New World screwworm rearing and germ-line transformation

The J06 wild type strain of C. hominivorax was collected in Jamaica in 2006 and is the parental strain that is reared routinely at the COPEG biosecurity plant. Adult females are stimulated to lay eggs by presenting them with warm containers of raw ground meat mixed with an attractant made from spent larval media [36]. Eggs are collected from adult females on the sixth day and are seeded in artificial larval diet (containing dry blood, dry egg, dry fishmeal protein, and cellulose fiber) [36] and kept at 39 °C and 80 % humidity for 3 days, adding more food daily, until they have reached the third instar stage. On the fourth day of development, the larvae are placed in a room at 31 °C and 80 % humidity for pupation into containers of sawdust. On the eighth day, the pupae are sieved out of the sawdust and placed into cages for emergence of adults in a colony room at 25.5 °C and 55 % humidity. Insects were reared in a 12 h/12 h light/dark cycle. The J06 strain was used for piggyBac-mediated germ-line transformation using a protocol similar to the one developed for L. cuprina [37]. Specifically, pre-blastoderm embryos were injected with a mix of pBac [FL] plasmid (800 ng/μL), Lchsp83-pBac helper plasmid (400 ng/μL), and pBac RNA helper (400 ng/μL). A DNA template was prepared and in vitro synthesis of piggyBac RNA helper was performed as described previously [11]. First instars showing transient expression of the ZsGreen or DsRedex2 marker were selected and raised on diet supplemented with 150 μg/mL tetracycline. G 0 adults were crossed to wild type flies and their offspring were screened for expression of the fluorescent marker as first instars. Homozygous C. hominivorax individuals were selected as crawl-off third instars based on fluorescence intensity and bred to create a stable line.

Female lethality tests

Homozygous lines were tested by setting up a cage with 100 flies with food and water without tetracycline. Females were induced to deposit eggs on the sixth day and eggs were collected and divided into two groups of the same weight. One group was seeded in larval diet containing 150 μg/mL tetracycline and the other group was seeded in diet without tetracycline. Larvae were reared to adults and the number of pupae, males, and females was counted. To measure dominant lethality, 10 homozygous transgenic males were crossed with 20 J06 virgin females in a cage with food and water without tetracycline. Larval offspring were raised as described above and the number of emerged adults counted. Three or more independent tests were performed for each line for both tests of female lethality. For testing the effect of tetracycline concentration on female viability, the strains were also tested on a larval diet that was identical to the standard diet except the dry fishmeal protein was replaced with soy flour [15].

Fitness tests

General fitness tests were performed for all the transgenic lines and for the J06 parental wild type strain, according to protocols used regularly in the COPEG biosecurity facility for quality control [38]. All tests were replicated at least three times unless otherwise indicated.

Survival from eggs to pupae

For each transgenic line and for the J06 strain, 75 mg of eggs were seeded in larval diet and raised until the pupal stage. The total volume of pupae was measured using a graduated cylinder and the total number of pupae in 25 mL was counted. From this, the total number of pupae and the average pupal weight was calculated. Regular testing has established that 75 mg of eggs is equal to 1875 eggs, and therefore the percentage of eggs that develop into pupae can be calculated.

Fertility

To measure the fertility of each line, eggs were collected and the egg mass dissociated into individual eggs by incubating them in a 4 % w/v sodium hydroxide solution for 2 min with constant stirring and then rinsing them with abundant distilled water [39]. For each line, 300 individual eggs were placed in a petri dish containing a damp paper towel and black filter paper on top. The petri dishes were incubated at 37 °C overnight and, the following morning, the number of hatched larvae were counted and the percentage egg hatch was calculated.

Fecundity

For each transgenic line and the J06 strain, a cage was set up with 50 males and 50 females with food and water containing tetracycline. On the sixth day after emergence, the females were induced to lay eggs and the total weight of the egg masses was measured. The average total number of eggs laid per female was calculated by dividing the total egg weight in mg by the number of females in the cage and then multiplying by 25 as 100 mg of eggs equals approximately 2500 eggs.

Adult emergence and sex ratio

For each transgenic line and a J06 control, 100 pupae were placed in a closed container and adults were allowed to emerge for 3 days after the emergence of the first insect. Males and females were counted and percentage of emergence and sex ratio calculated.

Male mating success assays

Transgenic and J06 wild type strains were reared under the same conditions of diet without tetracycline, temperature, and humidity. When adults emerged, males and females were immediately collected and placed in separate cages for 4 days to allow them to attain sexual maturity. On the fourth day, 15 virgin J06 females and five transgenic or five J06 males were placed in a rectangular metal cage (15 cm width, 15 cm height, and 25 cm length) for 8 h and the males were then removed from the cage. The following morning, the spermatecae of the females was dissected and observed with the light microscope to assess the presence of sperm as an indication of mating. The number of mated females was counted and the percentage calculated. This experiment was replicated at least three times for each line.

Male competitiveness assays

Transgenic and J06 wild type strains were reared under the same conditions of diet without tetracycline, temperature, and humidity. When adults emerged, males and females were immediately collected and placed in separate cages for 4 days to allow them to attain sexual maturity. On the fourth day, 10 transgenic males, 10 J06 males, and 10 J06 virgin females were placed in rectangular metal cages (60 cm height, 300 cm width, 60 cm length) for 20 h. The next day, the males were removed from the cage and the females were left until the sixth day. Females were then placed individually in 50 mL Falcon™ tubes containing a small piece of warm raw meat with attractant at the bottom to induce egg laying. The Falcon™ tubes were put in a 37 °C incubator for 2 h and then the egg masses were collected from each tube. The individual egg masses were placed in small petri dishes containing damp paper towel and a small volume of larval diet, labeled, and incubated at 37 °C until hatching. The first instars of each petri dish were observed with a stereo fluorescence microscope to assess paternity. Fluorescent larvae were consistent with mating of females to transgenic males while non-fluorescent larvae were the result of females mating to J06 males. No mixed populations of larvae were observed in this assay, as the limited time of 20 h given to the insects for mating is enough to prevent re-mating of the females to a second male. These tests were replicated at least three times.

To quantify male sexual competitiveness, the MCI was calculated using the formula from [40]:

$$ MCI=\frac{TW}{TW+WW} $$

Here, the mating of transgenic males to J06 wild type females (TW) and the mating of J06 wild type males to J06 wild type females (WW) was evaluated and values of MCI varied between 0 and 1, where 0 indicates that all the J06 wild type females mated to J06 wild type males, 1 indicates that they all mated to transgenic males, and 0.5 indicates that half mated with J06 wild type males and half mated with transgenic males, and that transgenic males are equally competitive to J06 wild type males.

Male longevity at lower temperatures

For the FL12-56 and FL11-15 transgenic lines and for the J06 control, two cages were set up with 50 males. In one cage, the flies were kept at 25 °C and in the other they were kept at 19 °C. The flies were given fresh food every 4 days. Every day the cages were checked for dead flies and these were counted until there were no live flies left in the cages. Two independent assays were performed at both temperatures.

Interspecific crossing to C. macellaria

Transgenic C. hominivorax lines and a wild type C. macellaria strain (collected in Panama) were reared under the same conditions of diet, temperature, and humidity. When adults emerged, males and females were immediately collected and placed in separate cages for 4 days, to allow them to attain sexual maturity. On the fourth day, five transgenic C. hominivorax males and 15 wild type C. macellaria virgin females were placed in rectangular metal cages (15 cm width, 15 cm height, and 25 cm length) for 3 days; separate control cages holding five wild type C. macellaria males with 15 C. macellaria females as well as five transgenic C. hominivorax males with 15 J06 C. hominivorax females were concurrently used. After this time, the spermatecae of the females was dissected and observed with a light microscope to assess the presence of sperm as an indication of successful mating. For each experiment, three replicates were set up for each line of transgenic males. Three independent experiments were performed.

Heterozygous female lethality in diverse genetic backgrounds

Crosses were performed using 10 males of the FL11-18B transgenic line and 30 virgin females of the different wild type strains collected in South America and the Caribbean: J03, J06, Brazil, Valledupar, and P-95. Males were removed from the cages on the fifth day and females were induced to lay eggs on the sixth day. The egg masses laid by each cage were weighed and half of the egg mass was seeded in diet containing tetracycline and the other half was seeded in diet without tetracycline. The larvae were fed each day with the corresponding diet and reared to adults. After emergence of all the adults, the number of males and females was counted. A single replicate experiment was performed for each strain.

Statistical analysis

To test the effect of tetracycline on sex, flies were aggregated for each line (between 3 and 8 bottles, for a total of approximately 500–1000 flies per line, per tetracycline treatment, see Additional files 1 and 2). Fisher’s exact test indicated a highly significant association (P values all less than 0.002) between sex and tetracycline, with female proportions equal to or very near 0 for each line. When data are aggregated over lines, the P value is < 0.0001. To test the effect of tetracycline level and female viability, a three-factor (strain, diet, tetracycline level) analysis of variance (ANOVA) was used to analyze the percentages of survivors that were female. The ANOVA table, along with an interaction plot of means, suggested that strain and tetracycline dosage level explain almost all of the variability in observed female survivorship. Separate tests of strain effects on survivorship were conducted at each level of tetracycline, both for fixed diet. For the general fitness parameters, six response variables of potential interest were measured on each of three replicate assays per line. Simple one-factor linear models were fit to each and all pairwise comparisons with the control (J06) were carried out using Dunnett’s procedure for control of experiment-wise error rate.

For both male mating success and male competition, the data were analyzed with generalized linear models for observed count variables, which were assumed to follow binomial distributions. These models were fit using the GLIMMIX procedure of the SAS statistical software package. For the mating success data, the overall hypothesis of equal probabilities among all 10 lines was tested. Additionally, all pairwise comparisons among the estimated probabilities for the 10 lines were carried out (with Tukey adjustment for control of experiment-wise error rate). For the competition data, the hypothesis of full competition was tested simply by a statistical comparison of the observed frequency with the hypothetical value of 50 %. Again, a generalized linear model was fit to the mating frequencies using PROC GLIMMIX. Cage/experiment effects were included in the model in order to enable investigation of over-dispersion. In the absence of over-dispersion, mating frequencies were aggregated over cages to conduct the test.

For the analysis of female viability in different genetic backgrounds, Fisher’s exact test was used to test the association between sex and strain in surviving adults from the crosses of FL11-18B with strains from different geographic regions, with between 100 and 600 flies sampled from each region. For analysis of the longevity data, Kaplan–Meier survival curves were estimated for each combination of temperature and strain after combining replicates, so that for each of the eight combinations of temperature and strain, 100 flies were at risk on day 0. At each temperature, Log-rank and Wilcoxon statistics were computed to tests for equality of survivor functions across the four strains. In the evidence of strain effects, subsequent pairwise comparisons were also made, again using log-rank and Wilcoxon tests, with Tukey adjustment for multiplicity of comparisons. Additionally, estimates of the mean and median survival time were computed for each strain and temperature. All of these computations were carried out using the LIFETEST procedure in SAS.