DOI: 10.5061/dryad.k6djh9wf4
title: Data from: Transinfection of Wolbachia wAlbB into Culex quinquefasciatus mosquitoes does not alter vector competence for Hawaiian avian malaria (Plasmodium relictum GRW4)

description: Avian malaria is expanding upslope with warmer temperatures and driving
 multiple species of Hawaiian birds towards extinction. Methods to reduce
 malaria transmission are urgently needed to prevent further declines.
 Releasing Wolbachia-infected incompatible male mosquitoes could suppress
 mosquito populations and releasing Wolbachia-infected female mosquitoes
 (or both sexes) could reduce pathogen transmission if the Wolbachia strain
 reduced vector competence. We cleared Culex quinquefasciatus of their
 natural Wolbachia pipientis wPip infection and transinfected them with
 Wolbachia wAlbB isolated from Aedes albopictus. We show that wAlbB
 infection was transmitted transovarially, and demonstrate cytoplasmic
 incompatibility with wild-type mosquitoes infected with wPip from Oahu and
 Maui, Hawaii. We measured vector competence for avian malaria, Plasmodium
 relictum, lineage GRW4, of seven mosquito lines (two with wAlbB; three
 with natural wPip infection, and two cleared of Wolbachia infection) by
 allowing them to feed on canaries infected with recently collected field
 isolates of Hawaiian P. relictum. We tested 73 groups (Ntotal = 1176) of
 mosquitoes for P. relictum infection in abdomens and thoraxes 6-14 days
 after feeding on a range of parasitemias from 0.028% to 2.49%, as well as
 a smaller subset of salivary glands. We found no measurable effect of
 Wolbachia on any endpoint, but strong effects of parasitemia, days post
 feeding, and mosquito strain on both abdomen and thorax infection
 prevalence. These results suggest that releasing male wAlbB-infected C.
 quinquefasciatus mosquitoes could suppress wPip-infected mosquito
 populations, but would have little positive or negative impact on mosquito
 vector competence for P. relictum if wAlbB became established in local
 mosquito populations. More broadly, the lack of Wolbachia effects on
 vector competence we observed highlights the variable impacts of both
 native and transinfected Wolbachia infections in mosquitoes.
description: This dataset comes from a series of experiments with mosquitoes,
 wolbachia, and avian malaria.
description: # Transinfection of Wolbachia wAlbB into Culex quinquefasciatus mosquitoes
 does not alter vector competence for Hawaiian avian malaria (Plasmodium
 relictum GRW4)
 [https://doi.org/10.5061/dryad.k6djh9wf4](https://doi.org/10.5061/dryad.k6djh9wf4) This dataset includes R code and data for: * Vector competence experiments * Salivary gland malaria DNA comparison * Mosquito Feeding success * Cytoplasmic incompatibility/egg viability * Mosquito Fecundity * Mosquito Survival * Avian malaria infection in canaries * Inheritance of Wolbachia ## Description of the data and file structure **In all .csv files empty cells indicate no data.** **Data files and column descriptions:** Note that when .csv files are loaded into R, blank spaces in column names are replaced with a ".", so "Male Strain" becomes "Male.Strain" ### **2023 Verily Bloodfed Mosquito Sample Tracker -** **Bloodfed Mosquito Tracker 12\_29\_23.csv** The column names and explanation are: \[1] "UNQ_ID" - ID for the sample \[2] "Bird" - ID of the bird that was fed on using leg color band combination \[3] "Bird.DPI" - Days post infection for the bird \[4] "Parasitemia_percent" - Malaria parasitemia in percent \[5] "Bird.CT.Score" - Malaria DNA amount in bird measured by qPCR, in CTs \[6] "Mosquito.ID" - ID for the mosquito \[7] "Date.of.blood.meal" - date mosquito fed on bird \[8] "Incubator.Temp" - Temperature of incubator \[9] "DPE.tested" - Days after feeding that the mosquito was killed, dissected and frozen \[10] "Date.collected" - Date that the mosquito was killed, dissected and frozen \[11] "Date.tested" - Not used; all blank \[12] "Sample.type" - Part of mosquito; we only analyzed THorax or ABdomen \[13] "Initials" - Initials of person who dissected mosquitoes; II - Isaiah Ipsaro; CG - Chris Garrison \[14] "Strain" - Mosquito line using Verily's original nomenclature; link b/w these and those used in paper: "Palmyra" = Palm-wPip; "Wild" - Field; "Palmtet" - Palm-None; "DQB3" - Palm-wAlbB; "DQB3-Oahu" - Oahu-wAlbB; "Oahu WB-" - Oahu-None; "Oahu WT" - Oahu-wPip;\ "Unknown" - not identified & not used \[15] "Cohort" -Group or batch of mosquitoes \[16] "Experiment.." - Experiment # \[17] "Spray" - Spray color \[18] "Cq.score" - qPCR CT score; NaN indicates sample never crossed threshold; sample is negative for Plasmodium DNA \[19] "Well" - qPCR plate well; mostly blank; not used \[20] "Notes" \[21] "GRW4..ve.Droplet.count" - ddPCR malaria droplet count; not used \[22] "Positive.Reference.counts" - ddPCR mosquito droplet count; not used \[23] "Ratio..Titer" - ratio of two previous columns; not used \[24] "Accepted.droplets.counts" - ddPCR droplet count; not used \[25] "Copies.per.20µL.well" - copies per 20 microliter well; not used ### **Innoculation sheet.csv** This file read in and merged with data from the file above (2023 Verily Bloodfed Mosquito Sample Tracker - Bloodfed Mosquito Tracker 12_29_23.csv) to examine whether the isolate influenced vector competence The column names and explanation are: \[1] "Bird" - ID of the bird that was fed on using leg color band combination \[2] "Isolate.ID" - name of the Plasmodium isolate; only two were used in the experiments; PUWA and KONACO \[3] "Innoc_vol_uL" - Amount of infected blood injected intramuscularly into canary \[4] "Innoc_paras_perc" - Parasitemia of infected blood injected intramuscularly into canary \[5] "Deglyc_Fresh" - Whether injected blood was from another bird (FRESH) or frozen and thawed (THAW) \[6] "Success" - Whether or not the canary that blood was injected into became infected \[7] "Ct" - qPCR score in CT values of blood that was injected into canary ### **colorfeedsucc.csv** \[1] "Bird" - ID of the bird that was fed on using leg color band combination \[2] "Parasitemia"- parasitemia of bird (not in percent; in fraction of cells infected) when fed on; not used \[3] "Strains_old" - Original names for mosquito lines \[4] "Generation" - generation of mosquitoes \[5] "Spray" - spray color \[6] "N_exp" - Number of mosquitoes that attempted to feed \[7] "N_fed" - Number that fed \[8] "FracFed" - Fraction that fed \[9] "Date" - Date of feeding (sometimes a date range) \[10] "Strains" - Line of mosquito as used in paper (e.g. Palm-wAlbB) \[11] "Order" - A column used for sorting this data frame; not used in analyses \[12] "Line" - The strain of mosquito (e.g. Palm) \[13] "WB" - The wolbachia type (e.g. wAlbB) ### **DQB3 maternal transmission ddPCR G7\_11.csv** \[1] "dd.plate...CSV" - The plate # for ddPCR \[2] "Generation" - The generation of mosquito \[3] "Sex" - Mosquito sex \[4] "Sample.ID" - Sample ID \[5] "wAlbB..ve.Droplet.count" - Droplet count from ddPCR for wAlbB Wolbachia \[6] "Positive.Reference.counts" - Droplet count from ddPCR for mosquito gene \[7] "Ratio" - Ratio of two previous columns \[8] "Accepted.droplets.counts" - Total droplet counts; not used \[9] "Copies.per.20µL.well" - Copies per well; not used ### **Salivary\_Gland\_Results.csv** \[1] "Order" - A column used for sorting this data frame; not used in analyses \[2] "Wolbachia" - The type of Wolbachia strain (note: DQB3 = wAlbB) \[3] "Sample" - Sample ID \[4] "GRW4.Positive.Droplet.count" - ddPCR Plasmodium DNA droplets \[5] "Positive..Housekeeping.Reference.counts" - ddPCR mosquito DNA droplets \[6] "GRW4_RPL5.Ratio" - Ratio of two previous columns \[7] "GRW4.Accepted.droplets.counts" - Total droplet counts; not used \[8] "GRW4.Copies.per.20uL.well"- Copies per well; not used ### **verily\_canary\_infections.csv** \[1] "Bird" - ID of the bird that was fed on using leg color band combination \[2] "Date" - date blood was taken \[3] "Weight" - weight of bird \[4] "DPI" - days post infection \[5] "Smear" - Smear made? Y/N \[6] "N_parasites" - # of Plasmodium parasites seen \[7] "Description" - Types of Plasmodium parasites seen \[8] "Field_viewed" - # of microscope fields viewed \[9] "Cells.per.field" - # of cells viewed per microscope field \[10] "Cells.Examined" - Total cells examined - the product of the two previous columns if cells.per.field has one value \[11] "Parasitemia.by.Slide" - Parasitemia - # of Plasmodium parasites seen divided by number of cells examined; this is value used for analyses \[12] "Blood.for.qPCR" - Was blood set aside for qPCR \[13] "qPCR.results" - qPCR results for Plasmodium DNA by qPCR if sample was tested (which wasn't always the case); not used in analyses ### **CI data 2.csv** \[1] "Row" - abbreviation used in Fig 1 x-axis \[2] "BRep" - Biological replicate \[3] "Male Strain" - Male mosquito strain \[4] "Female Strain" - Female mosquito strain \[5] "Females" - # of individual females laying eggs \[6] "Eggs" - # of eggs laid \[7] "Hatched" - # of eggs hatched \[8] "Unhatched" - # of eggs that didn't hatch \[9] "Fraction" - fraction of eggs that didn't hatch \[10] "Male Wolbachia" - Wolbachia inside male mosquito \[11] "Female Wolbachia" - Wolbachia inside female mosquito \[12] "UniBi" - Type of cross \[13] "Strain" - Mosquito genetic strain of the mosquito w/ wPip Wolbachia \[14] "Treatment" - Wolbachia cross ### **Fecundity.csv** \[1] "Isovial" - Vial # \[2] "Eggs" - Eggs laid \[3] "Hatched" - Eggs hatched \[4] "Male Strain" - Male mosquito strain \[5] "Female Strain" - Female mosquito strain \[6] "Male Wolbachia" - Wolbachia in male mosquito \[7] "Female Wolbachia" - Wolbachia in female mosquito \[8] "UniBi" -Cross type \[9] "Strain" - Mosquito genetic strain of the mosquito w/ wPip Wolbachia except in wAlbB-wAlbB crosses \[10] "Treatment" - Wolbachia cross ### **MosqSurvival.csv** \[1] "Row" - Row \[2] "Bird Tested + DPI" - Color band combination of bird and day post infection, in R this is converted to "Bird.Tested...DPI" \[3] "Parasitemia" - Parasitemia of the bird when mosquitoes fed \[4] "Incubator " - Incubator mosquitoes were kept in \[5] "Date" - Date incubators were checked \[6] "Time " - Time of inspection \[7] "Initials" - Person who checked mosquitoes \[8] "Fridge Temp (C)" - Temp in incubator \[9] "Humidity()" - Humidity in incubator \[10] "DPE Date" - Date mosquitoes fed on bird \[11] "Experiment number" - Experiment ID \[12] "Source" - Strain of mosquitoes (strain-wolbachia nomenclature is in old format: DQB3-Oahu (Oahu-wAlbB), Oahu WT (Oahu-wPip), Oahu WB- (Oahu-None), Palmyra (Palm-wPip), Palmtet (Palm-None), DQB3 (Palm-wAlbB) \[13] "DPE" - Days since mosquitoes fed (days post exposure) \[14] "Age" - Age of mosquitoes \[15] "wAlbB" - # of wAlbB mosquitoes alive \[16] "wPip" - # of wPip mosquitoes alive \[17] "None" - # of None mosquitoes alive \[18] "total Females" - Total females (all three groups) including dead mosquitoes \[19] "DwAlbB" - Dead mosquitoes in wAlbB group \[20] "DwPip" - Dead mosquitoes in wPip group \[21] "DNone" - Dead mosquitoes in None group \[22] "total F Dead" - Total dead mosquitoes (all three groups) \[23] "Total Adults" - Total live mosquitoes (all three groups) ### **Images files used for Figure 1** (Zenodo) MosqFemaleYE.png MosqMaleYE.png MosqFemaleGR.png MosqMaleGR.png CircleGR.png CircleYE.png UpBracket.png DownBracket.png FemSymb.png MaleSymb.png ## Code/Software There are three R code files, which we ran with R v.4.3.1 (2023-06-16 ucrt) and are stored in Zenodo: ### **Vector competence Final\_dryad.r** This code uses these three data files (above): * **2023 Verily Bloodfed Mosquito Sample Tracker - Bloodfed Mosquito Tracker 12_29_23.csv** * **Innoculation sheet.csv** * **Salivary_Gland_Results.csv** ### **FeedSucc\_MI\_CanParas\_dryad.r** This code uses these data files: * **colorfeedsucc.csv** * **DQB3 maternal transmission ddPCR G7_11.csv** * **verily_canary_infections.csv** * **Innoculation sheet.csv** ### **CI\_fecund\_survival\_dryad.r** This code uses these data files: * **CI data 2.csv** * **Fecundity.csv** * **MosqSurvival.csv**