香京julia种子在线播放

In the summer of 2018, five D. suzukii wild-type flies (2 females and 3 males) were collected from cherries from a cherry tree in a private garden in Tübingen, Germany. With these five flies, we established a D. suzukii stock, that we named “Tübingen 2018.” In 2019, we harvested several dozens of blackberries in a private garden in Tübingen; around 40 flies eclosed from these fruits and were used to set up a new line that was called “Brombeere 2019.” A third D. suzukii wild-type stock was established in 2018 starting from around 20 flies eclosed from several dozens of cherries from the city Bad Wimpfen, Germany, that is about 90 km to the north of Tübingen. The wild-type D. melanogaster stock was established from five female and five male flies collected in a wine orchard in Dijon in 2000 (“Dijon 2000”). All flies were raised in polysterol bottles containing standard corn meal food supplied with fresh baker’s yeast. A filter paper was stuck into D. suzukii culture bottles. The identical laboratory environment for both species ensures comparability of the data.

Ten male or female 5-days old flies were weighed on a micro-balance before drying for 2 h at 90°C. They were weighed for a second time after drying. The difference between the fresh and the dry weighs was used to evaluate the amount of water.

In each assay, groups of ten six to 10 days old flies were collected on ice and incubated in a petri dish containing silica (Sigma Aldrich) at 22°C. The petri dish was sealed with parafilm during the experiment. The experiment was repeated at least three times. Control flies survive for several hours in the petri dish without silica.

According to our recently published protocol (Wang et al., 2016), ten to twenty flies of each sex and species were incubated for 20 min with Eosin Y (0.5%, Sigma-Aldrich) at different temperatures. After staining, flies were washed with tap water at room temperature. Wings were cut off using micro-scissors, observed under a Leica EZ4 stereomicroscope and imaged using the in-built Leica camera and software.

Ten flies of each species were incubated in vials that contained either 0, 0.05 or 0.1 μg chlorpyriphos (stock solution 1 mg/ml acetone). Lethality was recorded during 4 h of incubation at the end of which all flies died when exposed at the highest insecticide amounts. This experiment was repeated at least three times.

To design qPCR primers, D. melanogaster, and D. suzukii transcript sequences were retrieved from flybase (flybase.org). Primers (Table 1) amplifying 100–120 bp were designed using the online primer3 software¹. Total RNA was extracted (RNEasy kit, Qiagen) from five freshly eclosed male or female flies (0 to 3 h old) to prepare the cDNA template (Omniscript RT kit, Qiagen) for the qPCR reaction (FastStart Essential DNA Green Master, Roche) on a Roche Nano LightCycler. This experiment was repeated four times. Data were analyzed with the inbuilt software of Roche and Microsoft Excel. The 2^–ΔΔCT method was applied to calculate the gene expression levels. The transcript levels of the reference gene RpS20 coding for ribosomal protein S20 were used to normalize gene expression.

To extract CHs, 6-h or 5-day old flies were frozen for 5 min at −20°C, prior to the extraction procedure. For wing analysis, wings were cut off using micro-scissors. Each pair of wings was immersed for 10 min at room temperature in vials containing 20 μl of hexane. For whole fly extraction, each individual was immersed under similar conditions in 30 μl of hexane. In all cases, the solution also contained 3.33 ng/μl of C26 (n-hexacosane) and 3.33 ng/μl of C30 (n-triacontane) as internal standards. After removing the wings or the fly bodies, the extracts were stored at −20°C until analysis. All extracts were analyzed using a Varian CP3380 gas chromatograph fitted with a flame ionization detector, a CP Sil 5CB column (25 m × 0.25 mm internal diameter; 0.1 m film thickness; Agilent), and a split–splitless injector (60 ml/min split-flow; valve opening 30 s after injection) with helium as carrier gas (velocity = 50 cm/s at 120°C). The temperature program began at 120°C, ramping at 10°C/min to 140°C, then ramping at 2 C/min to 290°C, and holding for 10 min. The chemical identity of the CHCs was checked using gas chromatography-mass spectrometry system equipped with a CP Sil 5CB column (Everaerts et al., 2010). The amount (ng/insect) of each component was calculated based on the readings obtained from the internal standards. For the sake of clarity we only show the sum (in ng) of desaturated CHs (ΣDesat), the sum of linear saturated CHs (ΣLin), and the sum of methyl-branched CHs (ΣBr) and their respective percentages calculated based on the overall CHs sum (ΣCHCs).

To learn about ecological similarities and differences between D. suzukii and D. melanogaster, we first compared their desiccation resistance (Figure 1). D. suzukii males were dead within 4 h after exposing them to dry conditions, while most D. melanogaster males survived until 5 h of exposition to dryness. They died between 6 and 8 h under this condition. D. suzukii females started to die after 5 h of exposure to dryness and after 10 h, their lethality reached 90%. The D. melanogaster females reached the 90% lethality rate after 15 h under the same condition. In conclusion, D. suzukii and D. melanogaster females live longer under dry conditions (<10% air humidity) than males. However, both D. suzukii males and females are more sensitive to dryness than D. melanogaster males and females.

Body mass and body water content are reported to be positively correlated to desiccation resistance (Ferveur et al., 2018). We determined these two parameters in D. suzukii and D. melanogaster males and females (Table 2). D. suzukii males are 58% heavier than D. melanogaster males, while D. suzukii females are 75% heavier than D. melanogaster females. These differences are statistically significant (see Table 2). Water represents 76% and 82% of the D. suzukii female and male body mass, respectively, while in D. melanogaster females and males it represents 80% and 87%, respectively. These differences have a low statistical significance (see Table 2). Thus, D. suzukii flies weight more than D. melanogaster flies, but, by trend, contain proportionally less water.

Our results suggest that the outward barrier is less efficient in D. suzukii flies than in D. melanogaster flies. Using an Eosin Y incubation assay (Wang et al., 2016, 2017), we tested whether the inward barrier function also differs between D. suzukii and D. melanogaster (Figure 2). In particular, we measured the permeability of the wing cuticle, given that this flat organ allows to produce an unambiguous scoring under light microscopy. The Eosin Y penetration temperature in wings lies between 37 and 40°C in both D. suzukii sexes, whereas its range is 55 to 60°C in both D. melanogaster male and female flies. Thus, the inward barrier of the D. suzukii wing cuticle is more permeable than the D. melanogaster wing cuticle.

To further evaluate the inward barrier efficiency, we also compared the sensitivity of D. suzukii and D. melanogaster to the contact insecticide Chlorpyrifos (Figure 3). In general, D. suzukii flies are more susceptible to Chlorpyrifos than D. melanogaster flies. We also observed that females of both species were more resistant against Chlorpyrifos than males. This finding is consistent with the assumption that the inward barrier is weaker in D. suzukii than in D. melanogaster.

The function of both the outward and inward barriers relies on the presence of surface CHCs. To test whether barrier differences between D. suzukii and D. melanogaster are reflected by the CHC composition, we performed GC/MS analysis of surface lipids of the whole body and of dissected wings (Figure 4). Our results are consistent with studies reporting CHC profiles in D. suzukii (Snellings et al., 2018) and D. melanogaster (Everaerts et al., 2010). On average, 1115 ng CHCs per male and 1404 ng CHCs per female were extracted from the body surface of D. suzukii. The body surface extracts of each D. melanogaster male contains 1506 ng CHCs on average, while each female D. melanogaster has 2095 ng CHCs on its body surface. The desaturation rates of D. suzukii male and female body CHCs are 63.1% and 59.1%, respectively. The desaturation rates of D. melanogaster male and female body CHCs are 59.3% and 71.1%, respectively. Around 6.5% of body CHCs on D. suzukii males and 10.4% on D. suzukii females are methylated. The methylation rates on D. melanogaster male and female body CHCs are 21.3% and 19.4%, respectively. We conclude that both the total amounts and the composition of body CHCs considerably differ between D. suzukii and D. melanogaster. In a nutshell, D. melanogater males and females have more branched, desaturated and total, but less linear CHCs than their D. suzukii counterparts. The CHC relative compositions of dissected D. suzukii and D. melanogaster wings are similar to the respective whole body CHC relative compositions.

To learn about the molecular mechanisms underlying CHC composition in D. suzukii and D. melanogaster, we monitored the expression levels of genes that code for enzymes involved in CHC formation (Figure 5). We used quantitative real-time PCR (qPCR), to record the expression levels of fas1, fas2, fas3, cyp4g1, farO, acc, desat1, and app transcripts in newly hatched flies. Compared to D. melanogaster females, the expression of genes encoding the first key enzymes of fatty acid synthesis FAS1 and ACC were low in D. suzukii females. However, these genes showed similar expression levels in D. melanogaster and D. suzukii males. Likewise, the expression of farO was similar in D. melanogaster and D. suzukii males and females. While fas3 expression was higher in D. suzukii males than in D. melanogaster males, it did not differ between D. melanogaster and D. suzukii females. The expression of the gene coding for the terminal CHC producing enzyme Cyp4g1 was lower in D. suzukii males as compared to D. melanogaster males whereas it did not differ between females of the two species. In both sexes, app transcript levels were higher in D. suzukii, while the expression levels of both fas2 and desat1 were around 100 times higher in D. suzukii than in D. melanogaster.

We also examined the expression levels of oskyddad (osy), snustorr (snu), and snustorr snarlik (snsl), all three coding for proteins required for the construction of the cuticle barrier (Figure 5). The expression of snsl did not differ significantly in both sexes of the two species. Levels of osy transcripts were higher in D. suzukii females and males than in D. melanogaster. However, snu expression was dramatically reduced in both D. suzukii females and males compared to same-sex D. melanogaster flies. We obtained identical results when we compared the expression levels of snu in both D. suzukii and D. melanogaster female and male dissected wing buds (Supplementary Figure S3).

Initially, snu was characterized in D. melanogaster as a key gene involved in barrier efficiency in a genetic approach in the laboratory (Zuber et al., 2018; Wang et al., 2020). Indeed, mutations in snu are lethal underlining its role in barrier function. Here, the quantitative variation of its expression correlating with cuticle barrier efficiency in D. suzukii and D. melanogaster suggests that it is also important in nature. To underpin this notion, we repeated the analysis of its expression changing two parameters. First, in order to rule out line-specific gene expression, we quantified snu transcript levels in two additional D. suzukii wild-type lines (see section “Materials and Methods”). Second, to exclude primer-specific effects, we designed a new set of D. suzukii snu-specific primers for these experiments (Table 1). The expression of snu was significantly reduced by more than 80% in D. suzukii compared to D. melanogaster in these experiments (Supplementary Figure S3).

Overall, the expression of some key genes coding for proteins and enzymes involved in lipid-based barrier construction and function show a substantial variation between D. suzukii and D. melanogaster. Especially, the expression divergence of snu is intriguing as its transcript levels have been shown to correlate with barrier efficiency in D. melanogaster (Wang et al., 2020).

Desiccation resistance is conveyed by a combination of the behavioral repertoire and physiological and physical properties of the organism. In case of incipient drought, an animal, for instance, may respond by taking up and storing more water and initiate fortification of its barrier against evaporation. These responses and their amplitude depend, of course, on the genetic constitution of the organism. Hypothetically, the micro-environment of an insect has a decisive impact on the quality of a responsive trait and the expression dynamics of the underlying genes. Under identical conditions, the response may vary at several levels. In dimorphic species, a difference in this regard may prevail between sexes. Response variation is probably more accentuated between species than within species.

In our inter-species comparative approach, we found that D. suzukii is more sensitive to low air humidity than D. melanogaster. This is in agreement with a work published by Terhzaz et al. (2018). Since all flies were reared under similar laboratory conditions, we can rule out possible experimental bias for this observation. Indeed, both species were shown to be responsive to environmental cues. D. suzukii appears in two morphs, i.e., the summer and the winter morph with distinct ecological traits. The winter morph, for instance, is more robust at low humidity conditions than the summer morph (Toxopeus et al., 2016; Fanning et al., 2019). D. melanogaster as a cosmopolitan species also shows geographical variation (Rajpurohit et al., 2017; Dong et al., 2019). We believe that there are two physiological explanations for the inter-species differences. The first one implies that the difference in water shortage tolerance between these two species relies on total body water content. Indeed, we found that the overall body water content in D. suzukii is lower than in D. melanogaster. However, the water-content hypothesis is not very plausible given that D. suzukii males which contain relatively more water than females are less robust than females at lower air humidity. The second physiological explanation underscores that the water loss rate through the three major routes respiration, excretion or cuticular transpiration may be higher or faster in D. suzukii than in D. melanogaster. For a number of different Drosophila species including D. melanogaster (D. suzukii was not included in that study), it was proposed that especially cuticular transpiration accounts for the highest water loss rate, whereas excretion (<6%), and respiration (<10%) are rather negligible in this regard (Gibbs et al., 2003).

According to the assumption of Gibbs et al. (2003), we think that a weaker outward barrier in D. suzukii may explain the observed difference in desiccation resistance between these species. Indeed, we found that D. suzukii flies have less CHCs on their surface than D. melanogaster flies suggesting that the CHC-based barrier is weaker in D. suzukii. However, Gibbs et al. (2003) reported that the quality of the cuticle barrier does not seem to depend on the CHC amounts in the Drosophila species studied. On the other hand, several recent works suggested that reduced CHC levels do cause enhanced desiccation (Qiu et al., 2012). Gibbs et al. (2003) also argued that the CHC melting temperature Tm that depends on CHC composition did not correlate with the water loss rate in their work. This is, however, in conflict with more recent findings. Higher proportions of desaturated CHCs confer increased desiccation resistance to D. melanogaster flies in experimentally selected lines (Ferveur et al., 2018). In another study, Chung et al. (2014) demonstrated that higher amounts of branched CHCs in D. serrata (29%) entail a higher desiccation resistance compared to D. birchii (3%). In analogy, our study reveals that D. suzukii has proportionally less branched CHCs (6% in males and 13% in females of the total amounts of CHCs) than D. melanogaster (23% in males and 19% in females of the total amounts of CHCs). We conclude that these traits may contribute to the difference in desiccation resistance observed between D. suzukii and D. melanogaster.

Remarkably, the CHC profiles in D. suzukii and D. melanogaster did not correlate with expression profiles of genes coding for lipid synthesis or modification enzymes. For instance, FASN2 that codes for a microsomal fatty acid synthase catalyzing the formation of methyl-branched fatty acids, is expressed at higher levels in D. suzukii than in D. melanogaster, while the relative amounts of branched CHCs is reduced in D. suzukii. Moreover, the expression of Desat1 that codes for a fatty acid desaturase responsible for fatty acid desaturation is enhanced in D. suzukii although the relative amounts of desaturated CHCs are lower in this species. Either the gene expression levels do not reflect the enzyme levels, or the catalytic activity of enzymes in D. suzukii is lower than in D. melanogaster. Alternatively, the limiting process of CHC amounts may not be their production but their externalization and deposition on the cuticle. Consistent with this interpretation, we found that the expression of snu coding for an ABC transporter involved in barrier construction and function (Zuber et al., 2018; Wang et al., 2020) is reduced in D. suzukii compared to D. melanogaster. Thus, it is possible that less CHCs accumulate on the cuticle of D. suzukii because of reduced Snu activity that should be tested in biochemical experiments. This interpretation is in line with our recent findings that in D. melanogaster flies with low snu expression in the wing have decreased CHC levels and compromised wing cuticle barrier function (Wang et al., 2020). Possibly, the expression divergence between D. melanogaster and D. suzukii relies on a species-specific regulatory network and involves more target genes than snu alone (Wittkopp, 2007). One such target gene may be osy, also coding for an ABC transporter needed for cuticle barrier efficiency and acting independently from Snu (Wang et al., 2020). The expression of osy is, in contrast to snu, slightly but significantly upregulated in D. suzukii compared to D. melanogaster. Obviously, this upregulation is not sufficient to equate the cuticle barrier efficiency in D. suzukii with the one in D. melanogaster. Admittedly, this is a compelling but simplified scenario as we do neglect an important point. The experiments presented in this work were done in a constant laboratory environment. Thus, we did not analyze the gene x environment (GxE) interaction in our desiccation assay. In other words, in nature, a complex, changing environment, gene expression variation may be different (Gibson, 2008; Hodgins-Davis and Townsend, 2009). For instance, the barrier of D. suzukii flies might be more robust under natural conditions than in the laboratory. Whether these differences may account for lifestyle differences between D. suzukii and D. melanogaster remains to be investigated.

To what extent can we apply our view on the situation of the outward barrier in D. suzukii and D. melanogaster to the situation of the inward barrier? Our experiments showed that the weaker outward barrier function of D. suzukii flies is paralleled by their weaker inward barrier function. Xenobiotics including both the inert dye Eosin Y and the insecticide Chlorpyrifos did penetrate the cuticle of D. suzukii more efficiently than in D. melanogaster. Thus, in a simple scenario, the CHC composition that defines the outward barrier function, would also be responsible for the inward barrier function in both species.