The Kruskal–Wallis analysis of variance, a non-parametric equivalent of ANOVA, was used to compare groups across species. When significant, this test was followed by pairwise Mann–Whitney U-tests, and the p-values were corrected for multiple comparisons using the Bonferroni technique. In this and subsequent analyses, the Bonferroni adjustment multiplies the p-values we obtained by the number of comparisons performed . For the morphological comparisons, we tested the following models: number of bristles ¼ f and ovipositor area ¼ f. The Kruskal –Wallis analysis of variance was used to compare groups across strains. When significant, this test was followed by pairwise Mann–Whitney U-tests, and the p-values were corrected for multiple comparisons using the Bonferroni technique.We sought to determine the susceptibility of four types of fruit to each of the four species of flies. They included a fruit with relatively soft skin and fruits with much tougher skin . Our aim was to discover whether females of each species were capable of penetrating the intact skin of ripening fruit. We were aware, however, that the failure of females of a given species to lay eggs in a fruit could be a sign of either their inability to puncture the skin or a general aversion to fruit of that variety.
To distinguish between these possibilities, we counted separately the number of eggs in the exposed region of the fruit, blueberry plant pot which included the pit and any regions where the skin had naturally broken, and the undamaged fruit body. The absence of eggs in both the easily accessible exposed region and the unexposed area would indicate an aversion to the fruit, while the presence of eggs in the exposed area, and their absence wherever the skin was intact, would be consistent with the hypothesis that the skin was acting as a barrier. We found that all four species of flies laid eggs in the exposed portion of the four fruit varieties , indicating that none of the species had a strong aversion to using these fruit as larval food sources. The eggs that were laid in this region were found in a number of positions. The egg was sometimes embedded in the fruit with the respiratory filaments pointing outwards, but it could also be found in the opposite orientation or lying sideways . In the raspberry assay, a significantly larger number of eggs were laid in the exposed region by D. mimetica and D. biarmipes females when compared with D. suzukii and D. subpulchrella . This may be a consequence of the fact that the latter two species appear to have had no difficulty puncturing intact raspberry skin , and therefore did not gravitate to the far smaller exposed area in the pit.
These results contrast with the data from both varieties of grapes, where all four species laid eggs in the exposed portion, but D. suzukii laid over three times as many as the other three species. In the case of cherries, both In the raspberry and cherry assays, only D. suzukii and D. subpulchrella laid eggs in areas of the fruit where the skin was intact . There was no significant difference between these two species in the number of eggs laid in either fruit. We also found cases of punctures that lacked egg filaments , a possible indication that an egg-laying attempt was unsuccessful. These were relatively rare in both the raspberry and cherry assays , suggesting that neither species had much difficulty laying their eggs in these fruits. The finding that D. subpulchrella flies are capable of puncturing the skin of cherries and raspberries raises the question of whether this species, like D. suzukii, could be a threat to soft fruit industries.We therefore endeavoured to determine whether D. subpulchrella larvae could survive and develop in the pulp of these fruits. For the cherry assays, we observed an average of 19.9 living larvae in each fruit 5 days after the assays were set up . For the raspberry assays, we observed the bottles 14 days after they were set up and found that an average of seven adult flies had emerged from pupae. This indicates that larvae can survive in both of these fruits and, at least in raspberries, can develop to adulthood. No punctures of any form were found in the intact regions of either raspberries or cherries exposed to D. mimetica and D. biarmipes. In conjunction with the results of §3a, the findings are consistent with the hypothesis that these species rarely or never puncture intact skin even in soft-skinned fruits and that the propensity to do so evolved in the ancestor of D. suzukii and D. subpulchrella .
The results of the two grape assays differed strongly from the other fruits. Only D. suzukii laid eggs through the intact skin of any grapes, but these were relatively rare in the Thompson grapes, and entirely absent from the red grapes. However, grapes exposed to D. suzukii showed numerous punctures without the telltale sign of egg filaments . The egg-free punctures were often found in clusters.We determined, in a separate experiment , that punctures without protruding egg filaments were considerably smaller than those with filaments . These findings are consistent with the hypothesis that D. suzukii females tried to lay their eggs in the grape bodies and often succeeded in making small holes in the skin, but in most cases failed to insert an egg. However, from a pest-management perspective, it is important to note that punctures without filaments can be sites of secondary infections. By contrast, the intact body regions of grapes exposed to flies of other species never showed punctures. However, in a few cases, we found evidence of ‘slashes’ in the bodies of grapes in bottles with either D. subpulchrella or D. suzukii . The slashes, which were not found in control fruits that were not exposed to these flies, may represent unsuccessful attempts to puncture the skin. Our results demonstrate that while all species have an affinity for grapes and will lay their eggs in exposed regions of the fruit, they rarely penetrate the intact skin of the two varieties we tested. The evidence of egg-laying attempts, plastic gardening pots especially in D. suzukii, suggests that a property of the skin may make the insertion of eggs difficult. The D. suzukii findings are consistent with the results of previous research.The fruit susceptibility experiments suggest that the ability to puncture fruit skin first evolved in, or at least was strongly enhanced in, the ancestor of D. suzukii and D. subpulchrella . We tested whether this capacity was accompanied by concomitant phenotypic changes in the structure of the ovipositor. We separated, mounted and imaged ovipositor plates from two strains of each of the four species and compared them on a number of traits. One categorical distinction between ovipositors in the D. suzukii and D. subpulchrella clade, on the one hand, and D. mimetica and D. biarmipes, on the other, is the presence of thick, pigmented bristles on the former, explaining why this type of ovipositor has been referred to as ‘serrated’ . These modified bristles are found close to the distal tip of the ovipositor , which comes into contact with fruit, while the more proximal bristles are unmodified and resemble the homologous bristles present on the ovipositors of other species, referred to in the literature as ‘thorn bristles’.
The evolution of modified bristles in this clade was accompanied by a significant increase in the overall number of thorn bristles . Interestingly, the three to four marginal bristles of the D. subpulchrella ovipositor tip are generally of the thick, pigmented variety, whereas those in the equivalent region of the D. suzukii plate are almost always unmodified . However, on the lateral side of the ovipositor, D. suzukii has more modified bristles than D. subpulchrella, and the total number of modified bristles does not differ significantly between the two species . The evolution of the serrated ovipositor was accompanied by a considerable increase in the ovipositor size , with the two species with the serrated variety having ovipositor plates with approximately three to four times the area of the two other species’ plates. By contrast, comparing the same species on wing area, often used as a proxy for body size, shows an increase of only 1.6-fold to twofold in D. suzukii and D. subpulchrella . Therefore, while there has been an overall increase in body size in D. suzukii andD. subpulchrella, there has been a disproportionate increase in ovipositor area. In order to have a quantitative method of distinguishing ‘sharp’ ovipositors from ‘blunt’ ones, we measured the length towidth ratio .While this ratio does not vary significantly among D. mimetica, D. biarmipes and D. subpulchrella , it increased markedly in D. suzukii, giving the egg-laying organ a pointed, streamlined shape . We used EFA to compare the ovipositor shape among the four species, focusing on the distal half of the structure , because this is the portion of the egg-laying organ that comes into contact with the fruit during an attempted puncture. EFA produced a series of four Fourier coefficients for each of the 25 harmonics used in the analysis. Using the PCA, we calculated PCs for each of the ovipositors . The vast majority of the variation was captured by the first two PCs, which are plotted in figure 5a. Three groupings are apparent in this figure, each indicated with an ellipse: the D. mimetica and D. biarmipes strains; the D. subpulchrella strains; and the D. suzukii strains. To help to visualize what the individual PCs represent, we used the inverse Fourier transform to reconstruct ovipositor outlines on the basis of each PC . The outlines suggest that the first PC represents the difference between a sharp and blunt ovipositor. Indeed, we found that there is a strong negative Pearson product correlation between the first PC and length to width ratio. Therefore, the horizontal axis of figure 5a shows that D. biarmipes and D. mimetica, the two species that did not puncture the intact skin of any of the fruits tested, both have relatively blunt ovipositors and do not differ significantly on this variable . Drosophila subpulchrella has a sharper distal ovipositor, whereas the ovipositor of D. suzukii is the sharpest of all. A positive value for the second PC is indicative of an ovipositor with a bulb at the tip, as is seen in D. subpulchrella . Not surprisingly, there is a strong, significant difference between the D. subpulchrella strains and those of the other three species on this PC , while the latter do not vary significantly among each other on this variable . The third PC , which only explains about 4% of the variation , appears reflective of the asymmetry in the direction in which the tip is pointed. The remaining PCs each accounted for less than 1% of the variation, and the use of an omnibus Kruskal–Wallis test found no evidence of significant differences between strains. We present a model for the evolution of a modified ovipositor in figure 6 . The appearance of enlarged bristles was accompanied by a change in the shape of the ovipositor tip, which became significantly sharper. Following this event, in one species, D. subpulchrella, a distal bulb appeared, while in the second species, D. suzukii, the ovipositor became even sharper and increasingly streamlined. The latter is associated with the ability of D. suzukii to puncture the skin of the grape varieties we assayed, which was not shared by the other species in this study.While there were numerous Japanese studies of D. suzukii infestations during the first half of the twentieth century , these reports were not widely disseminated among Western researchers. When this species was first collected in California in 2008, it was not readily identified and was initially mistaken for D. biarmipes. At the time, the only known drosophilid pest in the Western Hemisphere was the distantly related Zaprionus indianus, which had first been reported in North America in 2006, 7 years after its identification in Brazil . Although Z. indianus is a formidable threat to agriculture , it can only colonize exposed fruit. Other distantly related species, however, such asthe leaf-mining fly Scaptomyza flava, possess serrated ovipositors. In this species, the ovipositor is used to puncture a leaf, and the female then feeds on its contents.