communities in response to invasion by the fire ant 67 Solenopsis invicta. In: Meer RKV, Jaffe K, Cedeno A (eds) Applied myrmecology: a world 68 perspective. Westview Press, Boulder, Colorado, USA., pp 190-198 69 Jusino-Atresino R, Phillips Jr SA, Williams D (1994) Impact of red imported fire ants on the ant fauna of 70 central Texas. In: Exotic ants Biology, impact, and control of introduced species Westview Press, 71 Boulder, Colorado, USA, pp 259-268 72 Morris JR, Steigman KL (1993) Effects of polygyne fire ant invasion on native ants of a blackland prairie in 73 Texas. Southwest Nat 38:136-140 74 Porter SD, Savignano DA (1990) Invasion of polygyne fire ants decimates native ants and disrupts 75 arthropod community. Ecology 71:2095-2106