Courtship and Other Behaviors Affected by a Heat-Sensitive, Molecularly Novel Mutation in the cacophony Calcium-Channel Gene of Drosophila

Corresponding author: Jeffrey C. Hall, Brandeis University, 415 South St., Waltham, MA 02454-9110., hall{at}brandeis.edu (E-mail)

Communicating editor: M. NOOR

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The cacophony (cac) locus of Drosophila melanogaster, which encodes a calcium-channel subunit, has been mutated to cause courtship-song defects or abnormal responses to visual stimuli. However, the most recently isolated cac mutant was identified as an enhancer of a comatose mutation's effects on general locomotion. We analyzed the cac^TS2 mutation in terms of its intragenic molecular change and its effects on behaviors more complex than the fly's elementary ability to move. The molecular etiology of this mutation is a nucleotide substitution that causes a proline-to-serine change in a region of the polypeptide near its EF hand. Given that this motif is involved in channel inactivation, it was intriguing that cac^TS2 males generate song pulses containing larger-than-normal numbers of cycles—provided that such males are exposed to an elevated temperature. Similar treatments caused only mild visual-response abnormalities and generic locomotor sluggishness. These results are discussed in the context of calcium-channel functions that subserve certain behaviors and of defects exhibited by the original cacophony mutant. Despite its different kind of amino-acid substitution, compared with that of cac^TS2, cac^S males sing abnormally in a manner that mimics the new mutant's heat-sensitive song anomaly.

THE normal forms of calcium channels exist in many forms. In vertebrates, for example, six classes of voltage-gated Ca²⁺ channels, which are distinguished by their voltage dependency and sensitivity to pharmacological agents, have been cloned (HOFFMAN et al. 1994 ; CATTERALL 1998 ). Several aberrant forms of calcium-channel polypeptides are also known. Most of these variants, the majority of which in turn are naturally occurring, exhibit unsurprising connections between altered genotypes and phenotypes. In part because functions of ion channels are ubiquitous among organisms and widely dispersed within a given animal, one imagines that mutations in the genes encoding them would obviously derange motor functions. In this regard, certain mammalian and Caenorhabditis elegans mutants speak to the whole-organismal and tissue-functional meaning of calcium-channel functions. However, the phenotypic defects associated with these channel variants are in the main neuropathologies: migraines, ataxia, lethargy, muscular dysgenesis, or myotonia (e.g., LEE et al. 1997 ; MILLER 1997 ; DOYLE and STUBBS 1998 ; JEN 1999 ; ASHCROFT 2000 ; WEINRICH and JENTSCH 2000 ). It is difficult to pit such phenotypes against discrete, measurable behaviors exhibited by wild-type humans, mice, or nematodes (although see SCHAFER and KENYON 1995 ).

Rare among calcium-channel mutants are variants of Drosophila that exhibit relatively enticing—or at least nonpathological—abnormalities of behavior. The mutations in question turned out to have occurred in an X chromosomal gene called cacophony (abbreviated cac). One of the first courtship mutants that was deliberately induced in Drosophila is the original cac variant (SCHILCHER 1976 , SCHILCHER 1977 ). This mutation (cac^S, whose allelic designation stands for "song") alters the patterns of sounds produced by the male as he extends a given wing and vibrates it near the female to produce his courtship song: Pulses of tone, instead of each containing on the order of three cycles as in wild-type songs of this species, contain approximately five cycles; pulse amplitudes are also higher than normal (SCHILCHER 1977 ; KULKARNI and HALL 1987 ; PEIXOTO and HALL 1998 ). However, the songs generated by cac males are not pathologically degraded or otherwise aberrant; they are still highly patterned (WHEELER et al. 1989 ; NEUMANN et al. 1992 ). For example, cac males produce their tone pulses at a rate of 30/sec as in wild type (BERNSTEIN et al. 1992 ), and the mutant's intrapulse "carrier" frequencies (baritone notes) are largely normal (WHEELER et al. 1989 ).

High-resolution mapping of the cac locus led both to a complicated phenogenetic picture and to a determination of the molecular etiology of the mutation: The cac-induced song defect is uncovered in flies heterozygous for this mutation and lethal genetic variants that had been independently mapped to the locus. These lethals in turn fail to complement nightblind-A (nbA) mutations, which also co-map and by themselves cause defects in visually mediated behavior and the light-elicited electroretinogram (ERG; KULKARNI and HALL 1987 ; HOMYK and PYE 1989 ; SMITH et al. 1998B ); however, cac/nbA heterozygotes are normal for courtship song and vision. (Complementation tests of songs generated by chromosomally female XX flies were permitted by turning them into males via introduction of the transformer mutation, whose general properties are reviewed by BAKER et al. 2001 .) Fine mapping of cac, nbA, and the lethal mutations permitted positional cloning of the mutated gene; it was found to encode an 1 subunit of a voltage-activated calcium channel, which was named Dmca1A (SMITH et al. 1996 ). Chromosomal lesions at the locus (which are among the lethal cac variants) rupture the open reading frame (ORF; SMITH et al. 1996 ). The original cac^S variant was shown by SMITH et al. 1998B to be a missense mutant that harbors an amino-acid substitution within a transmembrane region of the third intrapolypeptide repeat (see Fig 1B). A cac^nbA mutation was found to have suffered a premature stop mutation in an alternatively spliced cassette with respect to a cac mRNA isoform whose expression is enriched in the visual system (SMITH et al. 1998B ; see Fig 1B).

View larger version (31K): In this window In a new window Download PPT slide   Figure 1. Sequence analysis of cacophony. (A) Proximity of the cacTS2-defined proline (P) to Dmca1A's EF hand (see tall thin box three residues downstream from the C-terminal end of that motif). An excerpt of amino-acid sequence for the CAC polypeptide (GenBank accession no. U55776) depicts the region containing the deduced region of the EF hand (cf. SMITH et al. 1996 ). This Dmca1A sequence is compared (as in SMITH et al. 1996 ) to portions of amino-acid sequences for a rat-brain calcium-channel 1 subunit (rbE2) and a polypeptide of this kind from Drosophila (Dmca1D) encoded at a locus separate from cac (ZHENG et al. 1995 ); identical amino acids are indicated by dots. (B) Diagram of a generic calcium-channel 1 subunit used to depict sites of intra-Dmca1A changes associated with cacophony mutations or post-transcripitional modification of cac RNA. Thus Dmca1A contains four intrapolypeptide repeats (I IV), each of which courses back and forth between the cellular membrane (green) six times (S1 S6, numbered in the N- to C-terminal direction). The amino-acid substitution in cacTS2 (TS2) is as described for A (also see Table 1). The approximate site of the F I missense mutation in cacS (S) is depicted where this substitution occurred within IIIS6 (cf. SMITH et al. 1998B ). A cac/nightblind mutant (nbAH18) is accounted for by a nonsense mutation in a region of the cac ORF shortly 3' to that which encodes IS6, which is alternatively spliced (with respect to two mutually exclusive cassettes, thus "alt.cass."), such that only a "visual-system-enriched" CAC isoform would be eliminated by cacnbAH18 (SMITH et al. 1998B ). Among the RNA-edited nucleotides within cac's transcript that lead to amino-acid changes, with reference to the genomic ORF (see text), a newly defined such site is shown for a region of the transcript that encodes residues located between IS3 and IS4 (see Table 1).

Additional findings stemming from analyses of the original cacophony mutants—along with other of the older ones and a newly induced mutation at the locus—have further broadened the phenotypic significance of the gene's action: cac^S (but not cac^nbA mutations) causes convulsions and other locomotor anomalies at an intermediate/high temperature (37°) and much faster-than-normal loss of all motor functions at an extremely high one (46°; PEIXOTO and HALL 1998 ). Induction of mutations that enhance temperature-sensitive (TS) paralytic defects of a comatose mutation (comt^ST53) in a gene encoding an N-ethylmaleimide-sensitive fusion protein (NSF) led to recovery of a new cac allele (DELLINGER et al. 2000 ). It was subsequently shown that this cac^TS2 mutant is by itself heat sensitive for paralysis (at a temperature milder than that required to paralyze cac^S) and causes failure of synaptic function at neuromuscular junctions (KAWASAKI et al. 2000 ).

Additional molecular findings have been obtained from analysis of the cacophony gene and its expression: The primary cac transcript was inferred by analysis of multiple cDNAs to be subjected to RNA editing (SMITH et al. 1996 ), one of the first examples of this process in Drosophila and for a gene that encodes a voltage-sensitive ion channel (cf. SEEBURG 2000 ); the presumed adenosine-to-inosine edits (observed as a-to-g substitutions at the level of cDNA heterogeneity) were subsequently shown to occur in actual flies via extractions of RNA and RT-PCR analysis (SMITH et al. 1998A ; PALLADINO et al. 2000 ). Assessments of Dmca1A sequences in Drosophila derived from natural populations revealed a low level of polymorphism; five noncoding sites were found to be variable among Drosophila melanogaster lines, although no amino-acid substitutions in the channel polypeptide were observed between this species and its close relative, D. simulans, within 1 kb of genomic sequence. However, courtship-song analysis of the melanogaster lines revealed a significant association between pulse amplitude and one of the polymorphic cac sites (PEIXOTO et al. 2000 ). As this site is within an intron, we entertained the possibility that it could be in linkage disequilibrium with a nonsynonymous (amino-acid-changing) polymorphism elsewhere in the gene. These preliminary interspecific Dmca1A comparisons were made in light of the fact that the cac^S mutation changes the normal song such that visual traces of the mutant sounds look to the human observer's eye as if they could be those generated by males of another Drosophila species (e.g., COWLING and BURNET 1981 ; HOIKKALA and LUMME 1987 ; TOMURA and OGUMA 1994 ), given the "nonpathological" nature of the cac^S phenotype. This supposition about the possibility of naturally occurring calcium-channel variations contributing to song variations, and even their evolutionary divergence, may be more than empty speculation because certain components of the song differences between D. virilis and D. littoralis have been genetically mapped to a region of the X chromosome that includes the cacophony locus (PAALLYSAHO et al. 2001 ).

Against this background, we wondered whether the new cac^TS2 mutant would exhibit a "patterned" song abnormality or a phenotype that parts company with that kind of defect in either direction. cac^TS2 flies are defective in their gross locomotion by definition (DELLINGER et al. 2000 ), but this isolation phenotype means that the songs of cac^TS2 males could fall anywhere in the range from severely anomalous to normal. Whatever that behavioral outcome might be, along with those obtained from testing visually mediated responses, we also aimed to correlate the intragenic cacophony change with the new mutant's phenotypes, concentrating on behaviors that extend well beyond the doleful defect by which this calcium-channel variant was identified.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

D. melanogaster strains and basic fly handling:
Flies were raised on a sucrose/cornmeal/yeast medium supplemented with the mold inhibitor Tegosept. Most cultures were maintained in 12 hr:12 hr light:dark cycles (12:12 LD) at 25° and 70% relative humidity. Flies emerging from the cultures were collected as <1-day-old adults under ether anesthesia. Males for courtship or longevity tests were stored singly in food vials; females paired with males for such tests, and males subjected to other kinds of phenotypic characterizations, were stored 10–15 flies per vial. To determine whether cac^TS2 flies would be sensitive to temperature changes, they (and parallel controls) were also reared at 18° and 29° in incubators programmed for 12:12 LD.

The strains from which flies were taken for behavioral (and in some cases physiological) tests were Canton-S wild type, cac^TS2, cac^S, w comt^ST53 (the white-eyed, comatose-mutated, cac⁺ strain that had been mutagenized by DELLINGER et al. 2000 to induce cac^TS2), comt^ST53, comt^ST17, cac^TS2, comt^ST17, and cac^S comt^ST17. The cac^S strain was the original one, in which factor(s) causing mediocre mating-initiation latencies (SCHILCHER 1977 ) were still present but did not affect the mutant male's courtship song (KULKARNI and HALL 1987 ). The comt^ST17 and comt^ST53 mutants (for which "ST" stands for "sensitive to temperature") are also known as comt¹ and comt⁴ according to FlyBase (http://flybase.bio.indiana.edu), which provides a soft-copy paper trail about the origin and properties of these NSF mutants. The cac^S, comt^ST17, comt^ST53, and cac^TS2 strains (for the latter, one of them) are true-breeding stocks in which males are hemizygous for any one of these X chromosomal mutations and females are homozygous for it. True-breeding w comt^ST53, cac^TS2 comt^ST17, and cac^S comt^ST17 stocks, and a separate cac^TS2-including stock (a source of males for some of the song recordings) were each maintained in a situation in which hemizygous mutant males mated with attached-X females [C(1)DX, y f/Y].

Molecular characterization of cac^TS2: Obtaining DNA and cDNAs:
DNA corresponding to the 34 exons that are distributed over 45 kb at the cac locus was obtained in two ways:

• By cDNA synthesis, for which, in a given such operation, total w comtST53 or cacTS2 RNA was extracted from an homogenate of 50 whole adults (from 4- to 6-day-old males of either genotype) using TRIzol reagent according to the manufacturer's instructions (GIBCO BRL, Rockville, MD). A total of 5 µg of RNA (from a given extract) was reverse transcribed with random hexamer primers using the ThermoScript RT-PCR system (GIBCO BRL).

• By obtaining genomic DNA, for which 75 4- to 6-day-old whole-adult males (w comt^ST53 or cac^TS2) were collected and immediately frozen with liquid nitrogen. They were then homogenized in the following buffer: 5% sucrose, 80 mM NaCl, 100 mM Tris pH 8, 0.5% SDS, 50 mM EDTA.

The homogenate was treated with 8 M KOAc, and phenol extractions were performed to separate out the nucleic acids. DNA was precipitated from the supernatant using isopropanol and exposed to two 70%-ethanol washes. The pellet was resuspended in TE (10 mM Tris-Cl, 1 mM EDTA) overnight and then treated with RNase A for 3 hr at 50°–60°.

PCR and DNA sequencing:

Primers were designed to amplify 100- to 1000-bp segments of genomic DNA and cDNA products for sequencing; 26 such primer pairs were designed, which in sum covered the entirety of the 5.6-kb Dmca1A ORF within the cac locus (cf. SMITH et al. 1996 ; PEIXOTO et al. 1997 ). Primers were synthesized by and purchased from Integrated DNA Technologies (Coralville, CA). PCR was carried out in a PTC-100 (MJ Research, Waltham, MA) for 30 cycles (94° for 1 min, 55° or 60° for 1 min, and 72° for 1 min). Reactions were performed in 50-µl volumes that included 0.4 mM primers (0.2 mM each forward and reverse oligonucleotides), 0.2 mM dNTP mix, 10x PCR buffer, and Taq DNA polymerase (Roche Molecular Biochemicals, Indianapolis) or 0.4 mM primers, 0.2 mM dNTP mix, 10x PCR buffer without Mg²⁺, 1.5 mM MgCl₂, and Taq DNA polymerase from another source (Promega, Madison, WI). PCR products were analyzed on 1 or 1.2% agarose gels and then purified with the QIAQuick spin PCR purification kit (QIAGEN, Valencia, CA) for direct sequencing. Single-stranded sequencing reactions in both directions were performed in 20-µl volumes that included 0.2 mM primer (forward or reverse), 5 µl eluant containing the purified PCR product, and 8 µl Prism Dye-Deoxy terminator cycle sequencing kit mix (Perkin-Elmer/Applied Biosystems, Foster City, CA). Such reactions were carried out in the PTC-100 for 24 cycles (96° for 30 sec, 50° for 20 sec, and 60° for 4 min). They were then purified in 50 mg/ml Sephadex G-50 fine DNA grade columns (Pharmacia Biotech, Piscataway, NJ) and electrophoresed in a Perkin-Elmer/Applied Biosystems model 373 Stretch XL DNA sequencer. PCR products to be cloned before sequencing were purified with the QIAEX II gel extraction kit (QIAGEN). Overnight ligations at 4° were set up using the pGEM-T Vector System I (Promega). Subcloning-efficiency DH5-competent cells (GIBCO BRL) were used for bacterial transformations, as plated in Luria broth agar culture dishes containing 100 µg/ml ampicillin, 0.05 µg/µl X-gal (Fisher Scientific, Pittsburgh), and 110 mM isopropyl thiogalactoside. Colonies were isolated and grown in overnight cultures. DNA was then extracted and purified using the QIAPrep spin miniprep kit (QIAGEN). Restriction enzyme digest with EcoRI (Promega) was performed to ensure that the DNA contained the fragment of interest. During the first stage of obtaining DNA sequence data from various subsets of the cacophony gene, as carried by w comt^ST53 or cac^TS2 flies, PCR products were cloned before sequencing (see above). Direct sequencing was performed for certain intragenic regions only to confirm or to deny putative nucleotide differences (between the two genotypes just given) that were observed in the first-stage sequencing operation. MacVector 6.0 software (Accelrys, San Diego) was used for analyses of nucleotide sequences and alignments as well as for conceptual translations (to generate the Dmca1A polypeptides inferred to be produced by w comt^ST53 or cac^TS2 flies). The investigator performing these molecular operations (B. Chan) deliberately did not consult with R. W. Ordway and co-workers as various components of the sequence data were emerging in parallel at our and his institutions (see RESULTS).

Behavioral observations and analyses: General courtship and mating performances:
Males were reared and stored at 25° as usual (see above), except that approximately one-half of the flies of a given genotype had their wings completely clipped off with fine-tipped scissors; then they and their intact brothers were stored individually in food vials. Single pairs of flies were placed in a courtship-observation apparatus at 25°; this device, known as a "mating wheel" (HALL 1979 ), contains 10 chambers (diameter, 1 cm; height, 1 cm), which are formed by rotating separate discs of the wheel such that 20 "half-chambers," 10 containing an individual male, the rest an individual female, are merged at one moment. The times elapsing between the moment of pairing and initiations of courtship and of (subsequent) mating were recorded, as were durations of copulation.

Courtship-song recordings and analyses:

Males to be recorded for sound production in the presence of females were reared and stored individually as above. Before recording, the male-containing vials were pretreated at 20°, 25°, or 30° for 30–60 min before being paired with individual females at the same temperature used for a given pretreatment. Wild-type virgin females were reared and stored as noted in the previous subsection, except that their wings were clipped off upon collection so that only the wing-vibrational sounds generated by a courting pair would emanate from males. Single male-female pairs were recorded at a given temperature for 5 min or until they mated; in the latter case, <3-min recordings were excluded from subsequent analyses. Fly-produced sounds were picked up by an Insectavox (GORCZYCA and HALL 1987 ) and interfaced with a Sony Hi8 video/audio camera. The temperature was monitored inside the Insectavox, before and after each recording, to verify that the temperature did not fluctuate more than 1°; if it did, that record was not analyzed. Songs that passed muster were digitized and logged using LifeSong software (BERNSTEIN et al. 1992 ), essentially as in VILLELLA et al. 1997 and ANAND et al. 2001 ; singing bouts with 3 pulses were not logged. During the 30° recordings, some of the mutant males fell on their backs and exhibited seizure-like activities; these produced sounds that could be misinterpreted as song sounds. Thus, it was necessary to observe the video record while logging a song produced by such a male, to confirm that the sounds entered at the keyboard into the male's soft-copy file were exclusively song pulses.

For each logged song file the following parameters were computed: cycles per pulse (CPP), pulse amplitude (in arbitrary units, but scaled in the same way among files as in BERNSTEIN et al. 1992 ), intrapulse frequency (IPF), and interpulse intervals (IPI). IPI cutoffs were applied as in PEIXOTO and HALL 1998 ; thus, for example, a >90-msec interval of silence between a pair of pulses at 20° was defined as an interbout interval.

Distributions of song bout types, varying as to their CPP values, were generated as follows: Using the song file obtained from a given male, the average CPP for any bout that produced at least eight pulses was computed; five categories were defined in terms of average pulse cyclicity per bout (lowest: 2 CPP; highest: >5); then proportions of bouts falling into each category were determined for that individual's record (see Fig 4). Subsequently, the mean proportion (±SEM) of each category's content among males of a given genotype (e.g., what part of 100% is the "2" or ">5" category in the average male?) was computed (see Table 3).

View larger version (41K): In this window In a new window Download PPT slide   Figure 2. Examples of pulse trains produced by a (A) wild-type (WT) and a (B) cacTS2 male. Both flies had their courtship wing vibrations recorded at 30° (cf. Fig 3 and Fig 4). These snatches of song were generated by application of subroutines within the LifeSong software (elements of which, as referred to in MATERIALS AND METHODS, digitize the audio record of a given courtship). The abscissas show time values representing the actual recording time (starting at 0 sec for a given male-female pair). The two bouts shown each occurred over 0.5 sec of time. The ordinate represents arbitrary measurements of pulse loudness (against background/no-fly noise levels that fluctuated between -1 to +1). Average numbers of cycles per pulse for the WT and the mutant train: 2.9 and 5.0, respectively.

View larger version (24K): In this window In a new window Download PPT slide   Figure 3. Courtship-song parameters. With respect to the three different recording temperatures (see abscissas), the numbers of courtships recorded are in parentheses accompanying the genotype labels within A and are given in order according to the three recording temperatures. These results revealed the basic differences between cacTS2 and normal songs: higher CPP and amplitude of sounds for the mutant (see A and B), and slightly but significantly longer-than-normal IPIs (see D). (A) Cycles per pulse, an appreciation of which can be gleaned from Fig 2. Here, the gross overall average CPPs for songs of a given male were computed and used to generate the means (±SEM) plotted for males of a given genotype singing at a given temperature. A one-way ANOVA was performed on log-transformed CPP values, using the data from all three temperatures with genotype as the main effect, and it showed significant differences among genotypes (F[8,79] = 8.13, P < 0.0001). Subsequent planned pairwise comparisons revealed no significant differences between CPP at 20° vs. 30° for wild-type (WT) males or w comtST53 males (all P 0.05). However, cacTS2 males exhibited significantly greater CPPs at 30° compared with 20° (P < 0.05). At 20°, males of all three genotypes yielded the same CPP (per male) averages (all P < 0.05). At 25°, cacTS2 males were "already" generating higher CPPs compared with males of the cac+ "parental" strain (w comtST53) or with WT males (P < 0.05); however, the values for cacTS2 were not different when comparing the 25° with the 20° records. At 30° cacTS2 values were significantly different from both the w comtST53 and the WT ones (all P < 0.05). A two-way ANOVA was performed on the CPP data, with genotype (GENO) and temperature (TEMP) as the main effects; for CPP there were GENO and TEMP effects (P < 0.0001 and P < 0.0001, respectively), but no interaction effect (GENO x TEMP) between the two (P = 0.17). (B) Amplitude, an arbitrarily scaled parameter (as exemplified in Fig 2). A one-way ANOVA was performed on these pulse-loudness indicators, with genotype as the main effect, and it showed significant difference among genotypes (F[8,79] = 5.95, P < 0.0001). Subsequent comparisons revealed that all genotypes had the same song amplitude at 20° (all P 0.05), whereas, at 30°, the pulse amplitudes of cacTS2 male songs were significantly higher than those of w comtST53 or WT males (all P < 0.05). Note that amplitudes monotonically increased with temperature for the two mutant types but did not for WT songs. (C) Intrapulse frequency, representing the average carrier frequencies in Hertz, computed from Fourier analyses of all pulses within a given song record (cf. WHEELER et al. 1989 ). A one-way ANOVA was performed on the IPFs, with genotype as the main effect, and it showed significant difference among genotypes (F[8,79] = 6.70, P < 0.0001). Subsequent comparisons revealed that these carrier frequencies did not change with temperature for any of the three genotypes (all P 0.05), although the IPFs were different among the male types at 30° (all P < 0.05). (D) Interpulse intervals, representing the brief timespans of relative "silence" between a given pair of pulses within a song-bout train (see Fig 2). A one-way ANOVA was performed on log-transformed IPIs (in milliseconds), with genotype as the main effect, and it showed significant difference among genotypes (F[8,79] = 52.53, P < 0.0001). Subsequent pairwise comparisons revealed that all genotypes showed the same inverse relationship between IPI and temperature. All three male types sang with IPIs that were not different at 20° (all P 0.05) but were significantly different from each other at 30° (all P < 0.05). Increased CPPs at 30° contributed to these slightly longer-than-normal IPIs of cacTS2 and w comtST53 (higher such values reflecting longer pulses); this is because a given IPI was measured between the temporal centers of a pair of successive pulses. [LifeSong produces a pulse "envelope" from each such sound and computes the timespan between the peaks of two adjacent envelopes to specify that IPI (cf. VILLELLA et al. 1997 ).]

View larger version (28K): In this window In a new window Download PPT slide   Figure 4. Examples of cycles-per-pulse bout types in songs from individual males. These distributions are based on the percentages of pulses for a given song that fell into the following CPP categories: 2, >2–3, >3–4, >4–5, and >5 (see legend to Table 3 for details, and note that the abscissa labeling here gives shorthand indicators for the five CPP categories). Histogram examples are depicted for males of three genotypes, whose songs were recorded at 20° or 30°. For the wild-type (WT) males, the distributions at both temperatures (representing a total of four different song recordings) show that most of the pulses were between 3 and 4 CPP (cf. Fig 3A). For the cacS songs exemplified, the distributions are shifted toward higher CPPs, independent of temperature. For the cacTS2 examples, the distributions at 20° are similar to the WT histograms (particularly for the mutant case on the left), but are shifted substantially to the right at the higher temperature.

Nonreproductive behaviors and responses to stimuli: Phototaxis:
A Y-tube apparatus (KULKARNI and HALL 1987 ) was used to measure fly movements toward light. One clear plastic arm of the Y and its stem were thoroughly wrapped with black tape; the other arm was so wrapped except at the tip. An incandescent light source (at the end of a fiber-optics lead) was placed 2–3 cm from the open tip of that arm (resulting in an 2000-lux stimulus). Five to 10 males of a given genotype, which had been raised and stored as adults at 25° (see above), were placed in the stem of the Y and allowed to walk toward the light for 2 min at room temperature (22°–23°). At the end of this time, numbers of flies distributed in each arm and those remaining in the start tube were counted. A second set of phototaxis measurements was obtained after exposing a different set of flies (of the various genotypes) to 29° for 20 min and then testing them at room temperature immediately afterward.

Electroretinograms:

These light-elicited voltage changes were recorded extracellularly, basically as in RENDAHL et al. 1992 , RENDAHL et al. 1996 , as augmented and modified somewhat by STOWERS and SCHWARZ 1999 . Moreover, one set of cac^TS2 vs. cac⁺ comparisons was made at room temperature (22°–23°) and another set in a recording chamber preheated to 30°–31°.

Locomotor activity:

Individual males of a given genotype, which had been raised and stored as adults at 25° (see above), had their general locomotor activity measured at room temperature by placing a given fly in a cylindrical plastic chamber (diameter: 1 cm, height: 1 cm), which was divided across the diameter by a straight line. After introducing the fly into this chamber and allowing it a 3-min "accommodation" period, the number of times it crossed the line in the next 2 min was counted (cf. KULKARNI and HALL 1987 ; VILLELLA et al. 1997 ). Additional line-crossing counts were made (for two separate groups of males) at 25° and at that temperature immediately after exposure of males to 29° for 20 min.

Responses to mechanical shock:

Males that were 3- to 5-day-old Canton-S wild type, cac^TS2, and comt^ST53 and had been reared at 18°, 25°, or 29° (and stored at these respective temperatures) were individually placed in a series of empty culture vials at 25° and vibrated using a Vortex Genie 2 (Fisher Scientific) at top speed for 20 sec. Recoveries were measured by noting the amount of postvortexing time required for the first fly within a given genotypic group (n = 10 for each test) to regain its ability to crawl along the inside surface of the vial; the other flies within the group rapidly followed suit. The test for each genotype was repeated 10 times, using different sets of 10 flies each time. A second set of vortexings and recovery-time assessments was made after first exposing the flies to 29° for 30 min.

Statistics:
JMP Version 3.1.5 (SAS Institute, Cary, NC) software (for Macintosh) was used to analyze data from the behavioral tests and recordings. Statistical analyses were carried out for the different kinds of metrics after transforming them to approximate normal distributions by testing the Studentized residuals (cf. VILLELLA et al. 1997 ; ANAND et al. 2001 ). These data were then subjected to ANOVA to compare the numerical results influenced by the various genotypes. Subsequent planned pairwise comparisons were performed, and 's were adjusted appropriately for experiment-wise error (cf. SOKAL and ROHLF 1995 ). ERG parameters were analyzed nonparametrically using Kruskall-Wallis tests.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Dmca1A nucleotide-sequence divergences from the norm in the cac^TS2 mutant:
We sequenced cDNAs and segments of genomic DNA from the cac^TS2 mutant and compared the results to those obtained from the cac⁺ allele carried on the w-marked, comt^ST53-bearing X chromosome in the strain used to induce this cacophony mutation. Several differences were found (Table 1), most of them involving synonymous base-pair changes or disparities between the published sequence for Dmca1A coding information (SMITH et al. 1996 ; PEIXOTO et al. 1997 ; GenBank accession no. U55776) and both the cac^TS2 and cac⁺ (w comt^ST53) sequences. One mutant vs. normal difference was found for which the amino acid specified is different between cac^TS2 and cac⁺(w comt^ST53), and the codon in the latter (normal) sequence is identical to the published one at this position (Fig 1A). A tcg codon was found in cac^TS2, which would substitute serine for proline (ccg). This conclusion is fully consistent with mutant vs. normal sequence data generated independently by R. W. ORDWAY (personal communication), in whose laboratory cac^TS2 was induced (DELLINGER et al. 2000 ). The proline present in the relatively C-terminal region of CAC referred to above is three residues downstream of a calcium-channel motif known as the EF hand (Fig 1). This is a stretch of 30 amino acids (Fig 1A) found in many different kinds of calcium-binding proteins (LEWIT-BENTLEY and RETY 2000 ; BURGOYNE and WEISS 2001 ).

One further case of CAC sequence heterogeneity was encountered in conjunction with the molecular characterization of cac^TS2: An atg (methionine) codon was found in a mutant-derived cDNA (Table 1), which at first blush was different from ata (isoleucine) at the corresponding position in cac⁺(w comt^ST53) or in the archived sequence (GenBank accession no. U55776). However, this turned out to be a case of cDNA heterogeneity (see legend to Table 1), which corresponds to a site within the extracellular loop between the third and fourth transmembrane segments of the first intra-Dmca1A repeat (Fig 1B). This feature of the sequencing results implies an additional instance of adenosine-to-inosine RNA editing (observed as an a-to-g change at the level of cDNA analysis)—one that happened not to be encountered in the earlier cases of sequencing library-derived or RT-PCR'ed cDNAs involving the cac ORF (SMITH et al. 1996 ; PEIXOTO et al. 1997 ; SMITH et al. 1998A ; PALLADINO et al. 2000 ).

General features of reproductive performances:
Courtship-initiation latencies, mating-initiation ones, and mating durations were measured to assess the overall reproductive behavioral performances of cac mutant males, compared with those of genetically normal ones and males hemizygous for comt^ST53. Half of the tested males of a given type had their wings removed to assess the contribution of courtship song to female receptivity. For example, if an intact mutant male type exhibited a longer-than-normal mating-initiation latency (in part, a measure of the wild-type females' receptivity), this subnormality would not be solely the result of any singing abnormality that the male might exhibit (see Courtship song) if the wingless individuals were also less successful than wingless wild-type males (see below).

Proportions of males that courted and mated are noted in Table 2. The percentage-courted values are all rather high, although slightly lower overall for the wingless males and for the w comt^ST53 males of either type. The w mutation impairs Drosophila's optomotor behavior (by eliminating screening pigment from the compound eye), and this visual-response defect is reflected in subnormal "tracking" of females by white-eyed males (e.g., COOK 1980 ); so this comatose mutation may not be the cause of the mild decrements shown in Table 2. The mating success of cac^TS2 males alone was slightly subnormal for the intact flies and more markedly impaired for the wingless version of this mutant (Table 2), i.e., almost threefold lower than the percentage mated (within 60 min) for wild-type or comt^ST53 males (implying that the w⁺, wingless version of the latter mutant mated as well as the corresponding normal males). The results for this cacophony mutant are similar to those obtained in flies expressing the original mutation in this gene: Wingless versions of such males exhibited relatively poor mating performance when tested in parallel with similarly dewinged wild-type males (SCHILCHER 1977 ; KULKARNI and HALL 1987 ).

In the current experiments, the w comt^ST53 males performed worst of all when intact or dewinged: only one mating in 42 trials (which happened to be achieved by a wingless form of this double mutant). This speaks to the latency values presented in Table 2. Times elapsed between pairing the flies and initiation of courtship were low for all male types except w comt^ST53 (latency values for the latter males, in both winged and wingless forms, were significantly longer than those of wild type). With regard to mating initiation, cac^TS2 males performed well with respect to courting pairs that did copulate (latency values for the wingless form of this mutant "look long" in Table 2 but were not significantly different from wingless wild type). The longest mating-initiation latencies recorded (Table 2) were for wingless comt^ST53 males (although, in their w⁺ form, they did not perform significantly worse than wild type, and the 55-min value for the w-impaired version of this mutant is from an n of 1). As expected (GREENSPAN and FERVEUR 2000 ), winglessness on the part of courting males, whatever their genotype in these experiments (Table 2), led to relatively mediocre mating performances both in terms of percentages of copulations achieved and nominally stretched out latencies (the mating-initation times for wingless wild type or comt were significantly longer; that for wingless cac^TS2 was statistically equivalent to the corresponding wild type, notwithstanding the twofold longer value for these mutant males).

Measurements of copulation durations revealed certain mutant peculiarities. cac^TS2 males exhibited 20% shorter-than-normal durations (Table 2), which are typically in the range of 15–20 min (e.g., LEE et al. 2001 ). However, these mutant vs. wild-type values were not significantly different (see legend to Table 2), and the nominal subnormality for cac^TS2 is not a general one for variants involving the locus; cac^S males exhibited 30% longer mating times compared with matched controls (a significant difference in this case: Table 2). Mutant vs. wild-type males were "matched" experimentally (see Table 2 legend), but background genotypic differences may have been partly responsible for the disparities. In this regard, males carrying the comt^ST53-bearing X chromosome in which cac^TS2 was induced also showed shorter-than-normal mating durations (the former mutation alone leading to values that were significantly less than those timed for wild-type male-female pairs). Therefore, it could be that the nominal shortening associated with cac^TS2 is a genetic-background effect or that this comatose mutation (located on an X from which the new cacophony mutation had been crossed away) leads to a copulation phenotype similar to that nominally caused by cac^TS2. Incidentally, the matings performed by either cac^TS2 or comt^ST53 males were fertile in each case for which fecundity of the relevant female was monitored (n = 12 and 8, respectively).

Courtship song:
To ask whether cac^TS2 affects a refined behavioral character, compared with its gross paralysis at high temperature (DELLINGER et al. 2000 ) and subnormal features of aggregate courtship performances (this report), outputs from the wing vibrations of courting mutant males were recorded. Such flies were found to exhibit higher-than-normal numbers of intrapulse cycles (CPP) among their song sounds (as exemplified in Fig 2B). This mutant character was temperature dependent, in that cac^TS2 CPPs were 25% higher at 30° (the warmest condition employed) compared with 20° or 25°; at the two lower temperatures, the mutant's CPPs were 10–15% higher than those of wild-type males, but at 30° cac^TS2 values were 35% higher than normal (Fig 3A). Implicitly, the cac⁺ CPPs were largely invariant over the 10° range, as expected (PEIXOTO and HALL 1998 ). Other such controls came from recordings of comt^ST53 and comt^ST17 males, which yielded CPP values that varied only slightly as a function of temperature; actually, the values for comt^ST53 were 8% higher at 30° compared with those at 20° (as plotted in Fig 3A), while CPPs for comt^ST17 males changed not at all over this thermal range (see Fig 5). The pulse amplitudes of cac^TS2 males were also heat sensitive (Fig 3B), exhibiting a steady increase (overall, 50%) as the temperature was raised, compared with those of both control types (which varied almost not at all from 20° 25° and at 30° increased in a less-marked manner, compared with cac^TS2, for comt^ST53 only). Therefore, in warm temperatures cac^TS2 males show an aberrant song phenotype very similar to the song abnormalities exhibited by the cac^S mutant at all temperatures that have been applied (SCHILCHER 1977 ; KULKARNI and HALL 1987 ; PEIXOTO and HALL 1998 ; SMITH et al. 1998B ). That is, the original cacophony mutant gives higher-than-normal CPP and amplitude values in cool, mild, or relatively warm conditions.

View larger version (14K): In this window In a new window Download PPT slide   Figure 5. Cycles-per-pulse values from songs of single and double mutants. These songs were analyzed for the four song parameters, but only the CPP values are presented (cf. Fig 3A, from which the cacTS2 data are replotted here). CPPs of the cacS comtST17 and cacTS2 comtST17 double mutants were compared to those within songs of comtST17 and cacTS2 males and recorded at the different temperatures shown on the abscissa. A one-way ANOVA was performed on log-transformed CPP values (using the data from all three temperatures) and revealed significant differences among genotypes (F[11,85] = 9.11, P < 0.0001). Subsequent pairwise comparisons, with genotype as the main effect, showed that CPPs at 20° vs. 30° remained the same for all genotypes (all P 0.05) except for the increase exhibited by cacTS2 at that elevated temperature (P < 0.05). Songs of cacS comtST17 males had higher CPP values across all three temperatures, compared with comtST17 (all P < 0.05). In contrast, the cacTS2 comtST17 doubly mutant type generated CPPs higher than those of comtST17 at 30° but not significantly different at 20°; yet the songs of these two male types did not approach the polycyclicity exhibited by cacS comtST17 (P < 0.05 for all temperatures). The cacTS2 males gave CPPs at 30° that were similar to those of the cacS comtST17 double mutant (P 0.05).

Elements of these earlier studies showed that the carrier frequencies for cac^S pulses (IPF) were neither abnormal nor varied appreciably with temperature (PEIXOTO and HALL 1998 ). However, cac^TS2 males tended to give lower-than-normal IPFs; the disparity from wild-type values (25% lower for cac^TS2 IPFs) was greatest at 30° (Fig 3C). Rates of pulse production speed up as the temperature is raised, which harks back to the first song study performed on D. melanogaster (SHOREY 1962 ; also see PEIXOTO and HALL 1998 ). Against this background, cac^TS2 males, along with control wild-type and comt^ST53 males, were expected to (and did) exhibit shorter IPIs as the temperature was raised from 20° to 30°. At the latter temperature, only interpulse-interval values differed among genotypes (see legend to Fig 3), but IPIs for the mutants were not dramatically longer than normal.

With regard to the effects of genotype and temperature on cycles per pulse, a two-way ANOVA revealed effects of both variables but no "interaction effect" (see legend to Fig 3). For this reason, and owing to more general concerns about appreciating the effects of cacophony mutations on song-pulse qualities, it occurred to us that gross values for the CPP song parameter (normally in the range of 3–4) do not adequately reveal the anomalous pulse polycyclicity caused by cacophony mutations (which led to overall average CPPs in the range of 4.5–5.0, referring for cac^TS2 to courtship at the nonpermissive temperature). In this regard, while logging mutant vs. normal song records one perceives dramatically abnormal song bouts for cacophony males—the frequent occurrence of pulse trains that are largely polycyclic—whereas wild-type records rarely include such trains. Thus it is subjectively crystal clear when one logs a cac^S song (recorded at "any" temperature) or a cac^TS2 one (at 30°) that the visual display of courtship sounds came from recording the song of a mutant as opposed to a wild-type male. Nonetheless, we performed a newly conceived bout-distribution analysis on the songs stemming from effects of certain mutant vs. normal genotypes. For this, the computer extracted by objectively preset criteria the various "average CPP" bout types from a given song record, which had been initially digitized merely by marking pulse locations as opposed to indicating anything about their qualities at that stage of the operation (a remark made because of bias that could come into play, as implied above). As a result of these bout analyses, average cycles per pulse per song train were displayed as histograms whose abscissas varied from a mean-CPP category of 2 to one representing an average >5. Examples of wild-type and cac-mutant songs from individual males displaying intra-fly variations in CPP bout types are in Fig 4. The mutant distributions referring to cac^TS2 at 30° or cac^S at that temperature or 20° are substantially skewed to the right compared with the wild-type plots at both temperatures.

Table 3 shows bout-analysis results from all the mutant and normal song records for which proportions of bouts falling into the five CPP categories were averaged among males of a given type. (For example, what proportion of an individual cac^TS2 male's bouts fell into a given category? Then, what was the average proportion for that category among such mutant males whose songs were recorded at a given temperature?) Highlights of these tabulated findings are that only 7 and 6% of song bouts from the average wild-type male fell into the highest category (>5 CPP) from the 20° and the 30° recordings, respectively. In contrast, the corresponding values for cac^TS2 at these two temperatures were 11 and 36%. A meta-analysis of cac^S songs recorded over a wide temperature range (PEIXOTO and HALL 1998 ), but not then subjected to bout breakdowns, revealed that 53–55% of this mutant's bouts fell into the highest (>5 CPP) category after 20° and 30° recordings were analyzed appropriately (Table 3).

Recall that cac^TS2 was induced via its interaction with a comatose mutation. We therefore tested males whose X chromosomes were mutated at each locus. Interestingly, cac^TS2 comt^ST17 males generated song pulses whose CPP values were squarely within the normal range (cf. Fig 3A) at all temperatures (Fig 5), i.e., a suppressive effect of the latter mutation's effect vis à vis that of the former alone (cf. Fig 3A). No interaction could be gleaned between cac^S and this comt mutation (Fig 5), because CPPs of the doubly mutant males were relatively high and cacophony-like at all temperatures (cf. PEIXOTO and HALL 1998 and Fig 3A).

Visually mediated responses of flies or parts thereof:
Given the visual-response abnormalities exhibited by certain cacophony mutants, cac^TS2 was tested for phototaxis by applying a Y-tube device that had previously been used to uncover the photophobic behavior (alluded to in the Introduction) that is caused by cac^nbA mutations (KULKARNI and HALL 1987 ; SMITH et al. 1998B ). However, cac^TS2 was revealed to be photophilic in tests for which the flies either were chronically at low temperature or were returned to that condition after exposure to 29° (Table 4). The latter experiment caused relatively high numbers of cac^TS2 flies to remain in the phototaxis "start tube" (see MATERIALS AND METHODS); this would seem to reflect generally poor locomotion caused by the higher temperature (see General features of locomotion) as opposed to a cac^TS2 phototactic defect, let alone a photophobic one. In any case the proportions of cac^TS2 vs. wild-type flies that did not move toward either light or darkness (upon postheating light stimulation) were not significantly different (see legend to Table 4). In contrast, a severe locomotor impairment was observed in a negative-control test performed on the semisick comt^ST53 mutant (Table 4): 40–90% of such flies remained at the start at low temperature, and all of them did after exposure to the higher one (however, this comatose mutant was not paralyzed after 29° treatment).

With regard to another kind of light-induced response, room-temperature ERG recordings from cac^TS2 flies (n = 3) gave normal-looking tracings (compared to three wild types) in terms of the shapes of light-on transient spikes, light-coincident photoreceptor potentials (LCRPs), light-off transients, and repolarization times (data not shown). Measuring the magnitudes of these three components (interfly averages in millivolts) revealed the mutant on-spike to be half normal and the LCRP and off-spike 60% normal; only the light-on transient was significantly lower (P < 0.05). Magnitudes of the three kinds of control values (in millivolts) were squarely in the range of those tabulated for wild-type ERGs in our earlier studies (RENDAHL et al. 1992 , RENDAHL et al. 1996 ; SMITH et al. 1998B ). For the 30°–31° recordings, the corresponding metrics (for cac^TS2 compared with wild type) were 50, 100, and 80% (n = 3 and 5, respectively); none of the mutant values were significantly lower than those of the control ones. Therefore flies expressing this cacophony mutation, even when heat stressed, exhibited nowhere near the marked deficits of these spike and LCRP amplitudes that had been observed in recordings of cac^nbA mutants (HOMYK and PYE 1989 ; SMITH et al. 1998B ). Moreover, we suspect that the reasonably robust light-on and light-off transient spikes reflect visual system functioning that would be sufficient to allow for the fairly solid behavioral response to visual stimuli that was exhibited by cac^TS2 (see above), which is nothing like the anomalous photophobic behavior that is caused by cac^nbA mutations (KULKARNI and HALL 1987 ). Having said this, the ERGs for cac^TS2 involved a readily observable anomaly when recorded at high temperature: Instead of a quick repolarization to baseline voltage after the light went off (an average of 0.4 sec in the current controls, which is characteristic of wild-type records), the mutant individuals exhibited relatively prolonged repolarization times (an average of 2.3 sec, which was significantly longer than normal: P < 0.05). This ERG anomaly is reminiscent of what was found for flies expressing one of the nightblind alleles (nbAP73)—the one that causes relatively mild visual-system malfunction compared with the other two cac^nbA mutants analyzed in this manner by SMITH et al. 1998B (and Fig 3 of that report).

General features of locomotion:
To determine whether the sluggishness inferred to be associated, if only marginally, with cac^TS2's courtship performances and phototaxis would be exhibited in a situation devoid of specific sensory stimuli, generic locomotor behavior was observed. After exposing flies to 29° and quantifying locomotion at 25° in an arena test, or monitoring such movements of nonheated flies at the latter temperature, cac^TS2 males were found to be approximately one-quarter to one-third as active as wild type (Table 5). The mutant's activity improved somewhat (giving approximately half-normal counts) when tested at 22°–23°. comt males (in the two genetic backgrounds noted in Table 5) tended to be more sluggish than cac^TS2 flies, especially in the 25° or 29° 25° tests.

After exposing mutant and normal flies to mechanical shock (Table 6), cac^TS2 males exhibited longer-than-normal recovery times in various iterations of these experiments (e.g., low-temperature rearing test at intermediate temperature or rearing in the latter condition high-temperature exposure). This stress test was the one kind for which comt males were less severely impaired than those carrying cac^TS2 (except in one respect, because w comt^ST53 animals were killed during development at 29°, as implied in the third subsection of Table 6).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Correlations between Dmca1A-encoding genotypes and cacophony-mutant phenotypes:
The newest cacophony mutant (DELLINGER et al. 2000 ) is a courtship variant, as was (and is) the original cac mutant (SCHILCHER 1977 ). Thus, cac^TS2 males are somewhat impaired in their overall courtship performance, including mating ability. However, cac^TS2 males courted more vigorously and effectively (Table 2) than one might expect from monitoring their generic locomotor activity (Table 5 and Table 6). One component of the courtship performance of cac^TS2 males implies a behavioral problem that goes beyond the nature of the sounds they communicate to females. They performed worse than wingless wild-type males did (Table 2), which indicates that this mutant is more pleiotropically defective than a "song only" variant.

Nevertheless, the most sharply defined courtship defect exhibited by a cac^TS2 male is its heat-sensitive anomalies of tone pulses that emanate from the wing vibrations it directs at a female (Fig 2 Fig 3 Fig 4, Table 3). These abnormalities of cycles per pulse and pulse amplitude were found to be similar to the nonconditional courtship-song peculiarities exhibited by the original cac^S mutant (e.g., PEIXOTO and HALL 1998 ). That the respective mutant phenotypes are alike is important, because cac^TS2 males did not have to exhibit any kind of singing eccentricity: Inasmuch as the isolation of this mutant involved behavioral criteria that had nothing to do with courtship (DELLINGER et al. 2000 ), the outcome of song-testing cac^TS2 could have left cac^S as the only singing variant associated with this gene. But both the original and the newest cacophony mutations cause courtship-song peculiarities, and it is interesting that the anomalously loud and polycyclic pulses produced by both cac^S and cac^TS2 males do not involve an appreciable derangement of such sounds: Each mutant type remains nicely patterned with respect to the qualities of individual "clicks" and their rate of production (as exemplified in Fig 2). Once again, if cac^TS2 turned out to be song defective it was not a foregone conclusion that such males would produce these sounds in a manner more salutary than that of other singing variants, such as those expressing slowpoke (slo) mutations. In this regard, slo potassium-channel mutants were identified using generic behavioral criteria (as was cac^TS2) and were found later to sing aberrantly (rare among ion-channel variants: KULKARNI and HALL 1987 ; PEIXOTO and HALL 1998 ) and to exhibit erratic patterns of anomalous tone pulses (PEIXOTO and HALL 1998 ).

This brings us to the question of why it might be that the songs of cac^S and of cac^TS2 (30°) males are not only song defective, but also similarly so in their tone-pulse qualities. As was introduced in conjunction with documenting cac^TS2's intragenic site change (Fig 1, Table 1), this amino-acid substitution is very near the EF hand within Dmca1A, directly C-terminal to the aforementioned IVS6 transmembrane domain (Fig 1B). The highly conserved EF hand and adjacent residues among calcium-channel 1 subunits of various species are involved in channel inactivation mediated by Ca²⁺ binding (e.g., ZHANG et al. 1994 ; ZHOU et al. 1997 ; DOUGHTY et al. 1998 ). Thus, this form of inactivation involves a calcium-influenced conformational change that occurs via cation binding within the EF hand's helix-loop-helix (PETERSON et al. 2000 ). Given the P-to-S substitution in cac^TS2 immediately C-terminal to the EF hand (Fig 1)—where this evolutionarily conserved proline (e.g., Fig 1A) is changed to a polar serine that has more conformational freedom—one imagines that the local three-dimensional structure in which the EF hand finds itself is altered in the mutant. The function of this domain would be altered accordingly but not ruined at permissive temperatures. Thus, the amino-acid substitution in cac^TS2 near the EF hand suggests that this protein change could cause the Dmca1A calcium channel to exhibit altered inactivation kinetics. Whereas inactivation features of the 1 subunit encoded by cac are unknown, it is reasonable to speculate that that process becomes less robust than normal in the cac^TS2 mutant as the flies are heated from 20° to 30°. Why the dynamics of inactivation may be subtly heat sensitive over the temperature range just stated is difficult to surmise, although perhaps it is the case that this process can barely occur at all at 37°, accounting for the grossly subnormal synaptic neurotransmission that occurs at that extreme temperature (KAWASAKI et al. 2000 ).

This hypothesis, as it relates to cac^TS2's behavioral phenotype within a "physiological" range of temperatures, goes on to suggest that anomalously polycyclic pulses in the songs of males expressing this mutation smack of a channel-inactivation change that would alter the contribution of calcium currents to the overall behavioral process in question. Thus, the repetitive-pattern phenotype, which is a reasonable descriptor for trains of Drosophila song pulses, would not have the intrapulse cycles inactivated as "tightly" as in wild type.

What about the songs of cac^S males, whose pulses are similarly polycyclic (albeit without the temperature sensitivity that accompanies the cac^TS2 phenotype)? The cac^S mutant is accounted for by an amino-acid substitution within the sixth membrane-embedded region of the penultimate intra-Dmca1A repeat (SMITH et al. 1998B ), a.k.a. IIIS6 (Fig 1B). Certain types of calcium channels prevent excessive influx of calcium when the channel opens by voltage-mediated inactivation (HERING et al. 2000 ). Pore-forming S6 transmembrane domains play a role in modulating voltage-dependent calcium-channel inactivation (HERING et al. 2000 ). This has been revealed (1) by creating chimeric 1-subunit polypeptides in which portions of IIIS6 from fast-inactivating channels replaced those of a slow-inactivating one, leading to inactivation kinetics characteristic of the donor calcium-channel type (TANG et al. 1993 ) and (2) by physiological disruptions of channel functions that are pointed to by the etiology of certain patho-physiological mutants in humans (reviewed by JEN 1999 ); certain such S6 mutations slow and others accelerate the development of inactivation (HERING et al. 2000 ). Therefore, a mnemonic device for apprehending the song abnormality exhibited by cac^S mutant males is, again, subnormal inactivation of intratone-pulse sounds, owing to their inappropriate polycyclicity (SCHILCHER 1977 ; KULKARNI and HALL 1987 ; PEIXOTO and HALL 1998 ). However, in this case the putative inactivation defect would have a different mechanistic etiology compared with that hypothesized for the cac^TS2-mutated polypeptide.

That channel inactivation can be mutationally altered in more than one way makes it nonmiraculous that the two different sites and kinds of amino-acid alterations in the two song-defective cacophony mutants are similarly non-wild type. But what if any change within the cac-encoded Dmca1A polypeptide would lead to the same kind of altered channel function insofar as song regulation is concerned? The cac^S and cac^TS2 tone-pulse phenotypes could represent some sort of default mutant phenotype. This possibility (in its extreme form) will not wash, however, because the gene has been mutated to a variety of different phenotypes. Some cac mutations are embryonic lethals (e.g., KULKARNI and HALL 1987 ; SMITH et al. 1998B ), as are mutations at another locus in this species that encodes a similar function (EBERL et al. 1998 ), although it is not obvious a priori that they would have this property: Certain ion-channel-encoding genes in this species are nonvital (e.g., TANOUYE et al. 1981 ; ZHOU et al. 1999 ), and there is not only more than one (cf. EBERL<