Stalk-eyed flies carrying a driving X chromosome compensate by increasing fight intensity

Exaggerated ornaments provide opportunities for understanding how selection can operate at different levels to shape the evolution of a trait. While these features aid their bearer in attracting mates or fending off competitors, they can also be costly and influenced by the environment and genetic variation. Eyestalks of the stalk-eyed fly, Teleopsis dalmanni, are of interest because eyestalk length is the target of both intra-and intersexual selection and is also reduced by loci on a highly-diverged sex ratio X chromosome (XSR), a meiotic driver accounting for up to 30% of wild X chromosomes. Male stalk-eyed flies fight to control access to females and over food using a combination of low intensity displays and high intensity physical fights. We staged, filmed and scored contests among eyespan-matched male pairs to evaluate whether X chromosome type impacts the behavior and outcome of aggressive interactions. While our results broadly match expectations from previous studies, we find that XSR males used more high intensity behaviors than males carrying a non-driving, standard X chromosome (XST), in particular when their eyestalks were of similar size or smaller than their opponents. Additionally, we find that when XSR males use high intensity behaviors they win more bouts than when they use low intensity behaviors. Taken together these results suggest that XSR impacts male aggressive behavior to compensate for the shorter eyestalks of XSR males, and may help to explain how this selfish chromosome is maintained.

Methods
Following previous studies of fighting in stalk-eyed flies (Panhuis and Wilkinson 1999; Egge et al. 2011; Bubak et al. 2014), we quantified male aggressive behavior in two sets of trials conducted either at SUNY Geneseo (outbred pair trials) or the University of Maryland, College Park (standardized opponent trials). We conducted trials with outbred and inbred flies as opponents to confirm that the results were robust to the opponents’ genetic background. Sexually mature males at least four weeks post-eclosion were anesthetized using carbon dioxide, eyespan and body length were measured from video-captured images to the nearest 0.01 mm using ImageJ. All trials were conducted in transparent plastic arenas (10 x 3.5 x 6 cm) lined with moist blotting paper and initially divided into three sections by opaque plastic barriers.  Each male was placed into an outer compartment of the arena and fasted 20-24 h.  At the beginning of the trial, a piece of agar gel made with corn juice was placed into the center compartment as a food resource and the barriers were removed. Trials were video recorded for ten minutes. Scoring of each video was done blind to genotype. Occurrence of aggressive behaviors performed by each fly in each trial were scored from video recordings following a published ethogram. Within a trial, we defined fighting “bouts” to begin once either fly performed an aggressive behavior and end when the flies were more than a body length apart or not facing each other for 3 s without exhibiting any aggressive behavior. A male was scored as losing a bout if he moved away from his opponent at the end of a bout either slowly (away) or quickly (retreat) and his opponent did not. A mutual away or retreat was considered a tied bout. Most trials involved multiple bouts. Aggressive behaviors were categorized as either high intensity (HI) behaviors that involved physical contact (tussle, attack/lunge, jump attack) or low intensity (LI) behaviors that involved males mutually displaying their eyestalks in close proximity (approach, flex, and extend, line-up eyestalks). Escalations were tallied for each individual as a transition from a LI to a HI behavior with no other behaviors or the start or end of a bout separating them.  Similarly, de-escalations were defined as a transition from HI to LI behavior. Males were collected immediately after each trial for DNA extraction and genotyping for X chromosome type (XSR or XST) using polymerase chain reaction (PCR) to amplify either of two markers (Table S1) that each predict drive phenotype in this population with 95% accuracy.

夸张的装饰性特征为解析选择如何在不同层级发挥作用以塑造性状演化提供了绝佳契机。这类特征既能帮助携带者吸引配偶、抵御竞争者，同时也会付出相应代价，并受到环境与遗传变异的共同影响。戴氏突眼蝇（Teleopsis dalmanni）的眼柄是研究热点：眼柄长度同时受到性内选择与性间选择的作用，同时还会受到高度分化的性比例X染色体（XSR，一种减数分裂驱动因子（meiotic driver），在野生X染色体中占比可达30%）上的基因位点所抑制。雄性突眼蝇（stalk-eyed fly）会结合低强度展示与高强度肢体对抗，争夺与雌性交配的机会以及食物资源。我们通过对眼展匹配的雄性个体进行配对竞赛，记录并评分其行为表现，以探究X染色体类型是否会影响攻击行为的模式与竞赛结果。尽管研究结果整体契合既往研究的预期，但我们发现，携带XSR染色体的雄性相比携带非驱动型标准X染色体（XST）的个体，会更多地展现高强度攻击行为，尤其是在其眼柄长度与对手相当甚至更短的情况下。此外，我们还发现，当XSR雄性使用高强度攻击行为时，其竞赛获胜率高于使用低强度行为时的情况。综合来看，这些结果表明XSR会影响雄性的攻击行为，以弥补自身眼柄较短的劣势，这或许有助于解释这类自私染色体如何得以在种群中维持。

方法
参照既往关于突眼蝇攻击行为的研究（Panhuis与Wilkinson 1999；Egge等人2011；Bubak等人2014），我们开展了两组试验以量化雄性攻击行为：一组在纽约州立大学杰纳西奥分校（SUNY Geneseo）进行（远亲配对试验），另一组在马里兰大学帕克分校（University of Maryland, College Park）开展（标准化对手试验）。我们使用远亲与近交个体作为对手开展试验，以验证研究结果不受对手遗传背景的影响。将羽化至少4周的性成熟雄性个体用二氧化碳麻醉，通过视频采集图像，利用ImageJ软件测量其眼展与体长，精度可达0.01 mm。所有试验均在透明塑料竞技场（10 × 3.5 × 6 cm）中进行，竞技场底部铺有湿润的吸水纸，初始时由不透明塑料挡板划分为三个区域。将每只雄性个体放置于竞技场的外侧隔间，禁食20~24小时。试验开始时，在中央区域放置一块玉米汁琼脂凝胶作为食物资源，随后移除挡板。试验全程录制10分钟视频。所有视频的评分均采用单盲法，不告知样本的基因型。参照已发表的行为谱（ethogram），从录像中对每只果蝇在每场试验中的攻击行为发生情况进行评分。在每场试验中，我们将“竞赛回合（bout）”定义为：任意一只果蝇发起攻击行为后开始，至两只果蝇相距超过1个体长，或连续3秒未面对面且未展现任何攻击行为时结束。若某只雄性在回合结束时主动远离对手（缓慢逃离或快速撤退），而对手未逃离，则该雄性被记为回合失利。若双方均逃离，则记为平局。多数试验包含多个竞赛回合。攻击行为可分为两类：高强度（HI）行为涉及肢体接触，包括扭打、攻击/突袭、跳跃突袭；低强度（LI）行为则为雄性在近距离内互相展示眼柄，包括接近、屈曲与伸展、对齐眼柄。个体的行为升级次数统计为：从低强度行为直接转换为高强度行为，且无其他行为间隔，或仅以竞赛回合的起止作为间隔。同理，行为降级被定义为从高强度行为转换为低强度行为。每场试验结束后，立即采集雄性个体样本用于DNA提取，并通过聚合酶链式反应（polymerase chain reaction, PCR）扩增两种标记物（表S1）以鉴定X染色体类型（XSR或XST），这两种标记物对该种群驱动表型的鉴定准确率可达95%。