key: cord-0748073-7k94y114
authors: Reis, Micael; Siomava, Natalia; Wimmer, Ernst A.; Posnien, Nico
title: Sexual dimorphism and plasticity in wing shape in three Diptera
date: 2021-02-19
journal: bioRxiv
DOI: 10.1101/135749
sha: 6e058775e66fb51fa033b705f61f0a3a3bc2eb9c
doc_id: 748073
cord_uid: 7k94y114

The ability of powered flight in insects facilitated their great evolutionary success allowing them to occupy various ecological niches. Beyond this primary task, wings are often involved in various premating behaviors, such as the generation of courtship songs and the initiation of mating in flight. These specific functions imply special adaptations of wing morphology, as well as sex-specific wing morphologies. Although wing morphology has been extensively studied in Drosophila melanogaster, a comprehensive understa nding of sexual wing shape dimorphisms and developmental plasticity is missing for other Diptera. Therefore, we raised flies of the three Diptera species Drosophila melanogaster, Ceratitis capitata and Musca domestica at different environmental conditions and applied geometric morphometrics to analyze wing shape. Our data showed extensive interspecific differences in wing shape, as well as a clear sexual wing shape dimorphism in all three species. We revealed an impact of different rearing temperatures wing shape in all three species, which was mostly explained by plasticity in wing size in D. melanogaster. Rearing densities had significant effects on allometric wing shape in D. melanogaster, while no obvious effects were observed for the other two species. Additionally, we do not find evidence for sex-specific response to different rearing conditions in all three species. We determined species-specific and common trends in shape alterations, and we hypothesize developmental and functional implications of our data. Contribution to the Field Statement The size and shape of organisms and organs must be tightly controlled during development to ensure proper functionality. However, morphological traits vary considerably in nature contributing to phenotypic diversity. Such variation can be the result of evolutionary adaptations as well as plasticity for example as reaction to changing environmental conditions during development. It is therefore a major aim in Biology to unravel the processes that control differences in adult morphology. Insect wings are excellent models to study how organ size and shape evolves because they facilitate basic tasks such as mating and feeding. Accordingly, a tremendous variety of wings sizes and shapes evolved in nature. Additionally, plasticity in wing morphology in response to different rearing conditions has been observed in many insects contributing to phenotypic diversity. In this work we applied Geometric Morphometrics to study wing shape in the three Diptera species: the Mediterranean fruit fly Ceratitis capitata, the Vinegar fly Drosophila melanogaster and the housefly Musca domestica. Flies were raised in different temperature and density regimes that allowed us to study the effects of these environmental factors on wing shape. Additionally, in accordance with different mating behaviors of these flies, we observed a clear sexual shape dimorphism in all three species. Since the three studied species represent serious pests and disease vectors, our findings may contribute to existing and future monitoring efforts.

and Liria, 2017). However, it remains largely unknown whether wing shape sex-specifically responds to different 93 environmental cues and how such effects may be linked to wing size variation.

We have previously shown that the three dipteran species Drosophila melanogaster, Ceratitis capitata, and Musca 95 domestica exhibit a clear sexual wing size dimorphism and that the response of wing size to different rearing 96 conditions is sex dependent (Siomava et al., 2016) . Therefore, these three species represent excellent models to 97 test whether wing shape changes in a similar sex dependent manner. In this study, we applied geometric 98 morphometrics to compare and comprehensively describe variation in wing morphology between C. capitata, D. 99 melanogaster and M. domestica. We found clear evidence for sexual shape dimorphism in all three species with 100 major contribution of wing size on shape differences in D. melanogaster. Different rearing temperatures had a 101 strong effect on total and non-allometric wing shape in all three species, while density effects were most 102 pronounced in D. melanogaster. Eventually, we did not find strong arguments for sexual dimorphism in response 103 to the different rearing conditions, although M. domestica males showed slightly stronger differences than 104 females. We identified highly variable regions, the radio-medial (r-m) crossvein, the R2+3 radial vein and the 105 basal-medial-cubital (bm-cu) crossvein, which changed similarly among the three species in response to various 106 larval densities and temperature regimes. This finding suggests that these regions may represent developmentally 107 less robust wing compartments. We discuss our findings in the light of different mating behaviors in C. capitata To generate a range of sizes for each species, we varied two environmental factors known to influence overall 131 body sizetemperature and density. Prior to the experiment, D. melanogaster flies were placed at 25°C for two 132 days. On the third day, flies were moved from vials into egg-collection chambers and provided with apple-agar 133 plates. After several hours, we started egg collection by removing apple-agar plates with laid eggs once per hour.

Collected plates were kept at 25°C for 24 h to allow embryonic development to complete. Freshly hatched first-135 instar larvae were transferred into 50 ml vials with 15 ml of fly food. Three vials containing 25 larvae each (low 136 density) and three vials with 300 larvae each (high density) were moved to 18°C; the second set of six vials with 137 the same densities was left at 25°C.

C. capitata flies were kept at 28°C and allowed to lay eggs through a net into water. Every hour, eggs were 139 collected and transferred to the larval food. After 22 h, first-instar larvae were transferred into small Petri dishes 140 (diameter 55 mm) with 15 ml of the larval food in three densities: 25 (low density), 100 (m edium density) or 300 141 (high density) larvae per plate. Two plates of each density were moved to 18°C. The second set of six plates was 142 left at 28°C for further development.

Eggs of M. domestica were collected in the wet larval food at RT and after 24 h, all hatched larvae were removed 144 from food. Only larvae hatched within the next hour were transferred into 50 ml vials with 5 g of food. Collection 145 of larvae was repeated several times to obtain two experimental sets with three replicates of three experimental 146 densities 10 (low density), 20 (m edium density) or 40 (high density) larvae. One set of nine vials was moved to 147 18°C, the other was left at RT.

After pupation, individuals of C. capitata and M. domestica were collected from the food and kept until eclosion 149 in vials with a wet sponge, which was refreshed every other day.

The experimental temperature regimes were chosen for the following reasons. D. melanogaster is known to 151 survive in the range 10 to 33°C, but flies remain fertile at 12 to 30°C with the optimum at 25°C (Hoffmann, 2010 (Hewitt, 1914) with the optimum 154 between 24 and 27°C (Hafez, 1948; Chun-Hsung, 2012) . The low temperature for our experiment was chose as 155 the one above the survival and fertile minimums for all three species -18°C. The warm temperature was aimed 156 to be optimal for each species. 

Wing shape was analyzed using landmark-based geometric morphometric methods (Rohlf, 1990; 165 1991). We digitized 10 anatomically homologous landmarks on wings of the three species (Fig. 1A) . The 166 landmarks were the following (nomenclature is given after (Colless and McAlpine, 1991 

Wing images were digitized using tpsUtil and tpsDig2 (Rohlf, 2015) to obtain raw x and y landmark coordinates.

Using superimposition methods, it is possible to register landmarks of a sample to a common coordinate system 176 in three steps: translating all landmark configurations to the same centroid, scaling all configurations to the same 177 centroid size, and rotating all configurations until the summed squared distances between the landmarks and their 178 corresponding sample average is a minimum scaling (Slice, 2005; Mitteroecker et al., 2013) . To follow these three 179 steps, we applied the generalized Procrustes analysis (GPA) (Dryden and Mardia, 1998; Slice, 2005) as 180 implemented in MorphoJ (version 1.06d) (Klingenberg, 2011) . The wings were aligned by principal axes, the 181 mean configuration of landmarks was computed, and each wing was projected to a linear shape tangent space.

The coordinates of the aligned wings were the Procrustes coordinates. To evaluate wing asymmetry, we used the (Table S1 ), we averaged the coordinates for the right and left wings for further analyses. Next, we used Type III

Procrustes ANOVA (with Randomization of null model residuals and 1,000 permutations) as implemented in 188 Geomorph (v. 3.3.1) (Adams et al., 2021) to determine the effect of the species, sex, temperature, and density as 189 well as potential interactions between these explanatory variables and shape (Table 1) Since most of the variation in shape was explained by differences between species (see Results), we split the 197 analysis to further evaluate the effects of sex, rearing temperature, and density as well as potential interactions on 198 wing shape within each species using Procrustes ANOVA in Geomorph (v. 3.3.1) (TableS2). Magnitudes of 199 sexual shape dimorphism were estimated using the Discriminant Function Analysis (DFA) and expressed in units 200 of Procrustes distance using MorphoJ (version 1.06d). DFA identifies shape features that differ the most between 201 groups relative to within groups and it can only be applied to contrast two experimental groups. Therefore, we 202 used this method to define sexual shape dimorphism (males and females), as well as effects of the rearing 203 temperature (high and low) and density (high and low) in each species. Wing shape changes were visualized using 204 wireframe graphs. To test for the significance of the observed differences, we ran a permutation test with 1,000 205 random permutations (Good, 1994) for each test using MorphoJ (version 1.06d).

Estimation of the non-allometric component of shape

To evaluate the impact of wing size on shape variation, we estimated wing centroid size (WCS) that was computed 210 from raw data of landmarks and measured as the square root of the sum of squared deviations of landmarks around 211 their centroid (Bookstein, 1996; Dryden and Mardia, 1998; Slice, 2005) in MorphoJ (version 1.06d). The WCS 212 values were corrected for differences in magnification and resolution among photos before being used as 213 covariate. Since a Type III Procrustes ANOVA for all three species including WCS as covariate revealed 214 significant interaction between species and WCS (TableS3, sheet "All Species_total"), we estimated the non-215 allometric shape component for each species independently. The non-allometric Procrustes coordinates were 216 obtained as the residuals of the multiple regression of the Procrustes coordinates onto WCS pooling the samples 217 by sex, temperature, and density, as implemented in MorphoJ (version 1.06d) (Klingenberg, 2011 (Klingenberg, , 2016 . To 218 evaluate the success of the size correction, we ran a Type II ANOVA testing for additive effects of explanatory 219 variables prior to and after the size correction. This analysis revealed that WCS had no impact on wing shape after 220 size correction (TableS3, compare sheets "*_total" to "*_non-allometric"), suggesting that the approach 221 accounted for most of the impact of size on shape. Note that species-specific Type III Procrustes ANOVAs 222 including WCS as covariate revealed significant interactions for C. capitata and D. melanogaster (TableS3, sheets 223 "C. capitata_total", "D. melanogaster_total"), suggesting complex relationships between wing size and shape.

Since the regression approach to estimate allometry across all explanatory variables (i.e. sex, temperature and slopes. Therefore, the distinction between allometric and non -allometric shape differences may not be fully 231 accurate. However, due to a lack of a better method, we decided to proceed with the analysis and to interpret the 232 results cautiously.

Magnitudes of non-allometric shape differences for each explanatory variable and for sex-specific effects of 234 different temperature and density conditions were estimated using DFA as described before. Since wing size and 

Wing shape variation in dipteran species

To characterize wing shape variation among Ceratitis capitata, Drosophila melanogaster, and Musca domestica, 243 we applied geometric morphometrics based on 10 landmarks (Fig. 1A) . Procrustes ANOVA revealed that most 244 of the shape variance was caused by differences among species (Table 1) . This result was also reflected by the 245 Principal Component Analysis (PCA) which clearly distinguished the three species with the first two Principal 246 components (PCs) accounting for almost 98% of the variation (Fig. 1B) . The main shape difference along PC1 247 (77.3% of the variation) reflected the ratio between the proximal and distal parts of the wing as well as win g 248 length. The wireframe graphs showed that C. capitata wings were broad in the proximal part (landmarks 1-5) and 

1B, wireframe graphs along PC2). Together, we found evidence for extensive wing shape variation among C. 

Sexual dimorphism in wing shape

Our interspecific shape analysis revealed a clear sexual shape dimorphism which was most pronounced for C.

275 capitata (Fig. 1B 279 (TableS2). Therefore, we split the analysis by species and ran a Discriminant Function Analysis (DFA) to 280 determine the total shape differences caused by sex ( Fig. 2A-C) . For all three species we found a highly significant 281 sexual shape dimorphism ( Fig. 2A-C) . Male wings were broader than those of females in C. capitata ( Fig. 2A) 282 and D. melanogaster (Fig. 2B) , while the opposite trend was observed in M. domestica (Fig. 2C) . Wings of C.

283 capitata females were slightly longer than male wings ( Fig. 2A) and in D. melanogaster and M. domestica male 284 wings were longer (Fig. 2B, C) . The radio-medial crossvein (r-m) defined by landmarks 4 and 5 was different 285 between males and females in C. capitata ( Fig. 2A) . In D. melanogaster we observed clear sexual differences in 286 the radial vein R2+3 defined by landmarks 2 and 9 and in the basal-madial-cubital crossvein (bm-cu) defined by 287 landmarks 6 and 7 (Fig. 2B ). In summary, we found a clear total sexual shape dimorphism in all three studied 288 species.

Influence of sexual size dimorphisms on wing shape

Since wings of the three studied species exhibit a clear sexual size dimorphism (Siomava et al., 2016) we next 293 asked how much of the shape differences between sexes was explained by differences in wing size . Plots of the 294 shape component that was most correlated with wing size against wing size implied a minor impact of size on 295 wing shape in C. capitata and M. domestica, while wing shape was clearly associated with differences in wing 296 size in D. melanogaster (Fig. S2) . To analyze the non-allometric shape differences between sexes in more detail, 

Effect of different rearing conditions on wing shape 316 317

To evaluate the effect of wing size on shape in the three species in more detail, we raised flies at different 318 temperatures and densities (Bitner-Mathé and Klaczko, 1999), which have been shown to influence body size.

The Procrustes ANOVA for all three species revealed significant interactions between species and density and 320 temperature, respectively (Table 1) , suggesting species-specific effects of the rearing conditions on wing shape.

Since temperature and density contributed additively to wing shape differences (TableS2), we therefore split the 322 analyses by species and performed species-specific DFA to study the total shape differences caused by different 323 rearing temperatures (Fig. 3A -C) and densities (Fig. 4A) , respectively. Additionally, we calculated the non-324 allometric component of shape (see Materials and Methods for details) and analyzed the differences using DFA 325 ( Fig. 3D -F for temperature and Fig. 4B for density).

In accordance with the species-specific Procrustes ANOVA testing for effects of rearing conditions on total win g 327 shape variation (TableS2), the DFA clearly assigned wings to one of the two rearing temperatures (Fig. 3A-C) .

The most obvious effect of different rearing temperatures on wing shape was observed in D. melanogaster (Fig.   329 3B). Wings of D. melanogaster flies raised at higher temperatures were wider in the distal-central region defined 330 by landmarks 7-9 and distally shortened (i.e. displacement of landmark 10) (Fig. 3B) . The distal contraction 331 remained after size correction, while the width was much less affected (Fig. 3E) . This observation suggests that 332 temperature-dependent plasticity in wing width is predominantly caused by differences in wing size. Different 333 rearing temperatures also affected distal-central wing width in C. capitata (Fig. 3A) and M. domestica (Fig. 3C ).

Narrower wings were observed at higher temperatures in M. domestica, while wings were narrower at lower 335 temperatures in C. capitata (Fig. 3A,C) . M. domestica wings were longer at higher rearing temperatures (i.e.

displacement of landmark 10) (Fig. 3C) . Only minor changes were present between total and non-allometric shape 337 differences in C. capitata and M. domestica (Fig. 3D,F) , suggesting that temperature-dependent size differences 338 had only small effects on wing shape in these two species. In all three species, we observed temperature-dependent 339 plasticity in the placement of the radio-medial (r-m) crossveins (defined by landmarks 4 and 5), the basal-medial-340 cubital (bm-cu) crossveins (defined by landmarks 6 and 7), the radial vein R2+3 (defined by landmarks 2 and 9) 341 and the anterior cubital (CuA1) veins (defined by landmarks 7 and 8) (Fig. 3) . 

As suggested by the Procrustes ANOVA (TableS2), the DFA showed that different rearing densities only 351 significantly affected total wing shape in D. melanogaster ( Fig. 4A ; Table S5 ). Wings of flies raised at high 352 densities were wider in the central-distal region (defined by landmarks 7-9) and elongated (i.e. displaced landmark 353 10) (Fig. 4A) . We observed plasticity in the placement of the basal-medial-cubital (bm-cu) crossveins (defined by 354 landmarks 6 and 7), the anterior cubital (CuA1) veins (defined by landmarks 7 and 8), the radial vein R2+3

(defined by landmarks 2 and 9) and the costal (C) vein (defined by landmarks 3 and 9) (Fig. 4A ). All these 356 differences were gone after size correction (Fig. 4B) , suggesting that wing shape differences in response to rearing 357 densities were predominantly associated with variation in size. The displacement of landmark 3 was consistent in 358 both analyses (compare Fig. 4A to 4B), implying that this region of the wing may be affected by different rearing 359 densities independent of wing size. Despite a weak effect of density on C. capitata wing shape in our Procrustes 360 ANOVA (TableS2), the DFA did not reveal significant shape differences for flies raised at high and low densities 361 ( 382 capitata and D. melanogaster a DFA revealed very similar non-allometric shape differences between high and 383 low temperatures (Fig. S3 , Table S5 ) and densities (Fig. S4, Table S5 ), respectively for both sexes. Despite TableS2; "*_non-allometric"-sheets). Interestingly, the DFA revealed a significant effect of temperature ( Fig.   387 5A,B, Table S5 ) and density (Fig. 5C ,D, Table S5 ) on non-allometric shape differences in males, while females 388 were unaffected. 

In summary, we found no evidence for a sexual dimorphism in the response to different rearing conditions in C. and it has been shown that males and females respond differently to different rearing conditions, which affect 410 wing size (Siomava et al., 2016) . Therefore, the major aim of this study was understanding the relationship 411 between wing size and wing shape across sex and rearing conditions. In the following we will discuss our results

with respect to size and shape relationships and we hypothesize developmental and functional implications of our 413 findings.

Total shape variation and size and shape relationships 

2003), we detected a slightly stronger plasticity in proximal landmarks (Fig. 3B , see e.g. landmarks 1 and 3) in D.

melanogaster. This discrepancy may be due to the range of temperatures chosen in the different experiments. In 449 the previous work, stressful conditions (12°C and 14°C as the cold temperature and up to 30°C as the high 450 temperature) were included and wings of flies reared at extreme temperatures were clearly separated along CV2 451 in a Canonical Variate Analysis (CVA) (Fig. 3 in (Debat et al., 2003) ). In contrast, we applied intermediate non-

stressful regimes (18°C and 25°C), suggesting that the temperature effects on the distal wing region may partially 453 be a stress response. It is important to note that we realized during our analysis, that room temperature (generally 454 between 22 and 25°C) was likely lower than optimal for M. domestica (Siomava et al., 2016) . Therefore, the 455 obtained results for this species should be treated with som e caution because we may not have covered entire 456 range of optimal rearing temperatures and specifically for the Italian strain we used , the low temperature may 457 elicit some stress response.

Different rearing temperatures had clear effects on wing size in C. capitata and D. melanogaster and no effect in (Fig. S2) . Accordingly, we observed differences between the non-allometric 460 and the total shape differences across rearing temperatures in C. capitata and D. melanogaster, while wing shape 461 plasticity was unaffected after size correction in M. domestica. For instance, we did not observe the displacements 462 of the CuA1 and R2+3 veins after size correction, suggesting that these shape changes are associated with variation 463 in wing size. Interestingly, our data suggests more pronounced shape differences after size correction in C.

capitata (e.g. compare Fig. 3A to B) . This finding is counterintuitive in the light of a partitioning of total shape 465 variation into an allometric and a non-allometric component (Gidaszewski et al., 2009; Klingenberg, 2016) . Given 466 that the regression-based size correction applied in this work may not be optimal, the increased shape variation 467 after accounting for wing size could be an artifact. However, a study in the Leporinus cylindriformis group of ray- bm-cu and R2+3 veins were not observable in the non-allometric shape component (compare Fig. 4A to B).

Although we did not detect total wing shape differences in C. capitata and M. domestica, the non-allometric 

In addition to changes in wing shape along the proximal-distal axis, our shape analysis revealed a high variation 508 in the positioning of the r-m (landmarks 4 and 5), R2+3 (landmarks 2 and 9) and bm-cu (landmarks 6 and 7) veins 509 that was common for all three species. Displacement of these veins was found in all temperature and density 510 groups, suggesting that this region represents a very plastic aspect of wing patterning. Further support for this 511 suggestion comes from the loss of buffering against environmental perturbations in high altitude Ethiopian D. 

Our morphometrics analysis revealed that M. domestica have wings that are longer and narrower than those of C. revealed that male wings were wider than female, shorter in case of C. capitata, and radial veins were more spread 556 apart making wings more compact (Fig. 2) . The allometric component of shape additionally increased this 557 difference in D. melanogaster. The short and wide rounded wings of males in these species are likely to displace 558 more air and repeat calling song pulses more quickly than long narrow wings, and the wing moment of inertia 559 could be low enough to buzz. Interestingly, these flies produce two different types of wing vibration during the 

what could be achieved when the wing mass is concentrated near the axis of rotation (Berg and Rayner, 1995) .

Intriguingly, our shape analysis revealed major differences between C. capitata and D. melanogaster exactly in 574 this regionvariation in the width in proximal vs. distal regions (Fig. 1, PC1 axis) . Thus, C. capitata had wider 575 wings in the proximal part appropriate for high frequency buzzing and in D. melanogaster this part was narrower 576 but perhaps wide enough for low frequency buzzing. Although this hypothesis remains to be tested, it is tempting 577 to speculate that these shape differences may be linked to the mating behavior of the flies and specific properties 578 of their courtship songs. 

Competing Interests

The authors declare that they have no competing interests. 597 598

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