Source: NORTH CAROLINA STATE UNIV submitted to
DEVELOPMENT AND EVALUATION OF TRANSGENIC INSECT STRAINS FOR GENETIC CONTROL PROGRAMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1005012
Grant No.
(N/A)
Project No.
NC02550
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Nov 19, 2014
Project End Date
Sep 30, 2019
Grant Year
(N/A)
Project Director
Scott, MA, .
Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Entomology
Non Technical Summary
One the greatest accomplishments of the USDA in the 20th century was the eradication of the New World screwworm (NWS) fly from all of North and Central America. NWS females lay their eggs in open wounds or a natural orifice. The hatched larvae then feed on the animal's living tissue. Animals with severe screwworm infestations may die if untreated. NWS was eradicated using the sterile insect technique, which involved the mass rearing of NWS, sterilization by irradiation and distribution of sterile flies over the targeted area. Currently, sterile NWS are released in a " buffer zone" in southern Panama to prevent re-infestation from South America. Transgenic "male-only" NWS offer the potential of more efficient genetic suppression, reduced diet costs, higher plant capacity, improved monitoring and reduced risk should (in the very unlikely event) any NWS that escape the plant. Transgenic NWS lines have been obtained carrying a single-component tetracycline-repressible female-lethal system. Females die at the pupal stage. These strains will be further evaluated for characteristics that influence production such fertility and fecundity. Additional experiments that will provide data needed for a risk-benefit analysis by regulators (e.g. outcrossing potential, longevity). A two-component female-specific embryo lethal genetic system is also being developed at North Carolina State University. The most efficient gene system will be sent to Panama for evaluation in NWS. The most promising strains carrying the early lethal system will be selected for additional experiments to obtain data for a potential field release.Drosophila suzukii (commonly called spotted wing Drosophila or SWD) is a serious pest of berries and other soft fruits that has recently invaded the West and East coasts of the United States. Female SWD use their highly developed serrated ovipositor to pierce the skin of soft fruits and lay their eggs inside the fruit. Unlike the larger native fruit flies (e.g. blueberry maggot) that have only one generation a year, SWD has a short generation time and multiple generations per year. Thus it is potentially a much more serious pest. Any fruit that contain developing SWD larvae (maggots) can cause an entire shipment to be rejected. Growers are currently using broad spectrum insecticides to protect fruit from damage caused by SWD. We are developing transgenic SWD "male-only" strains that could be used for genetic suppression programs. Similar to the NWS strains, female SWD will die unless tetracycline is added to the diet. A male-only release is significantly more effective in suppressing a population than releasing both sexes.
Animal Health Component
70%
Research Effort Categories
Basic
20%
Applied
40%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Knowledge Area


Subject Of Investigation


Field Of Science

Goals / Objectives
MALE-ONLY NEW WORLD SCREWWORM PROJECTThe New World screwworm (NWS), Cochliomyia hominivorax is a devastating pest of livestock. Females lay their eggs in open wounds or a natural orifice and the hatched larvae then feed on the animal's living tissue. If untreated, the animal can die for a NWS infestation. The eradication of NWS from North and Central America using the sterile insect technique (SIT) has been widely recognized as one of the major accomplishments of the USDA in the 20th century. Several potential advantages of "male-only" NWS strains have been identified. These include more efficient population suppression, improved surveillance, savings in diet costs if females die early in development and potential elimination of the need for sterilization by radiation. We have made transgenic NWS lines that carry a single-component, tetracycline-repressible, female-lethal system. Ten of these lines show high female lethality at the pupal stage on diet that lacks tetracycline.GOALS: We have two main goals. Firstly, with existing transgenic strains, thoroughly evaluate the biological characteristics that influence production and field effectiveness. Secondly, develop and evaluate transgenic NWS strains that carry a female embryo lethal system. Objectives: Single component, pupal lethal.1. Production. Measure various fitness parameters that influence production. These include biological yield, egg hatch, and longevity. 2. Regulatory approval. A) Measure biological parameters of importance for a risk-benefit analysis. For example, examine the potential for outcrossing to secondary screwworm (C. macellaria) and longevity at a range of temperatures. B) Characterize, at a molecular level (e.g. number of transgene copies, flanking site sequences), one or two of the most promising lines. Stabilize lines selected for molecular analysis by excision of one piggyBac end, eliminating the potential for horizontal gene transfer and reducing the risk of a field release.3. Regulatory approval and strain effectiveness Examine the influence of genetic background on the level of female lethality by crossing transgenic males with females collected from different locations.4. Strain-effectiveness. Perform a proof-of-principle population reduction experiment in large field cage. This will involve establishing a stable population of NWS in the cage and then test if regular releases of males from the transgenic strain can cause eradication of the population.Objectives: Two component embryo female lethal1. Development. Evaluate which combination of "driver (DR)" and "effector (EF)" are most effective at inducing early female lethality in Lucilia cuprina at NCSU. L. cuprina is a close relative of NWS and is used a model species for testing genetic constructs, as we cannot work with NWS in North Carolina. 2. Development. Make transgenic NWS with the most promising "driver" and "effector" gene constructs. Perform lethality test by crossing DR and EF lines on diet that lacks tetracycline. If necessary, modify the gene constructs to achieve higher expression of the cell death genes in NWS.3. Development. Make "double homozygous" NWS lines that carry the most effective combinations of DR and EF. Confirm high female lethality is at the embryo stage and is tetracycline repressible.4. Production, regulatory approval and strain effectiveness. Do the same experiments as outlined for the single component system (objectives 1-4) MALE-ONLY SPOTTED WING DROSOPHILADrosophila suzukii (commonly called spotted wing Drosophila) is a serious pest of berries and other soft fruits that has recently invaded the West and East coasts of the United States. Female D. suzukii use their highly developed serrated ovipositor to pierce the skin of soft fruits and lay their eggs inside the fruit. Unlike the larger native fruit flies (e.g. blueberry maggot) that have only one generation a year, D. suzukii has a short generation time and multiple generations per year. Thus it is potentially a much more serious pest. Any fruit that contain developing D. suzukii larvae (maggots) can cause an entire shipment to be rejected. Growers are currently using broad spectrum insecticides to protect fruit from damage caused by D. suzukii. Consequently, growers in North Carolina are using more insecticide to manage this invasive fruit fly but the effectiveness of these treatments is weather dependent. It is also anticipated that D. suzukii will develop resistance to some of the more commonly used insecticides. Non-chemical means for controlling D. suzukii are needed. D. suzukii is a close relative of the model genetic organism D. melanogaster. Thus it is likely that many genetic tools that have been developed for D. melanogaster could be applied to D. suzukii.GOAL: To develop transgenic strains of male-only strains of D. suzukii for use in genetic control programs.Objective 1: To develop and evaluate a "male-only early lethal" genetic system in D. melanogaster.Objective 2: To make transgenic D. suzukii that carry the genetic construct developed under objective 1.A male-only transgenic SWD strain has many of the same advantages as described above for a male-only NWS strain (e.g. more effective population suppression)
Project Methods
Male-only New World Screwworm (NWS)Single component Pupal lethalThe ten transgenic lines that show 100% female lethality unless fed tetracycline are being currently evaluated in the biosecure facility in Panama.Production. Fitness parameters that will measured for the male-only lines include average pupal weight, ratio of males to emerged adults, average fertility, fecundity, larval productivity and longevity at 26 °C using well established protocols.Regulatory approval. We will measure biological parameters of importance for a risk-benefit analysis. These include:Adult longevity at cooler temperatures. 50 adults will be collected and incubated at 26 °C. Additional cages, each containing 50 adults, will be incubated at 16 and 22 °C. Three cages will be set for each temperature condition. Adult food and water will be provided as needed. Dead adults will be counted daily until all are dead and then mean life span will be calculated.Potential for Outcrossing. C. macellaria is found in the areas that contain NWS. Mating cages will contain equal numbers of NWS males and C. macellaria females. Control cages will contain equal numbers of C. macellaria males and females. We will measure the egg production, % egg hatch rate (if any), % of larvae that develop to adults (if any) and adult fertility. Mated females will be dissected and examined for the presence of sperm stored in spermathecae. Larvae (if any) from the test cross will be examined for GFP expression to confirm they are descended from the NWS transgenic male. Lastly, DNA will be extracted from the larvae or adults (if any) and analyzed to confirm hybridization.Development and assessment of "stabilized" lines. Embryos from the line will be injected with plasmid DNA that contains a Lchsp83-DsRed marker gene, piggyBac right end and phiC31 attB recombination site. The embryos will be co-injected with helper RNA that encodes phiC31 integrase. Lines will be selected based on co-expression of the DsRed and ZsGreen markers. Genomic DNA will be analyzed to confirm site-specific integration into the attP site. To stabilize the transgene, embryos from the recombinant line will be injected with helper DNA that encodes piggyBac transposase. G0 offspring will be screened for excision of the element by loss of the DsRed marker but retention of the ZsGreen marker. Molecular analyses will be used to confirm excision. The remaining transgene should be stabilized as only one piggyBac end will remain.Influence of different genetic backgrounds. A potential risk is that the efficacy of the female-killing system varies in different genetic backgrounds. To assess this potential background-effect by crossing transgenic males to females from wild-type strains that have been collected from various locations. The % female offspring will be measured.Strain-effectiveness. Perform a proof-of-principle population reduction experiment in large field cage. This will involve establishing a stable population of NWS in the cage and then test if regular releases of males from the transgenic strain can cause eradication of the population.Objectives: Two component embryo lethalDevelopment. The two-component system will use the hid cell death gene as the "effector". The hid gene will contain the sex-specific intron from the NWS tra gene. Only females will splice hid transcripts correctly and thus females will die. Expression of hid will be controlled with tTA. To ensure embryo lethality, a promoter from a gene expressed early in development will be used to drive tTA expression. This is the "driver". We have isolated the Lucilia sericata hid gene and shown it is very effective at inducing cell death in Drosophila embryos. We have also isolated the promoter for L. sericata bottleneck cellularization gene and shown that the promoter is very active in Lucilia embryos. The "driver" and "effector" constructs have been made and are being evaluated in L. cuprina. If successful, they will be used to make transgenic NWS in Panama. If not successful, we will isolate and characterize additional blowfly gene promoters that are strongly active in the embryo and have low activity in adult flies.tTA expression in the NWS "driver" lines will be examined by RT-PCR using RNA from early embryos (2-3h). The highest expressing tTA line(s) will then be crossed to lines that have the tetO-hid transgene marked with a different color fluorescent protein. The %female offspring on diet without tetracycline will be measured. The most effective "driver" and "effector" combinations will be crossed and recombinant strains made that are homozygous for both transgenes.Production, regulatory approval and strain effectiveness. The same experiments as outlined for the single component system will be performed with the lines homozygous for both "driver" and "effector" transgenes.Male-only Spotted Wing Drosophila (SWD)We have developed "male-only" strains of D. melanogaster that carry a gene construct we called FL3. FL3 contains the sex-specific intron from the NWS tra gene to make a single component tetracycline-repressible female lethal construct. The intron interrupts the tTA gene. If the NWS tra intron is correctly sex-specifically spliced only females would make tTA protein which initiates a positive feedback loop leading to more tTA production. Females die at the pupal stage as it takes some time for tTA to accumulate to lethal levels. To achieve lethality at earlier stages the FL3 system is being modified. Adjacent to the tTA binding sites (21 copies, tetO) we will insert another minimal core promoter upstream of a cell death gene. Binding of tTA to tetO in the bidirectional construct will lead to activation of both tTA and the cell death gene. We will use a highly active version of the hid cell death gene called hid[ala5]. The constructs will be made in a piggyBac vector that carries a constitutively expressed fluorescent protein gene to identify transgenic flies. DNA will be microinjected into early SWD embryos and transformants will be identified by fluorescent protein expression and bred to homozygosity by standard means. We will measure male and female viability raised with and without tetracycline added to the diet. The expectation is that females will be fully viable on diet supplemented with tetracycline but die on diet that lacks the antibiotic.

Progress 11/19/14 to 09/30/19

Outputs
Target Audience:The project director gave several invited presentations over the period of this report as listed below. The target audience were mostly scientists working in the genetics or entomology fields. The annual BRAG meeting also included biotechnology regulators as did the meeting in Uruguay on using gene drives for control of screwworm. Joint meeting of the Associazione Genetica Italiana (AGI) and Società Italiana di Mutagenesi Ambientale e Genomica (SIMAG), Cortona, Italy, 26-28 September 2019. Invited talk on "Development of genetic systems for control of insect pests" Institute of Molecular Life Sciences, University of Zürich, Switzerland, September 17, 2019. Invited talk on "Development of genetic systems for control of insect pests". Annual conference of the Australian Society for Parasitology, Adelaide, Australia, 8-11 July, 2019. Invited plenary talk on "Development of genetic systems for control of New World screwworm and sheep blowfly". Gene Drive Modeling Conference, Washington DC, June 11-12 2019. Invited talk on "Potential Applications of Gene Drives for Control of Agricultural Insect Pests". Project Director's meeting for the Biotechnology Risk Assessment Grants (BRAG) Program (June 6, 2019), Riverdale, MD. Poster presentation: "Development and evaluation of safeguards for conditional suppressive gene drives for spotted wing Drosophila and the New World screwworm ". Advances in biotechnology and their potential use in the eradication of the New World screwworm, Montevideo, Uruguay, 15-16 January, 2019. Two invited talks "Genetic control of screwworm using transgenic male-only strains" and "Screwworm Cas9 gene drive". Annual meeting of the Entomological Society of America, Vancouver, Canada, November 11-14, 2018. One talk: "Development of Gene Drive Systems for Control of Spotted Wing Drosophila and New World Screwworm" Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Talks were given at several conferences including a meeting with stakeholders on spotted wing Drosophila research. Dr Scott presented a poster at the annual BRAG meeting (NIFA) for project directors. Several biotech regulators visited the poster and asked a number of questions about insect biotechnology. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? New World screwworm Single component female lethal strains. The minimum concentration of tetracycline or doxycycline required for maintenance of the single component male-only strains FL12#26 and FL12#56 was determined. The doxycycline used was an inexpensive, locally sourced (ie Panama city, Panama) veterinary grade product. We found tetracycline could be used at the lower dose of 50 ug/mL. We previously used 150 ug/mL for maintenance. Doxycycline could be used at a lower concentration of 25 ug/mL. These results will lead to significant savings for mass rearing of the transgenic strains. As the strains have now been maintained for over 100 generations, they were being backcrossed to the parental wild type Jamaica 06 strain to improve overall fitness. Two component and all-in-one female embryo lethal strains. Several transgenic lines were made with the DR6 and DR7 tTA driver constructs. In the related sheep blowfly, DR6 and DR7 lines express higher levels of tTA in the embryo and appear to have little leaky expression at later stages than tTA lines made previously (e.g. DR2). The screwworm DR6 and DR7 driver lines were crossed with EF1 effector lines and are being bred to homozygosity for both transgenes. The expectation is that, if tetracycline or doxycycline is not added to the parental adult diet, tTA expressed in the developing embryo will activate expression of the hid cell death gene (EF1), leading to female lethality. The hid gene contains the transformer sex-specific intron and consequently only the female transcripts encode functional HID protein. Spotted wing Drosophila. Male only strains. We tested the ability of males from the FL19 male-only strain to suppress a wild type population in large bottles with standard diet. At the start of the experiment, each bottle contained 20 wild type females, 20 wild type males and 60 FL19 males. The FL19 males were obtained by rearing FL19 for a generation on diet without tetracycline. Control bottles contained 20 wild type males and females but not FL19. Within 5 generations we found a significant decrease in the population in test bottles but not control. The test bottles were driven to extinction within 10 generations. We are now repeating the experiment in cages with a larger starting population of 200 wild type males and females. Five two component embryo-lethal strains have been extensively tested. Three strains are lethal to both sexes whereas for the other two strains only female embryos die. Of these 5 strains, one bisex-lethal and one female-lethal showed promising fitness characteristics (e.g. egg laying) and were selected for further testing. When these strains were shifted from tetracycline diet to regular diet, within 3 days the bisex strain failed to produce any eggs that hatched into larvae. For the female lethal strain, after 3 days on standard diet only males developed from embryos. Control flies continued to produce viable offspring for 10 days after transfer from tetracycline to standard diet. These strains have been selected for a population suppression experiment as was done for the FL19 strain. CRISPR/Cas9. Lines that express Cas9 in spotted wing Drosophila were crossed with strains that express guide RNAs for the Sex lethal gene. Sex lethal is essential for female development but has no essential function in males. From the crosses with lines that express high levels of Cas9, all female offspring died at the embryo or early larval stages. With lines that express lower levels of Cas9, females developed but all were sterile. Upon dissection, the females showed poor ovary development. In D. melanogaster, Sex lethal is required for germ line development in females. Sex lethal appears to play a similar role in ovary development in spotted wing Drosophila.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Paulo, D.F., Williamson, M.E., Arp, A.P., Li, F., Sagel, A., Skoda, S.R., Sanchez-Gallego, J., Vasquez, M., Quintero, G., Perez de Leon, A.A., Belikoff, E.B., Azeredo-Espin, A.M.L., McMillan, W.O., Concha, C. and Scott, M.J. (2019) Specific Gene Disruption in the Major Livestock Pests Cochliomyia hominivorax and Lucilia cuprina Using CRISPR/Cas9. G3 (Bethesda) 9: 3045-3055. https://doi.org/10.1534/g3.119.400544


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The relevant presentations are listed below. An important target audience are regulators of biotechnology. A poster was given at the annual BRAG meeting in Riverdale, MD and the PD gave a talk on how gene drive systems could be used for control of agricultural insect pests at a meeting in Montreal in 2018, which included regulators and representatives of funding agencies interested in gene drive. The audience from the meeting in Portland included several stakeholders who have akeen interest in better methods for control of spotted wing Drosophila. the PD gave an update on progress with D. suzukii male-only strains. Third Research Co?ordination Meeting (RCM) on the FAO/IAEA Co-ordinated Research Project "Comparing Rearing Efficiency and Competitiveness of Sterile Male Strains Produced by Genetic, Transgenic or Symbiont-based Technologies" in Bangkok, Thailand from 18-22 June, 2018. Presentation: "The no blokes gene is essential for male viability and X chromosome dosage compensation in the Australian sheep blowfly". "3rd Meeting of the Gene Drive Research Sponsors and Supporters Forum", Montreal, Canada, September 11-13, 2018. Presentation "Development of gene drive systems for control of spotted wing Drosophila and New World screwworm". Project Director's meeting for the Biotechnology Risk Assessment Grants (BRAG) Program (May 22, 2018), Riverdale, MD. Poster presentation: "Development and evaluation of safeguards for conditional suppressive gene drives for spotted wing Drosophila and the New World screwworm ". "Sustainable SWD Management", Portland, Oregon, February 5-6, 2018. Presentation "Progress towards genetic control of spotted wing Drosophila". Annual meeting of the Entomological Society of America, Denver, CO, November 5-8, 2017. One talk: "Use of genome and transcriptome information for the development of male-only strains of the New World screwworm" Changes/Problems:New World screwworm male only strains. We observed an unexpected decresase in male fitness for several strains in 2018. this led to testing if modifications to the larval diet and tetracycline concentration could imrpove fitness. Spotted wing Drosophila, male-only strains. We did not expect it would be problematic to reliably distinguish flies with 1 or 2 copies of the fluorescent protein marker gene. Consequently for each combination of driver and effector transgene, several single pair crosses were set with flies we thought were homozygous for each transgene. If we were incorrect after 2 or 3 generations the descendents of the single pair cross would produce some flies that lack the marker gene. These sublines would be discarded. The plan is to maintain the single pair crosses for 5 generations and then combine to make a single strain homozygous for both transgene. What opportunities for training and professional development has the project provided?Biotechnology class on "Insect Genome manipulation", Spring semester 2018. Dr. Melina Florez-Cuadros (postdoctoral fellow) and Megan Williamson (PhD student). This was the 2nd time we ran this new lab-based course on using CRISPR/Cas9 and RNAi tools for knocking out or knocking down genes. The lab was significantly revised following feedback from last year. We made more extensive use of existing transgenic lines and limited embryo injection to one species. Dr. Florez-Cuadros gave a new set of short talks before the start of each lab. The PD advised before the talks and provided feedback. Outreach. Dr Florez-Cuadros developed a one-day program called "HOLA BUG: Motivating Hispanic high school students to pursue higher education using insect outreach". This was a mix of short talks and hands on experience with insects. Dr Florez-Cuadros visited high schools in North Carolina with a high % Hispanic population. The program was in English and Spanish. How have the results been disseminated to communities of interest?Talks were given at several conferences including a meeting with stakeholders on spotted wing Drosophila research. Dr Scott presented a poster at the annual BRAG meeting (NIFA) for project directors. Several biotech regulators visited the poster and asked a number of questions about insect biotechnology. What do you plan to do during the next reporting period to accomplish the goals?New World screwworm male only strains. Determine if male fitness is improved by using a lower dose of tetracycline and modifications to the diet. Develop and evaluate new lines with the DR6 and DR7 driver constructs. Spotted wing Drosophila, male-only strains. Complete breeding of double homozygous female embryo lethal strains. Evaluate male production on diet without tetracycline. Test if male-only strains can be used for population suppression in small cage trials.

Impacts
What was accomplished under these goals? New World screwworm, single component female lethal strains. Approval was obtained from Panamanian authorities for an open field test. The selected strain (FL12#56) was reared at high numbers, radiation sterilized (biological containment) and released at the field test site in Panama. Males were collected from over dozen traps, which provided information on dispersal and longevity. A report for the regulatory body was being prepared at the end of this reporting period. During the reporting period a drop in male fitness for many of the established transgenic lines was observed. Our preliminary investigations indicate this was due to use of a high dose of tetracycline and use of poor quality ingredients in the larval diet. At the end of the reporting period we were testing if changes to the diet and lower tetracycline combinations helped improve male fitness. New World screwworm, two component and all-in-one female embryo lethal strains. The most promising strains made with the DR2 driver were evaluated for male fitness. For example, mating competition assays with a 1 J06 female:1 J06 male:1 transgenic male. J06 is the strain currently used for mass rearing. The transgenic males show generally a poor ability to compete with J06 males. As for the single component strains, at the end of the reporting period we were testing if changes to the diet and lower tetracycline combinations helped improve male fitness. Injection experiments with the new DR6 and DR7 tTA driver constructs were planned for late 2018. We are hopeful that these driver will lead to improved strains as in a related blowfly, the DR6 and DR7 lines express higher levels of tTA in the embryo and appear to have little leaky expression at later stages (previous report). Spotted wing Drosophila. Male only strains. FL19 produces only males on diet without tetracycline and an approx. equal mix of males and females on diet with 20 ug/mL tetracycline. To mimic what would happen in a field release, FL19 was reared on diet without tetracycline and the resulting males collected. The males were then mated with wild type females on regular diet and diet without tetracycline. Only males were produced on diet without tetracycline. Hemizygous FL19 females died at the late larval stage. A different genetic system was used to make strains that carry female embryo lethal genes. Many different driver and effector lines were made and tested in combination. Some combinations were ineffective, some combinations were effective but could not be maintained on diet with tetracycline (20 ug/mL). At the end of the reporting period, 7 combinations appeared to be effective and were viable on diet with tetracycline. However, as it was difficult to reliably distinguish flies with 1 or 2 copies of the fluorescent protein marker gene, several lines were set with single pairs. These will be maintained for 5 generations at which point we will be confident that the lines are homozygous for each transgene. CRISPR/Cas9. Homozygous Cas9 lines were evaluated by crossing with a strain homozygous for transgene that expresses a white gRNA. The strongest lines used the vasa promoter to drive Cas9 expression. Mutagenesis rates up to 80% were found as seen by production of white-eyed males. Although less active the nanos-cas9 lines appeared to be much more specific for the germline. These Cas9 lines can be used for efficient site-directed mutagenesis of genes of interest.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: 1. Davis, R.J., Belikoff, E.B., Scholl, E.H., Li, F. and Scott, M.J. (2018) no blokes is essential for male viability and X chromosome gene expression in the Australian sheep blow fly. Current Biology, 28: 1987-1992
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: 2. Scott, M.J., Gould, F., Lorenzen, M.D., Grubbs, N., Edwards, O.R. and O'Brochta, D.A. (2018) Agricultural Production: Assessment of the Potential use of Cas9-mediated Gene Drive Systems for Agricultural Pest Control. J. Responsible Innovation, 5 (sup1): S98-S120
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: 3. Dearden, P.K., Gemmel, N.J., Mercier, O.R., Lester, P.J., Scott, M.J., Newcomb, R.D., Buckley, T.R., Jacobs, J.M.E., Goldson, S.G. and Penman, D.R. (2018) The potential for the use of gene drives for pest control in New Zealand: a perspective. J. Royal Society of New Zealand, 48: 225-244
  • Type: Other Status: Published Year Published: 2018 Citation: Scott, M.J., Linger, R.J. and Belikoff, E.J. (2018) Genetically engineered larvae for wound healing. US patent application number 15/923,595 filed on March 16, 2018,US provisional patent application No. 62/473,704. Filed 3/20/2017 in the United States.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The relevant presentations are listed below. An important target audience are regulators of biotechnology. Two talks were given at an international meeting and a poster was given at the annual BRAG meeting in Riverdale, MD. The latter includes regulators from USDA, EPA and FDA. The international meeting drew regulators from a number of countries. Another important target audience are scientists who work on transgenic insects for sterile release programs. The PD attended and presented at a specialist meeting in Panama. Given the outbreak of screwworm in Florida, the PD was happy to accept an invitation to speak to a NC statewide meeting for those who work with animals. The audience was mostly composed of veterinarians in private practice. Lastly, the PD happily accepted invitations to speak to colleagues in the Dept of Entomology, Uni. Georgia and at a meeting on transgenic animals. The talks included work on screwworm and spotted wing Drosophila. Each audience was composed of students, postdoctoral fellows, scientists and faculty. The transgenic animal meeting included a number of scientists from industry. Transgenic animal research conference XI, Tahoe City, CA., USA 13-17 August 2017. Invited presentation on "Genetic control of screwworm using male-only strains". Third international workshop for regulation of animal biotechnology, Charlottesville, VA, June 26-30, 2017. Two invited presentations were given "Insect biotechnology: current uses and future developments" and "Transgenic strains for genetic control of the New World screwworm in Panama". North Carolina academy of laboratory animal medicine annual meeting 2017, September 21, 2017. Invited presentation on "Genetic control of screwworm using male-only strains". Project Director's meeting for the Biotechnology Risk Assessment Grants (BRAG) Program (May 23, 2017), Riverdale, MD. Poster presentation: "Development and evaluation of safeguards for conditional suppressive gene drives for spotted wing Drosophila and the New World screwworm ". Second Research Co?ordination Meeting (RCM) on the FAO/IAEA Co-ordinated Research Project "Comparing Rearing Efficiency and Competitiveness of Sterile Male Strains Produced by Genetic, Transgenic or Symbiont-based Technologies" in Panama City, Panama from 27-31 March, 2017. Presentation: "Transgenic sexing strains: evaluation of the influence of genetic background in Drosophila and new Lucilia cuprina gene promoters for driving tTA expression". Department of Entomology, The University of Georgia, Athens, GA., February 27, 2017. Invited talk on "Genetically modified insect strains for genetic control of pests" Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Industry Immersion Program Jul 30-Aug 4 2017. Dr. Rebecca Davis (Postdoctoral fellow), Sophia Webster (PhD student), Katherine Knudsen (PhD student) A week long intensive program. Highly competitive selection process for grad students and post docs in final stage of training looking to transition to industry. Learned networking skills and how to make themselves marketable to industry recruiters. Learned how to do a 1 minute elevator pitch- had sessions where industry professionals critiqued participants' pitches, resumes, and LinkedIn pages, and gave feedback. LIstened to sponsoring companies talk about their company culture, structural organization, and goals. Learned about different facets of biotech/pharma industry, for example regulatory, HR, R&D, Patent law/ intellectual property. Were provided structured networking sessions to make connections with local industry professionals. Had the chance to ask local industry professionals questions during panels about HR, entrepreneurship, non-bench careers in industry and other relevant topics. Students also participated in a week long team project in which they were required to define an unmet need in the industry and come up with a product to meet that need. Teams performed market analysis (cost, potential customer population etc), assessed regulatory hurdles, and pitched the product at a formal presentation at the end of the week. Biotechnology class on "insect Genome manipulation", Spring semester 2017. Dr. William Reid (postdoctoral fellow), Katherine Knudsen (PhD student). New lab-based course on using CRISPR/Cas9 and RNAi tools for knocking out or knocking down genes. Dr. Ried and Ms Kundsen assisted in running the class, including giving short talks before teh start of lab. The PD advised before the talks and provided feedback. Qiagen and GSL co-sponsored CLC Workbench Workshop Aug 30-31 2017. Dr. Rebecca Linger (postdoctoral fellow) Training was on usingCLCBiomedical Workbench (biomedical version ofCLCGenomics): variant calling, epigenetics (bisulfite sequencing), RNAseq analysis, and an advancedworkshopfor pluggingCLCGenomics data into Ingenuity Pathway Analysis (IPA). Training also covered using CLCGenomics Workbench:de novoassembly (Illumina & Pacbio), genome finishing, microbial genomics (microbiome, metagenomics), and RNAseq analysis. How have the results been disseminated to communities of interest?New World screwworm. Talks were given at several conferences including an international meeting for regulators of animal biotechnology, specialist meetings on transgenics anaimals and another on transgenic insects for SIT and a local meeting in North Carolina for people who work with animals, which was mostly veterinarians. T The PD also gave a poster on gene drive research at the annual BRAG meeting (NIFA) for project directors. This was mostly on spotted wing Drosophila. What do you plan to do during the next reporting period to accomplish the goals?New World screwworm, single component female lethal strains. Perform an open field test with sterlized males if regulatory approval is obtained. New World screwworm, female embryo lethal strains. Complete evaluation of new tTA drivers and evaluate male behavior of the best strains. Spotted wing Drosophila. Male only strains. Peform additional crosses to identify the best tTA driver and tetO-effector strains. Breed to homozygosity strains with both components. Begin evaluation of strain fitness. Spotted wing Drosophila, CRISPR/Cas9. Breed Cas9 lines to homozygosity. Perform crosses to measure activity (with the white gRNA strain) in triplicate to obtain publishable data.

Impacts
What was accomplished under these goals? New World screwworm, single component female lethal strains. Nothing to report from this period. The selected strain (FL12#56) was being reared at high numbers in anticipation of approval for open field testing. New World screwworm, two component and all-in-one female embryo lethal strains. Extensive lethality testing on diet with and without tetracycline was perfromed on strains with the DR2 driver lines. Several strains are 100% lethal when homozygous and some dominant lethal. The DR2 all-in-one strain TD1 was made homozygous again after extensive backcrossing to various wild type strains. The new TD1 strain has drammatically improved fitness characteristics (e.g. egg production). The best two component and the new all-in-one strains were selected for male behavior testing and evaluation under mass rearing conditions (20L tray). In Raleigh, evaluation of new DR6 and DR7 tTA driver lines was largely completed. These new driver lines express higher levels of tTA in the embryo and appear to have little leaky expression at later stages. They would appear to be better driver lines than DR2 and so constructs were shipped to Panama. Spotted wing Drosophila. Male only strains. We had made many transgenic lines with tTA driver and tetO-cell death effectors. Initial testing failed to identify driver/effector combinations that were lethal. However, late in the reporting period we found that a new tTA driver line was very promising as it gave 100% female lethality when crossed with a tetO-cell death gene effector construct. With this tTA line we can perfrom additional crosses and determine the best effector lines. Similarly with the effector line that did give 100% lethality we can perform additional crosses and identify the best driver lines. Spotted wing Drosophila, CRISPR/Cas9. Initial testing of transgenic lines that express Cas9 in the germline (Vasa or nanos promoter) identified several lines that were active. The lines were tested by crossing to a transgenic line that expresses guideRNA for the white gene (U6 promoter). Cas9-mediated mutagenesis of the white gene would lead to males with white eyes (wild type is red, white is X-linked). The Cas9 lines will be bred to homozygosity and tested in triplicate to provide data for a future publication.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Yan, Y., Linger, R.J. and Scott, M.J. (2017) Transgenic early-larval sexing systems for genetic control of the Australian sheep blow fly Lucilia cuprina. Scientific Reports, 7, 2538. doi:10.1038/s41598-017-02763-4.


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Talks International Conferences ICE 2016, XXV International Congress of Entomology, Orlando, FL, USA, September 25-30 on Development and evaluation of male-only strains of the New World screwworm National Conferences 2016 Annual Meeting ofWERA1021, Orlando, FL, September 29, 2016 on Male-only strains, Cas9 strains and gene drive in SWD "Roadmaps to Gene Drives", Raleigh, NC, February 24-26, 2016 on Assessment of the Potential use of Cas9-mediated Gene Drive Systems for Agricultural Pest Control" Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?For the BMC Biology paper, a press release was issued by NCSU as this paper describes the first sexing system developed for New World screwworm. The story was highlighted on several web sites. For example, https://entomologytoday.org/2016/09/06/genetically-modified-screwworm-flies-may-enhance-sterile-insect-technique/ In addition see talks listed under "target audience". What do you plan to do during the next reporting period to accomplish the goals?New World screwworm Perform population suppression experiments in large cages in containment with the best single component male-only strains. Complete evaluation of female lethality on diet without tetracycline for the two component strains. Select the best strains and evaluate for several characteristics that influence strain production (e.g. biological yield). Also perfrom similar fitness experiments with the TD1 strain. For L. cuprina, make double homozygous strains with the new DR6 and DR7 driver constructs and evaluate female lethality. Spotted wing Drosophila Cross driver and effector lines and evaluate female lethality on diet that lacks tetracycline. Make transgenic straisn that express Cas9 in the germline and evaluate. These strains will be subsequently used to make "docking sites" for site-specific recombination systems.

Impacts
What was accomplished under these goals? MALE-ONLY NEW WORLD SCREWWORM PROJECT Accomplishments: Single component, pupal lethal. 1. Production. Evaluation completed and the results published in BMC Biology. 2. Regulatory approval. After several meetings with my collaborators, regulators gave a preliminary indication that our approval for a small field test with radiation sterilized males from one of the transgenic strains would be approved. Formal approval had not been obtained by the end of the reporting period. 3. Strain effectiveness Conditions were determined for maintaining a stable population of screwworm in large cages within the biosecure production plant. Accomplishments: Two component embryo female lethal 1. Development. Evaluation of double homozygous L. cuprina strains carrying DR2 driver and EF1 or EF3 effector was completed. The results were published in Scientific Reports. We showed that this genetic system could be used to make a transgenic embryonic sexing system. However, the DR2 showed sufficient activity in adult females that it was necessary to feed females a low dose of tetarcycline for the first two days after emergene in order to have nornal levels of egg production. While not ideal, the system could be adapted for C. hominivorax. 2. Development. Since the DR2 driver was not ideal, we searched and found additional genes that showed high levels of expression in the embryo but low in adult. Promoters from these genes, Lcnullo and LcCG14427, were isolated and used to make the new tTA driver constructs DR6 and DR7. Transgenic lines carrying the DR6 and DR7 drivers were established. 3. Development. Double homozygous C. hominivorax strains were made with several DR2 and EF1 lines. As in L. cuprina, females require some tetracycline in their diet to produce eggs. The most effective strains produce only females on diet that lacks tetracycline. Lethality appears to occur at the embryo/first instar stages. At the end of the reporting period the most promising strain was DR2#17/EF1#16. 4. Development. A single C. hominivorax line was obtained with an "all-in-one" construct that combines both DR2 tTA driver and EF1 tetO-hid effector in a single construct. This line, TD1, shows 100% female lethality but had poor biological yield. At the end of the reporting period the TD1 strain was being backcrossed to the wild type strain to try to improve productivity. MALE-ONLY SPOTTED WING DROSOPHILA Development. One line, FL20 was made that carries a dominant tetracycline-repressible female lethal gene construct. With the FL19 line developed previously, we now have two lines with a late-acting female lethal gene. The lines are fully viable on diet with tetracycline. Development. With the aim of making a strain that carried a two-component tetracycline-repressible female embryo lethal system, 23 transgenic tTA driver lines were established for 4 different gene constructs that used D. suzukii gene promoters such as sry-alpha. Further, 23 transgenic effector lines were made for 8 different gene constructs that use the cell death genes reaper, grim and hid. The transgenic lines were being bred to homozygosity at the end of the reporting period. Development. Although a genome sequence is available for D. suzukii, there are a paucity of genetic tools for genome manipulation. To address this need, we evaluated guide RNA and Cas9 expression ssytems that were developed for D. melanogaster. We found that these sytems functioned effiecently in D. suzukii and we were able to use them to induce mutations in the white and sex lethal genes. The results were published in Biochemical and Biophysical Research Communications in 2016. We plan to use the CRISPR/Cas9 technology to create transgenic strains with "docking sites" for site-specific recombination. This will greatly simplify the development of transgenic strains.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Schwartz, S., Truglio, M., Scott, M.J., and Fitzsimons, H.L. (2016) Long-term Memory in Drosophila Is Influenced by the Histone Deacetylase HDAC4 Interacting with the SUMO-Conjugating Enzyme Ubc9. Genetics. 203: 1249-1264. doi: 10.1534/genetics.115.183194
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Yan, Y. and Scott, M. J. (2015) A transgenic embryonic sexing system for the Australian sheep blow fly Lucilia cuprina. Sci. Rep.5, 16090; doi: 10.1038/srep16090
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Linger, R.J., Belikoff, E.J. and Scott, M.J. (2015) Dosage Compensation of X-Linked Muller Element F Genes but Not X-Linked Transgenes in the Australian Sheep Blowfly. PLoS ONE 10(10): e0141544. doi:10.1371/journal.pone.0141544
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Anstead, C. A., Batterham, P., Korhonen, P. K., Young, N. D., Hall, R. S., Bowles, V. M., Richards, S., Scott, M. J. and Gasser, R. B. (2016) A blow to the fly - Lucilia cuprina draft genome and transcriptome to support advances in biology and biotechnology. Biotechnol Adv. 34: 605-620. doi: 10.1016/j.biotechadv.2016.02.009. PubMed PMID: 26944522.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Concha, C., Palavesam, A., Guerrero, F.D., Sagel, A., Li, F., Hernandez, Y., Pardo, T., Quintero, G., Vasquez, M., Phillips, P.L., McMillan, W.O., Skoda, S.R. and Scott, M.J. (2016) A transgenic male-only strain of the New World screwworm for an improved control program using the sterile insect technique. BMC Biology, 14: 72. DOI: 10.1186/s12915-016-0296-8
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Linger, R. J., Belikoff, E. J., Yan, Y., Li, F., Wantuch, H. A., Fitzsimons, H. L. and Scott, M. J. (2016) Towards next generation maggot debridement therapy: transgenic Lucilia sericata larvae that produce and secrete a human growth factor. BMC Biotechnol. 16:30. doi: 10.1186/s12896-016-0263-z. PubMed PMID: 27006073.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Li, F. and Scott, M. J. (2016) CRISPR/Cas9-mediated mutagenesis of the white and Sex lethal loci in the invasive pest, Drosophila suzukii. Biochem Biophys Res Commun. 469 (4): 911-916. doi: 10.1016/j.bbrc.2015.12.081. PubMed PMID: 26721433.


Progress 11/19/14 to 09/30/15

Outputs
Target Audience:On May 6, 2015, The PD gave a presentation entitled "Prospectus of Genetically Modified Male only Screwworm Strain" at the COPEG Stakeholders Meeting, Panama City, Republic of Panama. Stakeholders in attendance included: -Directors of the Commission for the Eradication and Prevention of screwworm (COPEG), which is charged with running the New World screwworm (NWS) eradication and prevention campaign in Panama. - representatives of growers in the USA, Mexico and Central America that supported the NWS eradication campaign. -Administrators and scientists at USDA-APHIS and USDA-ARS that have supported or have an interest in using genetic modification to improve genetic control of insect pests. -scientists from the International Atomic Energy Agency (IAEA) that supports area-wide sterile insect technique programs in many countries. The PD attended at a gave a presentation at the First Research Coordination Meeting (RCM) on the FAO/IAEA Co-ordinated Research Project "Comparing Rearing Efficiency and Competitiveness of Sterile Male Strains Produced by Genetic, Transgenic or Symbiont-based Technologies" held in Vienna, Austria from 6 to 10 July, 2015. The presentation was on "Transgenic sexing systems for genetic control of the New World screwworm and the Australian sheep blowfly". Stakeholders in attendance included: -scientists and academics that are actively pursuing genetic approaches for control of insect pests. -scientists from the IAEA The PD gave an invited plenary presentation to the annual American Association of Veterinary Parasitologists (AAVP) meeting held in Boston, MA, July 11-14, 2015. Title of talk: " Genetic control of ectoparasite dipteran livestock pests".? Stakeholders present included: -scientists and academics that have an interest in control of ecoparasites of animals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The PD gave a presentation at the COPEG Stakeholders Meeting, Panama City, Republic of Panama May 6, 2015, on " Prospectus of Genetically Modified Male only Screwworm Strain", with our collaborator Dr. Steven Skoda, USDA-ARS. What do you plan to do during the next reporting period to accomplish the goals?New World screwworm. 1) Develop methodology for evaluating strains for population suppression assays in large cages in containment. 2) Cross DR2 and EF1 homozygous lines and asses female lethality on diet without tetracycline. Select the most promising lines and cross and breed to make double homozygous strains on diet that contains tetracycline. Spotted wing Drosophila 1) Make new transgenic lines and breed to homozygosity 2) Evaluate by crossing DR and EF lines or simply testing sinle component lines on diet without tetracycline.

Impacts
What was accomplished under these goals? MALE-ONLY NEW WORLD SCREWWORM PROJECT Accomplishments: Single component, pupal lethal. 1. Production. Eight homozygous transgenic lines produce only males when raised in diet that lacks tetracycline and this effect is dominant. Indeed, the female lethal construct shows a high degree of penetrance across all eight transgenic lines, which produce very low numbers of females when heterozygous individuals are raised in diet lacking tetracycline. Further, two transgenic lines, FL11-2B and FL11-15, produce no heterozygous females and thus would be suitable for a fertile-release program. In these assays, flies were counted in numbers that ranged in the thousands and with six or seven replicates for each transgenic line. In all of the 8 transgenic lines, we observed very little evidence for reduced productivity relative to the control strain (J06) when raised in diet containing tetracycline. J06 is the current mass production strain. Hatching, survival and pupal weight were very similar to each other and the control strain. The only characteristic in which there was significant variation between the lines was egg production. The transgenic lines FL11-2B, FL11-15, FL11-18, FL12-30, FL12-31 and FL12-56 were all very similar to J06 in the number of eggs produced, while the other lines laid a significantly lower number of eggs. Importantly, both sexes survived to adulthood when raised in diet containing tetracycline and the sex ratios of the emerged adults did not differ significantly from those of the parental J06 strain, indicating that the addition of tetracycline to the diet is permissive for rearing both sexes. 2. Regulatory approval. For any planned transgenic release, the potential for outcrossing to closely related species in the field is a concern. Within Panama, Cochliomyia macellaria is a closely related species of Calliphorid. This species is broadly sympatric with C. hominivorax across Central and South America. Although the two species have not been reported to hybridize in the wild and previous studies showed that they mate very rarely in caged settings, producing no hybrid offspring, we studied the potential for transgenic C. hominivorax males to mate with wild type C. macellaria females in the laboratory. In six independent assays with the FL12-56 line, we found no evidence of cross mating. Control experiments, using wild type C. macellaria males resulted in over 80% of mated females. Likewise, FL12-56 males mated successfully with wild type C. hominivorax J06 females under the same experimental conditions. As the New World screwworm is a tropical insect, it is not expected to be able to disperse to or survive in colder or dryer climates in South America, particularly during winter. To evaluate the potential for our transgenic strains to thrive at lower temperatures and perhaps colonize new areas of North and South America, we studied the longevity of males from the FL12-56 and FL11-15 lines and the J06 parental strain at 25°C and 19°C in the laboratory (Fig. 5B). Results from two independent assays showed that although males from both J06 and the two transgenic lines live longer at the lower temperature, there was no statistically significant difference between the two transgenic lines and J06 at 19°C (log-rank and Wilcoxon tests on Kaplan-Meier survival curves). 3. Regulatory approval and strain effectiveness Males from the transgenic lines FL11-18B and FL11-2 were crossed with virgin females from several wild type strains. Small differences in female viability on diet lacking tetracycline were observed suggesting genetic background has only a minor influence on strain effectiveness. Accomplishments: Two component embryo female lethal 1. Development. Driver lines with tTA driven by the promoters from the L. cuprina genes, bnk (DR2), spitting image (DR3) and act5C (DR5) were crossed to EF1 and EF3 effector lines. EF1 and EF3 contain the tetO/hsp70 enhancer/promoter that is activated by tTA. EF3 contains the wild type sequence of the L. cuprina hid cell death gene interrupted by the sex-specific intron from the C. hominivorax tra gene. EF1 is similar but contains a mutant version of hid (his[ala2]) that cannot be repressed by MAPK and thus should be more active. All combinations produced 100% dominant female lethality on diet lacking tetracycline. However, only some EF3 lines showed 100% lethality. Further, only with the DR2 driver did females die at the embryo stage, as shown by RT-PCR analysis of embryo RNA using primers for the tra gene. Thus the most promising constructs for testing in screwworm are DR2 and EF1. 2. Development. Several transgenic C. hominovorax lines were made that carry the DR2 and EF1 gene constructs. These lines were being bred to homozygosity at the end of the reporting period. MALE-ONLY SPOTTED WING DROSOPHILA Objective 1. As transformation of D. suzukii was efficient we decided to evaluate gene constructs directly in D. suzukii. Objective 2. One line, FL19 was made that carries a dominant tetracycline-repressible female lethal gene construct. The line is fully viable on diet with tetracycline. Although no females are produced on diet lacking tetracycline, male productivity is significantly less than wild type. Consequently transgenic lines are being made with several other gene constructs, both single and two component systems.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Edman, R.M., Linger, R.J., Belikoff, E.J., Li, F., Sze, S.-H., Tarone, A.M. and Scott, M.J. (2015) Functional characterization of calliphorid cell death genes and cellularization gene promoters for controlling gene expression and cell viability in early embryos. Insect Molecular Biology, 24: 58-70. doi: 10.1111/imb.12135
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Scott, M.J., Morrison, N.I., Simmons, G.S. (2014) Transgenic approaches for sterile insect control of dipteran livestock pests and lepidopteran crop pests. In: Transgenic Insects: Techniques and Applications (Benedict, M.Q., ed.) pp 152-167, CABI, Wallingford, UK.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Scott, M. J. (2014) Development and evaluation of male-only strains of the Australian sheep blowfly, Lucilia cuprina. BMC Genetics, 15(Suppl 2):S3. doi:10.1186/1471-2156-15-S2-S3
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Sandeman, R. M., Levot, G., Heath, A. G., James, P. J., Greeff, J. C., Scott, M. J., Batterham, P. and Bowles, V. M. (2014) Control of the Sheep Blowfly - are we there yet? Int. J. Parasitol., 44:879-891 pii: S0020-7519(14)00231-8. doi: 10.1016/j.ijpara.2014.08.009