Drosophila females have an acoustic preference for symmetric males.

SignificanceTheoretically, symmetry in bilateral animals is subject to sexual selection, since it can serve as a proxy for genetic quality of competing mates during mate choice. Here, we report female preference for symmetric males in Drosophila, using a mate-choice paradigm where males with environmentally or genetically induced wing asymmetry were competed. Analysis of courtship songs revealed that males with asymmetric wings produced songs with asymmetric features that served as acoustic cues, facilitating this female preference. Females experimentally evolved in the absence of mate choice lost this preference for symmetry, suggesting that it is maintained by sexual selection.

Divergence of a speciation trait through artificial selection: Insights into constraints, by-product effects and sexual isolation.

Speciation and sexual isolation often occur when divergent female mating preferences target male secondary sexual traits. Despite the importance of such male signals, little is known about their evolvability and genetic linkage to other traits during speciation. To answer these questions, we imposed divergent artificial selection for 10 non-overlapping generations on the Inter-Pulse-Interval (IPI) of male courtship songs; which has been previously shown to be a major species recognition trait for females in the Drosophila athabasca species complex. Focusing on one of the species, Drosophila mahican (previously known as EA race), we examined IPI's: (1) rate of divergence, (2) response to selection in different directions, (3) genetic architecture of divergence and (4) by-product effects on other traits that have diverged in the species complex. We found rapid and consistent response for higher IPI but less response to lower IPI; implying asymmetrical constraints. Genetic divergence in IPI differed from natural species in X versus autosome contribution and in dominance, suggesting that evolution may take different paths. Finally, selection on IPI did not alter other components of male songs, or other ecological traits, and did not cause divergence in female preferences, as evidenced by lack of sexual isolation. This suggests that divergence of male courtship song IPI is unconstrained by genetic linkage with other traits in this system. This lack of linkage between male signals and other traits implies that female preferences or ecological selection can co-opt and mould specific male signals for species recognition free of genetic constraints from other traits.

A latitudinal cline in a courtship song character of Drosophila melanogaster.

The courtship song of male Drosophila melanogaster is generated by wing vibration and contains an interpulse interval (IPI) which is species-specific and usually falls in the mean range of 30-40 ms. The IPI is extremely temperature-sensitive, so we wondered whether flies collected along the eastern coast of Australia between latitudes 16.9°S and 42.9°S might have adapted to the different thermal conditions and show differences in mean IPI. We observe a significant correlation between IPI and latitude in addition to the well-known association between latitude and body size (Bergmannn's Rule). However, somewhat surprisingly we could not detect a significant association between body size and IPI. We also examined flies collected from the North and South-facing slopes of 'Evolution Canyon' in Israel and observed differences in IPI that support the view that thermal adaptation can shape this important song character. We also examined the songs of flies from Kenya and observed no correlation between altitude of collection and IPI. In all three experiments, body size did not correlate with IPI. A global analysis of all three sets of populations on three continents revealed a strong association between IPI and latitude. We speculate that IPI is shaped by thermal and sexual selection whereas body size is also shaped by natural selection.

Experimental and statistical reevaluation provides no evidence for Drosophila courtship song rhythms.

From 1980 to 1992, a series of influential papers reported on the discovery, genetics, and evolution of a periodic cycling of the interval between Drosophila male courtship song pulses. The molecular mechanisms underlying this periodicity were never described. To reinitiate investigation of this phenomenon, we previously performed automated segmentation of songs but failed to detect the proposed rhythm [Arthur BJ, et al. (2013) BMC Biol 11:11; Stern DL (2014) BMC Biol 12:38]. Kyriacou et al. [Kyriacou CP, et al. (2017) Proc Natl Acad Sci USA 114:1970-1975] report that we failed to detect song rhythms because (i) our flies did not sing enough and (ii) our segmenter did not identify many of the song pulses. Kyriacou et al. manually annotated a subset of our recordings and reported that two strains displayed rhythms with genotype-specific periodicity, in agreement with their original reports. We cannot replicate this finding and show that the manually annotated data, the original automatically segmented data, and a new dataset provide no evidence for either the existence of song rhythms or song periodicity differences between genotypes. Furthermore, we have reexamined our methods and analysis and find that our automated segmentation method was not biased to prevent detection of putative song periodicity. We conclude that there is no evidence for the existence of Drosophila courtship song rhythms.

Failure to reproduce period-dependent song cycles in Drosophila is due to poor automated pulse-detection and low-intensity courtship.

Stern has criticized a body of work from several groups that have independently studied the so-called "Kyriacou and Hall" courtship song rhythms of male Drosophila melanogaster, claiming that these ultradian ∼60-s cycles in the interpulse interval (IPI) are statistical artifacts that are not modulated by mutations at the period (per) locus [Stern DL (2014) BMC Biol 12:38]. We have scrutinized Stern's raw data and observe that his automated song pulse-detection method identifies only ∼50% of the IPIs found by manual (visual and acoustic) monitoring. This critical error is further compounded by Stern's use of recordings with very little song, the large majority of which do not meet the minimal song intensity criteria which Kyriacou and Hall used in their studies. Consequently most of Stern's recordings only contribute noise to the analyses. Of the data presented by Stern, only perL and a small fraction of wild-type males sing vigorously, so we limited our reanalyses to these genotypes. We manually reexamined Stern's raw song recordings and analyzed IPI rhythms using several independent time-series analyses. We observe that perL songs show significantly longer song periods than wild-type songs, with values for both genotypes close to those found in previous studies. These per-dependent differences disappear when the song data are randomized. We conclude that Stern's negative findings are artifacts of his inadequate pulse-detection methodology coupled to his use of low-intensity courtship song records.

GABAergic Local Interneurons Shape Female Fruit Fly Response to Mating Songs.

Many animals use acoustic signals to attract a potential mating partner. In fruit flies (Drosophila melanogaster), the courtship pulse song has a species-specific interpulse interval (IPI) that activates mating. Although a series of auditory neurons in the fly brain exhibit different tuning patterns to IPIs, it is unclear how the response of each neuron is tuned. Here, we studied the neural circuitry regulating the activity of antennal mechanosensory and motor center (AMMC)-B1 neurons, key secondary auditory neurons in the excitatory neural pathway that relay song information. By performing Ca2+ imaging in female flies, we found that the IPI selectivity observed in AMMC-B1 neurons differs from that of upstream auditory sensory neurons [Johnston's organ (JO)-B]. Selective knock-down of a GABAA receptor subunit in AMMC-B1 neurons increased their response to short IPIs, suggesting that GABA suppresses AMMC-B1 activity at these IPIs. Connection mapping identified two GABAergic local interneurons that synapse with AMMC-B1 and JO-B. Ca2+ imaging combined with neuronal silencing revealed that these local interneurons, AMMC-LN and AMMC-B2, shape the response pattern of AMMC-B1 neurons at a 15 ms IPI. Neuronal silencing studies further suggested that both GABAergic local interneurons suppress the behavioral response to artificial pulse songs in flies, particularly those with a 15 ms IPI. Altogether, we identified a circuit containing two GABAergic local interneurons that affects the temporal tuning of AMMC-B1 neurons in the song relay pathway and the behavioral response to the courtship song. Our findings suggest that feedforward inhibitory pathways adjust the behavioral response to courtship pulse songs in female flies.SIGNIFICANCE STATEMENT To understand how the brain detects time intervals between sound elements, we studied the neural pathway that relays species-specific courtship song information in female Drosophila melanogaster We demonstrate that the signal transmission from auditory sensory neurons to key secondary auditory neurons antennal mechanosensory and motor center (AMMC)-B1 is the first-step to generate time interval selectivity of neurons in the song relay pathway. Two GABAergic local interneurons are suggested to shape the interval selectivity of AMMC-B1 neurons by receiving auditory inputs and in turn providing feedforward inhibition onto AMMC-B1 neurons. Furthermore, these GABAergic local interneurons suppress the song response behavior in an interval-dependent manner. Our results provide new insights into the neural circuit basis to adjust neuronal and behavioral responses to a species-specific communication sound.

A single male auditory response test to quantify auditory behavioral responses in Drosophila melanogaster.

Many animals utilize auditory signals to communicate with conspecific individuals. During courtship, males of the fruit fly Drosophila melanogaster and related species produce a courtship song comprised of sine and pulse songs by vibrating their wings. The pulse song increases female receptivity and male courtship activity, indicating that it functions as a sexual signal. One song parameter, interpulse interval (IPI), varies among closely related species. In D. melanogaster, a song with a conspecific IPI induces a stronger behavioral response than heterospecific songs, indicating the ability of the flies to discriminate conspecific IPI. Traditionally, the fly's response to the song is measured under grouped conditions, in which the effect of sensory modalities other than audition cannot be excluded. Here, to quantify the individual ability to discriminate a conspecific song, we systematically analyzed the auditory response of single male flies to sound with various parameters. Moreover, we applied this method, termed SMART (Single Male Auditory Response Test), to two sister species for potential application in a comparative approach. By quantifying the locomotor activity of single D. melanogaster males during sound exposure, we detected increased locomotor activity in response to pulse songs, but not to white noise or pure tone. The conspecific song evoked stronger response than the heterospecific songs, and ablation of their antennal receivers severely suppressed the locomotor increase. A pulse song with a small IPI variation evoked a continuous response, while the response to songs with highly variable IPIs tends to be rapidly decayed. This provides the first evidence that fruit flies discriminate IPI variations, which possibly inform the age and social contexts of the singer. Sister species, D. sechellia, exhibited a locomotor response to pulse song, while D. simulans exhibited no behavioral response. This suggests that auditory and other stimuli that elicit this behavioral response are diversified among Drosophila species.

Flightin maintains myofilament lattice organization required for optimal flight power and courtship song quality in Drosophila.

The indirect flight muscles (IFMs) of Drosophila and other insects with asynchronous flight muscles are characterized by a crystalline myofilament lattice structure. The high-order lattice regularity is considered an adaptation for enhanced power output, but supporting evidence for this claim is lacking. We show that IFMs from transgenic flies expressing flightin with a deletion of its poorly conserved N-terminal domain (flnΔN62 ) have reduced inter-thick filament spacing and a less regular lattice. This resulted in a decrease in flight ability by 33% and in skinned fibre oscillatory power output by 57%, but had no effect on wingbeat frequency or frequency of maximum power output, suggesting that the underlying actomyosin kinetics is not affected and that the flight impairment arises from deficits in force transmission. Moreover, we show that flnΔN62 males produced an abnormal courtship song characterized by a higher sine song frequency and a pulse song with longer pulses and longer inter-pulse intervals (IPIs), the latter implicated in male reproductive success. When presented with a choice, wild-type females chose control males over mutant males in 92% of the competition events. These results demonstrate that flightin N-terminal domain is required for optimal myofilament lattice regularity and IFM activity, enabling powered flight and courtship song production. As the courtship song is subject to female choice, we propose that the low amino acid sequence conservation of the N-terminal domain reflects its role in fine-tuning species-specific courtship songs.

Delineation of a frequency-organized region isolated from the mouse primary auditory cortex.

The primary auditory cortex (AI) is the representative recipient of information from the ears in the mammalian cortex. However, the delineation of the AI is still controversial in a mouse. Recently, it was reported, using optical imaging, that two distinct areas of the AI, located ventrally and dorsally, are activated by high-frequency tones, whereas only one area is activated by low-frequency tones. Here, we show that the dorsal high-frequency area is an independent region that is separated from the rest of the AI. We could visualize the two distinct high-frequency areas using flavoprotein fluorescence imaging, as reported previously. SMI-32 immunolabeling revealed that the dorsal region had a different cytoarchitectural pattern from the rest of the AI. Specifically, the ratio of SMI-32-positive pyramidal neurons to nonpyramidal neurons was larger in the dorsal high-frequency area than the rest of the AI. We named this new region the dorsomedial field (DM). Retrograde tracing showed that neurons projecting to the DM were localized in the rostral part of the ventral division of the medial geniculate body with a distinct frequency organization, where few neurons projected to the AI. Furthermore, the responses of the DM to ultrasonic courtship songs presented by males were significantly greater in females than in males; in contrast, there was no sex difference in response to artificial pure tones. Our findings offer a basic outline on the processing of ultrasonic vocal information on the basis of the precisely subdivided, multiple frequency-organized auditory cortex map in mice.

Double meaning of courtship song in a moth.

Males use courtship signals to inform a conspecific female of their presence and/or quality, or, alternatively, to 'cheat' females by imitating the cues of a prey or predator. These signals have the single function of advertising for mating. Here, we show the dual functions of the courtship song in the yellow peach moth, Conogethes punctiferalis, whose males generate a series of short pulses and a subsequent long pulse in a song bout. Repulsive short pulses mimic the echolocation calls of sympatric horseshoe bats and disrupt the approach of male rivals to a female. The attractive long pulse does not mimic bat calls and specifically induces mate acceptance in the female, who raises her wings to facilitate copulation. These results demonstrate that moths can evolve both attractive acoustic signals and repulsive ones from cues that were originally used to identify predators and non-predators, because the bat-like sounds disrupt rivals, and also support a hypothesis of signal evolution via receiver bias in moth acoustic communication that was driven by the initial evolution of hearing to perceive echolocating bat predators.

No boundaries: genomes, organisms, and ecological interactions responsible for divergence and reproductive isolation.

Revealing the genetic basis of traits that cause reproductive isolation, particularly premating or sexual isolation, usually involves the same challenges as most attempts at genotype-phenotype mapping and so requires knowledge of how these traits are expressed in different individuals, populations, and environments, particularly under natural conditions. Genetic dissection of speciation phenotypes thus requires understanding of the internal and external contexts in which underlying genetic elements are expressed. Gene expression is a product of complex interacting factors internal and external to the organism including developmental programs, the genetic background including nuclear-cytotype interactions, epistatic relationships, interactions among individuals or social effects, stochasticity, and prevailing variation in ecological conditions. Understanding of genomic divergence associated with reproductive isolation will be facilitated by functional expression analysis of annotated genomes in organisms with well-studied evolutionary histories, phylogenetic affinities, and known patterns of ecological variation throughout their life cycles. I review progress and prospects for understanding the pervasive role of host plant use on genetic and phenotypic expression of reproductive isolating mechanisms in cactophilic Drosophila mojavensis and suggest how this system can be used as a model for revealing the genetic basis for species formation in organisms where speciation phenotypes are under the joint influences of genetic and environmental factors.

Courtship song differs between African and European populations of Drosophila melanogaster and involves a strong effect locus.

The courtship song of Drosophila melanogaster has long served as excellent model system for studies of animal communication and differences in courtship song have been demonstrated among populations and between species. Here, we report that flies of African and European origin, which diverged approximately 13,000 years ago, show significant genetic differentiation in the use of slow versus fast pulse song. Using a combination of quantitative trait mapping and population genetic analysis we detected a single strong QTL underlying this trait and we identified candidate genes that may contribute to the evolution of this trait. Song trait variation between parental strains of our recombinant inbred panel enabled detection of genomic intervals associated with six additional song traits, some of which include known courtship-related genes. These findings improve the prospects for further genetic insights into the evolution of reproductive behavior and the biology underlying courtship song.

New data on bioacoustics and courtship behaviour in grasshoppers (Orthoptera, Acrididae, Gomphocerinae) from Russia and adjacent countries.

The songs of seven grasshopper species of subfamily Gomphocerinae from Russia, Ukraine, Georgia, and Kazakhstan were studied. We analysed not only the sound, but also the stridulatory movements of the hind legs to more entirely describe the songs. In Mesasippuskozhevnikovi, Chorthippusmacrocerus, and C.hammarstroemi, the legs are moved in a relatively simple pattern; four other species, Myrmeleotettixpalpalis, Stenobothrusnewskii, C.pullus, and Megaulacobothrusaethalinus demonstrate more complex leg movements. In six of the seven species studied, the courtship songs contain more sound elements than the calling songs. The highest number of courtship sound elements was found in M.palpalis and M.aethalinus. The different parts of a remarkably long stridulatory file in M.aethalinus are thought to participate in the production of different sound elements. The songs in S.newskii are shown for the first time. This species produces sound not only by common stridulation but also by wing beats. A relationship of S.newskii to some other species of the genus Stenobothrus, which are able to crepitate, is discussed. We also analyse the frequency spectra of the songs. A maximum energy of the song power spectra in six species studied lies in ultrasound range (higher than 20 kHz). In only M.aethalinus, the main peaks in the song power spectra lie lower than 20 kHz. The courtship behaviour in M.palpalis, C.macrocerus, and C.hammarstroemi was shown to include conspicuous visual display (movements of antennae, palps and the whole body).

Changes in the cellular makeup of motor patterning circuits drive courtship song evolution in Drosophila.

How evolutionary changes in genes and neurons encode species variation in complex motor behaviors is largely unknown. Here, we develop genetic tools that permit a neural circuit comparison between the model species Drosophila melanogaster and the closely related species D. yakuba, which has undergone a lineage-specific loss of sine song, one of the two major types of male courtship song in Drosophila. Neuroanatomical comparison of song-patterning neurons called TN1 across the phylogeny demonstrates a link between the loss of sine song and a reduction both in the number of TN1 neurons and the neurites supporting the sine circuit connectivity. Optogenetic activation confirms that TN1 neurons in D. yakuba have lost the ability to drive sine song, although they have maintained the ability to drive the singing wing posture. Single-cell transcriptomic comparison shows that D. yakuba specifically lacks a cell type corresponding to TN1A neurons, the TN1 subtype that is essential for sine song. Genetic and developmental manipulation reveals a functional divergence of the sex determination gene doublesex in D. yakuba to reduce TN1 number by promoting apoptosis. Our work illustrates the contribution of motor patterning circuits and cell type changes in behavioral evolution and uncovers the evolutionary lability of sex determination genes to reconfigure the cellular makeup of neural circuits.

Single-cell type analysis of wing premotor circuits in the ventral nerve cord of Drosophila melanogaster.

To perform most behaviors, animals must send commands from higher-order processing centers in the brain to premotor circuits that reside in ganglia distinct from the brain, such as the mammalian spinal cord or insect ventral nerve cord. How these circuits are functionally organized to generate the great diversity of animal behavior remains unclear. An important first step in unraveling the organization of premotor circuits is to identify their constituent cell types and create tools to monitor and manipulate these with high specificity to assess their function. This is possible in the tractable ventral nerve cord of the fly. To generate such a toolkit, we used a combinatorial genetic technique (split-GAL4) to create 195 sparse driver lines targeting 198 individual cell types in the ventral nerve cord. These included wing and haltere motoneurons, modulatory neurons, and interneurons. Using a combination of behavioral, developmental, and anatomical analyses, we systematically characterized the cell types targeted in our collection. Taken together, the resources and results presented here form a powerful toolkit for future investigations of neural circuits and connectivity of premotor circuits while linking them to behavioral outputs.

Female preferences between incipient species of Drosophila athabasca reveal divergent closed functions, epistasis, and segregation of veiled preference alleles.

Theoretical models typically assume that speciation is driven by divergence in female preference functions for secondary sexual traits. However, little is known about how these functions diverge between incipient species. This study used song playback experiments and mate choice tests to characterize female preference functions for divergent male courtship songs between two incipient species of Drosophila athabasca complex; D. athabasca (WN) and Drosophila mahican (EA). The study focused on two male song parameters: pulses per burst (PPB) and inter-pulse interval (IPI). Preference functions for PPB were open-ended and lacked species-specificity. In contrast, preference functions for IPI revealed a closed shape, with peak preferences highly divergent between species. Thus, females imposed divergent and ongoing sexual selection on male IPI. Further work demonstrated variation in peak preferences and choosiness for IPI across genetic lines, while F1 hybrid females varied extensively in their mating preferences across different heterospecific line crosses. Finally, F2 female genotypes with foreign single-copy chromosome substitutions showed little to no change in preferences until three or four chromosomes were combined: indicating strong dominance epistasis. These results suggest "veiled" or hidden segregating female preference alleles for male speciation phenotypes. This may explain the rapid speciation observed in this species complex.

LIM-kinase 1 effects on memory abilities and male courtship song in Drosophila depend on the neuronal type.

The signal pathway of actin remodeling, including LIM-kinase 1 (LIMK1) and its substrate cofilin, regulates multiple processes in neurons of vertebrates and invertebrates. Drosophila melanogaster is widely used as a model object for studying mechanisms of memory formation, storage, retrieval and forgetting. Previously, active forgetting in Drosophila was investigated in the standard Pavlovian olfactory conditioning paradigm. The role of specific dopaminergic neurons (DAN) and components of the actin remodeling pathway in different forms of forgetting was shown. In our research, we investigated the role of LIMK1 in Drosophila memory and forgetting in the conditioned courtship suppression paradigm (CCSP). In the Drosophila brain, LIMK1 and p-cofilin levels appeared to be low in specific neuropil structures, including the mushroom body (MB) lobes and the central complex. At the same time, LIMK1 was observed in cell bodies, such as DAN clusters regulating memory formation in CCSP. We applied GAL4 × UAS binary system to induce limk1 RNA interference in different types of neurons. The hybrid strain with limk1 interference in MB lobes and glia showed an increase in 3-h short-term memory (STM), without significant effects on long-term memory. limk1 interference in cholinergic neurons (CHN) impaired STM, while its interference in DAN and serotoninergic neurons (SRN) also dramatically impaired the flies' learning ability. By contrast, limk1 interference in fruitless neurons (FRN) resulted in increased 15-60 min STM, indicating a possible LIMK1 role in active forgetting. Males with limk1 interference in CHN and FRN also showed the opposite trends of courtship song parameters changes. Thus, LIMK1 effects on the Drosophila male memory and courtship song appeared to depend on the neuronal type or brain structure.

Divergence and correlated evolution of male wing spot and courtship display between Drosophila nepalensis and D. trilutea.

Male-specific wing spots are usually associated with wing displays in the courtship behavior of Drosophila and may play important roles in sexual selection. Two closely related species, D. nepalensis and D. trilutea, differ in wing spots and scissoring behavior. Here, we compare male morphological characters, pigmentation intensity of male wing spots, wing-scissoring behavior, courtship songs, and reproductive isolation between 2 species. F1 fertile females and sterile males result from the cross between females of D. nepalensis and males of D. trilutea. The pigmentation of wing spots is significantly weaker in D. trilutea than in D. nepalensis and the F1 hybrid. Males scissor both wings in front of the female during courtship, with a posture spreading wings more widely, and at a faster frequency in D. nepalensis than in D. trilutea and the F1s. Males of D. trilutea vibrate wings to produce 2 types (A and B) of pulse songs, whereas D. nepalensis and the F1s sing only type B songs. The incidence of wing vibration and scissoring during courtship suggests that wing vibration is essential but scissoring is a facultative courtship element for successful mating in both species. The association between the darker wing spots with more elaborate scissoring might be the consequence of correlated evolution of these traits in D. nepalensis; however, D. trilutea retains wing scissoring during courtship despite having weaker pigmentation of wing spots. The genetic architecture of 2 traits differs in the F1s, consistent with maternal or sex-linked effects for spots but nonadditive effects for scissoring.

Songs and morphology in three species of the Chorthippusbiguttulus group (Orthoptera, Acrididae, Gomphocerinae) in Russia and adjacent countries.

Songs and morphology are compared between Chorthippusmiramae (Vorontsovsky, 1928) that was previously named as C.porphyropterus and two other closely related species, C.brunneus (Thunberg, 1815) and C.maritimus Mistshenko, 1951. We compare them because the calling song of C.miramae was previously shown to have song elements similar to those of other two species. One morphological character, the length of stridulatory file, appeared to be the best character to distinguish between all three species. For C.maritimus and C.miramae, we present the morphological descriptions since they are absent in the literature. We also establish the synonymy C.maritimus = C.bornhalmi Harz, 1971, syn. n. = C.biguttuluseximius Mistshenko, 1951, syn. n. In the song analysis, we analyse not only the sound but also the leg-movement pattern, which is very helpful to find a homology between various song elements. We show that the calling song of C.miramae usually contains two elements, one element being similar to the C.brunneus calling song, and another - to the C.maritimus calling song. Despite some similarities, the calling song elements in C.miramae have some peculiarities. The courtship song of C.miramae is similar to the C.brunneus song, whereas the rivalry songs of C.miramae comprise both the maritimus-like elements and the unique ones. C.miramae generally demonstrates a richer song repertoire than the other two species.

A Computational Error and Restricted Use of Time-series Analyses Underlie the Failure to Replicate period-Dependent Song Rhythms in Drosophila.

From 1980 to 1991, Kyriacou, Hall, and collaborators (K&H) reported that the Drosophila melanogaster courtship song has a 1-min cycle in the length of mean interpulse intervals (IPIs) that is modulated by circadian rhythm period mutations. In 2014, Stern failed to replicate these results using a fully automated method for detecting song pulses. Manual annotation of Stern's song records exposed a ~50% error rate in detection of IPIs, but the corrected data revealed period-dependent IPI cycles using a variety of statistical methods. In 2017, Stern et al. dismissed the sine/cosine method originally used by K&H to detect significant cycles, claiming that randomized songs showed as many significant values as real data using cosinor analysis. We first identify a simple mathematical error in Stern et al.'s cosinor implementation that invalidates their critique of the method. Stern et al. also concluded that although the manually corrected wild-type and perL mutant songs show similar periods to those observed by K&H, each song is usually not significantly rhythmic by the Lomb-Scargle (L-S) periodogram, so any genotypic effect simply reflects "noise." Here, we observe that L-S is extremely conservative compared with 3 other time-series analyses in assessing the significance of rhythmicity, both for conventional locomotor activity data collected in equally spaced time bins and for unequally spaced song records. Using randomization of locomotor and song data to generate confidence limits for L-S instead of the theoretically derived values, we find that L-S is now consistent with the other methods in determining significant rhythmicity in locomotor and song records and that it confirms period-dependent song cycles. We conclude that Stern and colleagues' failure to identify song cycles stems from the limitations of automated methods in accurately reflecting song parameters, combined with the use of an overly stringent method to discriminate rhythmicity in courtship songs.