Melanic pigmentation and light preference within and between two Drosophila species

Environmental adaptation and species divergence often involve suites of co‐evolving traits. Pigmentation in insects presents a variable, adaptive, and well‐characterized class of phenotypes for which correlations with multiple other traits have been demonstrated. In Drosophila, the pigmentation genes ebony and tan have pleiotropic effects on flies'' response to light, creating the potential for correlated evolution of pigmentation and vision. Here, we investigate differences in light preference within and between two sister species, Drosophila americana and D. novamexicana, which differ in pigmentation in part because of evolution at ebony and tan and occupy environments that differ in many variables including solar radiation. We hypothesized that lighter pigmentation would be correlated with a greater preference for environmental light and tested this hypothesis using a habitat choice experiment. In a first set of experiments, using males of D. novamexicana line N14 and D. americana line A00, the light‐bodied D. novamexicana was found slightly but significantly more often than D. americana in the light habitat. A second experiment, which included additional lines and females as well as males, failed to find any significant difference between D. novamexicana‐N14 and D. americana‐A00. Additionally, the other dark line of D. americana (A04) was found in the light habitat more often than the light‐bodied D. novamexicana‐N14, in contrast to our predictions. However, the lightest line of D. americana, A01, was found substantially and significantly more often in the light habitat than the two darker lines of D. americana, thus providing partial support for our hypothesis. Finally, across all four lines, females were found more often in the light habitat than their more darkly pigmented male counterparts. Additional replication is needed to corroborate these findings and evaluate conflicting results, with the consistent effect of sex within and between species providing an especially intriguing avenue for further research.     

The β-alanyl-monoamine synthase ebony is regulated by schizophrenia susceptibility gene dysbindin in Drosophila

The Drosophila homolog of schizophrenia susceptibility gene dysbindin(Ddysb)affects a range of behaviors through regulation of multiple neurotransmitter signals,including dopamine activity.To gain insights into mechanisms underlying Ddysb-dependent regulation of dopamine signal,we investigated interaction between Ddysb and Ebony,the Drosophilaβ-alanyl-monoamine synthase involved in dopamine recycling.We found that Ddysb was capable of regulating expression of Ebony in a bi-directional manner and its subcellular distribution.Such regulation is confined to glial cells.The expression level of ebony and its accumulation in glial soma depend positively on Ddysb activity,whereas its distribution in glial processes is bound to be reduced in response to any alterations of Ddysb from the normal control level,either an increase or decrease.An optimal binding ratio between Dysb and Ebony might contribute to such non-linear effects.Thus,Ddysb-dependent regulation of Ebony could be one of the mechanisms that mediate dopamine signal.     

N‐β‐alanyldopamine metabolism,locomotor activity and sleep in Drosophila melanogaster ebony and tan mutants

Drosophila melanogaster Meigen mutants for N‐β‐alanyldopamine (NBAD) metabolism have altered levels of NBAD, dopamine and other neurotransmitters. The ebony¹ mutant strain has very low levels of NBAD and higher levels of dopamine, whereas the opposite situation is observed in the tan¹ mutant. Dopamine is implicated in the control of movement, memory and arousal, as well as in the regulation of sleep and wakefulness in D. melanogaster. N‐β‐alanyldopamine, which is best known as a cuticle cross‐linking agent, is also present in nervous tissue and has been proposed to promote locomotor activity in this fly. The daily locomotor activity and the sleep patterns of ebony¹ and tan¹ mutants are analyzed, and are compared with wild‐type flies. The tan¹ mutant shows reduced locomotor activity, whereas ebony¹ shows higher levels of activity than wild‐type flies, suggesting that NBAD does not promote locomotor activity. Both mutants spend less time asleep than wild‐type flies during night‐time; ebony shows more consolidated activity during night‐time and increased sleep latency, whereas tan is unable to consolidate locomotor activity and sleep in either phase of the day. The daily level of NBAD‐synthase activity is measured in vitro using wild‐type and tan¹ protein extracts, and the lowest NBAD synthesis is observed at the time of higher locomotor activity. The abnormalities in several parameters of the waking/sleep cycle indicate some dysfunction in the processes that regulates these behaviours in both mutants.     

The genetic basis of adaptive pigmentation variation in Drosophila melanogaster

In a broad survey of Drosophila melanogaster population samples, levels of abdominal pigmentation were found to be highly variable and geographically differentiated. A strong positive correlation was found between dark pigmentation and high altitude, suggesting adaptation to specific environments. DNA sequence polymorphism at the candidate gene ebony revealed a clear association with the pigmentation of homozygous third chromosome lines. The darkest lines sequenced had nearly identical haplotypes spanning 14.5 kb upstream of the protein-coding exons of ebony. Thus, natural selection may have elevated the frequency of an allele that confers dark abdominal pigmentation by influencing the regulation of ebony.     

CRISPR/Cas9‐mediated ebony knockout results in puparium melanism in Spodoptera litura

Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S. litura embryos, we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.     

Divergent enhancer haplotype of ebony on inversion In(3R)Payne associated with pigmentation variation in a tropical population of Drosophila melanogaster

The pattern and intensity of pigmentation have direct impact on individual fitness through various ecological factors. In a Drosophila melanogaster population from southern Japan, thoracic trident pigmentation intensity of most of the strains could be classified into Dark or Light‐type. The expression level variation of the ebony gene correlated well with this phenotype and the allelic differences in expression indicated that the variation is partly due to cis‐regulatory changes. In the ～13 kb gene region, we identified 17 nucleotide sites and 2 indels that were in complete association with the thoracic trident pigmentation intensity. Interestingly, 11 out of 19 sites located within ～0.5 kb of the core epidermis enhancer. These sites had no obvious association with the abdominal pigmentation intensity in the previously analysed African populations from Uganda and Kenya, which suggested that multiple potential mutational pathways in the cis‐regulatory control region of a single gene could lead to similar phenotypic variation within this species. We also found that the Light‐type enhancer haplotype is strongly linked to a cosmopolitan inversion, In(3R)Payne, which is predominant in warmer climatic regions in both hemispheres. The sequence pattern suggested that the strong linkage may be due to selective forces related to thermal adaptation. The inferred selection for lighter pigmentation in the Japanese population is in the opposite direction of the previously reported case of selection for darker individuals in African populations. Nevertheless, both adaptive changes involved cis‐regulatory changes of ebony, which shows that this gene is likely to be a common target of natural selection.     

稀有的黑檀体基因(ebony)5''UTR自发突变导致黑腹果蝇的黑条体突变

由于果蝇Drosophila群体中有很多自发突变其中包括多种体色突变,因此它是一个研究自发突变的优秀的模式体系。本研究证实我们实验室发现的一个可以引起果蝇体色突变的自发突变(bsr)是一个黑檀体(e)的等位基因,将其命名为ebsr。序列分析显示ebsr的5′端缺失了953个碱基,其中包括外显子1后端的206个碱基及相连的内含子1的747个碱基。逆转录PCR结果显示5′端的缺失导致内含子1不能从mRNA中剪接掉,由此导致该mRNA的翻译起始密码子AUG前端增加了一个3.2kb的序列。该序列导致ebsr的mRNA的5′UTR(5′-untranslated region)区较野生型基因增加近3kb的长度。通过mRNA二级结构分析发现这个增加的3kb的片段可以形成复杂的颈环结构(stem-loop)。免疫印迹结果显示该突变基因没有基因产物产生。本研究进一步证实了由于mRNA的5′UTR序列结构的改变可以影响到蛋白质的翻译。     

黑腹果蝇黑条体突变型的基因定位研究

黑腹果的体色突变类型常见的有黄体（yellow,y）、黑体（black,b）和黑檀体（ebony,e）,分别位于X染色体,第二染色体和第三染色体上,.黑条体突变型是本实验室1991年9月从野外采集的黑腹果蝇野生型单雌系后代中发现的自发突变品系,为了探明黑条体突变型是原有黑体突变类型的再现还是新的突变,采用常规杂交方法和互补7实验技术对黑腹果蝇黑条体突变型的定位进行了探讨,互补测验的结果表明,黑条体与黑檀体杂交的子一代为反式排列的杂合体无互补,表现为突变型,子二代中,由于交换而产生重组类型的顺式排列的杂合体表现为野生型。因此确定黑条体突变基因（bsr）与黑檀体突变基因（e）是等位的,位于第三染色体的93D2区,但分别位于不同的位点上,属于同一顺反子的新的点突变,同时对于各体色间的相互作用及遗传传递方式的进行了讨论。     

稀有的黑檀体基因（ebony）5′UTR自发突变导致黑腹果蝇的黑条体突变

由于果蝇Drosophila群体中有很多自发突变其中包括多种体色突变, 因此它是一个研究自发突变的优秀的模式体系。本研究证实我们实验室发现的一个可以引起果蝇体色突变的自发突变（bsr）是一个黑檀体（e）的等位基因, 将其命名为e^bsr。序列分析显示e^bsr的5′端缺失了953个碱基, 其中包括外显子1后端的206个碱基及相连的内含子1的747个碱基。逆转录PCR结果显示5′端的缺失导致内含子1不能从mRNA中剪接掉, 由此导致该mRNA的翻译起始密码子AUG前端增加了一个3.2 kb的序列。该序列导致e^bsr的mRNA的5′UTR（5′-untranslated region）区较野生型基因增加近3 kb的长度。通过mRNA二级结构分析发现这个增加的3 kb的片段可以形成复杂的颈环结构（stem-loop）。免疫印迹结果显示该突变基因没有基因产物产生。本研究进一步证实了由于mRNA的5′UTR序列结构的改变可以影响到蛋白质的翻译。     

黑腹果蝇黑条体突变体的求偶行为

黑腹果蝇Drosophila melanogaster黑条体果蝇(e^bsr)与黑檀体果蝇(e)为同一个基因(ebony)的不同突变体, 两者具有相似的形态表型, 但行为特征表现出明显的差异。本研究以黑条体、 黑檀体和野生型果蝇为研究对象, 首先检测果蝇的视力和活跃度, 再采用不同交配组合进行求偶成功率、交配时间和求偶模式的分析。结果表明: 黑条体果蝇视力与活跃度与野生型果蝇比较无显著差异; 黑条体果蝇的交配成功率和交配潜伏期与野生型果蝇不存在显著的差异; 黑檀体果蝇的交配成功率和交配潜伏期与野生型果蝇存在极显著的差异（P<0.000）。黑条体果蝇表现出异于黑檀体果蝇的活跃度和交配活力, 可能是由于黑条体果蝇ebony基因的新突变导致了果蝇体内多巴胺水平异常, 从而形成了黑条体果蝇独特的求偶模式。