A new allele of engrailed, en^NK14, causes supernumerary spermathecae in Drosophila melanogaster

ABSTRACT

A spontaneous mutation, en^NK14, was a new allele of engrailed (en) in Drosophila melanogaster. Females of en^NK14 have three spermathecae, instead of two in wild type, under a wide range of developmental temperatures, while the males show no abnormal phenotype. Spermathecae of the mutant female can accept inseminated sperms, albeit with a delay of at least an hour until full acceptance compared with wild type. The time course of decrease in the number of stored sperms was thoroughly similar between the mutant and wild type. en^NK14 females produced fewer progeny than wild type females despite storing a larger number of sperms. The delay of sperm entry and lower fecundity suggested some functional defects in secretory products of the spermathecae. In addition, some spermathecae in the mutant were accompanied by a mass of brown pigments in the adipose tissue surrounding the capsule. Six contiguous amino acids, Ser340–Ala345, were replaced by one Thr in en^NK14. In another mutant, en^spt, Ser325 was also shown to be substituted by a Cys. These amino acid changes were located within a serine-rich region, in which Ser325, Ser340 and Thr341 were suggested as targets of Protein Kinase C by an in silico analysis. The splicing pattern of en mRNA did not differ between en^NK14 and wild type in embryo, larva, pupa or adult. Our results suggest that en plays an important role in determining the number of spermathecae as well as in sperm storage function in the Drosophila female.

INTRODUCTION

Sperm storage in females is necessary in animals in which insemination and fertilization are temporally separated, and occurs among a wide range of taxa from insects to mammals (Neubaum and Wolfner, 1998). Most insects have two kinds of sperm storage organs, spermathecae and ventral receptacles (Snodgrass, 1993; Pitnick et al., 1999). The spermatheca is a mushroom-shaped organ, which consists of a sclerotized capsule and a duct, arising from the anterodorsal portion of the uterus. The ventral receptacle is typically a blind-ended coiled tubule arising from the anteroventral portion of the uterus. A large proportion of sperms are stored in the ventral receptacle and utilized first for fertilization, and sperms in the spermatheca are used after depletion of the ventral receptacle (Schnakenberg et al., 2011, 2012). The number of spermathecae is variable among insects: one in most orders, two in all Drosophila species, and three in most other flies (Sturtevant, 1925; Fowler, 1973; Snodgrass, 1993; Pitnick et al., 1999). Sperm storage organs play important roles in female reproductive strategy, because the variation in their number and size is associated with differences in efficiency of sperm utilization, egg productivity and female longevity (Pitnick et al., 1999; Prokupek et al., 2008).

In D. melanogaster, both spermathecae originate from the A8 segment of the genital imaginal disc during the pupal stage (Epper and Sánchez, 1983; Chen and Baker, 1997; Sánchez et al., 1997; Keisman et al., 2001; Sánchez and Guerrero, 2001; Estrada et al., 2003; Sun and Spradling, 2012). The number of spermathecae is thought to be determined by a genetically controlled developmental program, but supernumerary spermathecae are often observed in D. melanogaster, for instance in a race carrying three spermathecae (Sturtevant, 1925) and in an isolated mutant whose females had three or four spermathecae (Wexelsen, 1928). Importantly, a mutation, spermatheca (spt), gives rise to supernumerary spermathecae in a temperature-dependent manner (Hadorn and Graber, 1946). By a series of complementation tests, spt was shown to be an allele of engrailed (en), en^spt (Chase and Baker, 1995). Recently, females having three spermathecae were also found in some P element-induced mutation lines (Bangham et al., 2003; Dhillon et al., 2020). En is well known as a homeodomain-containing transcription factor that plays important roles in boundary formation (Kornberg, 1981; Kornberg et al., 1985). En family proteins were recently revealed to be considerably pleiotropic, having three primary functions in many metazoans: transcription regulation, translation control and action in extracellular signaling pathways (reviewed by McGrath et al., 2013; Wizenmann et al., 2015; Punia et al., 2019; Cao et al., 2020). However, the molecular basis of numerical variation in spermathecae is yet to be elucidated.

Here we characterized a new mutation, which generates three spermathecae in females. As a first step for addressing the functionality of sperm storage organs and understanding the mechanism of spermatheca development, we characterized the abilities of acceptance, storage and usage of sperm in the mutant and revealed the genetic basis of the mutation. Our results lead us to propose that the gene responsible is a new allele of en, thus, en^NK14.

RESULTS

Supernumerary spermathecae in NK14 females

Females of NK14 were found to have three spermathecae, instead of two in wild type (Fig. 1). More than 98% of NK14 females have three spermathecae and the remainder have two or four (Table 1). Females carrying three spermathecae were also observed in Oregon R, but very rarely. After mating with males of dj−GFP, which is used as a fluorescent sperm marker, all spermathecae and ventral receptacles showed green fluorescence in NK14 and wild type females (Fig. 1C, 1D). This result showed that each spermatheca in NK14 was capable of accepting sperms similarly to those in wild type. No clear difference was found in the morphology of each spermatheca between NK14 and wild type. Some spermathecae were, however, accompanied with a mass of brown pigments (Fig. 1E, 1F). These anomalous structures were localized to the adipose tissue surrounding the capsule. Their shapes were amorphous and sizes variable (Supplementary Fig. S1). We found no morphological abnormality in males of NK14.

Fig. 1.

Female internal reproductive organs. (A, B, E, F) Observation with a stereo microscope. (C, D) Observation with a confocal laser scanning microscope after mating with males of dj−GFP. en^NK14 (A, C, E, F) and Oregon R (B, D) females. Arrowheads indicate spermathecae and arrows indicate an anomalous mass of brown pigments. The ventral receptacles (vr) are also indicated.

Table 1. Frequencies of numerical variation in spermathecae

Line	Chromosome(s)	Frequency (S.D.)						Number of tests	Number of females dissected
		two		three		four
NK14 (enNK14)		0.015	± 0.029	0.983	± 0.029	0.003	± 0.007	7	278
	iso-X	0.869	± 0.095	0.125	± 0.097	0.006	± 0.020	10	188
	iso-II	0.600	± 0.112	0.399	± 0.113	0.001	± 0.002	6	987
	iso-III	1.000		0.000		0.000		7	687
	iso-II and -III	0.393	± 0.094	0.607	± 0.094	0.000	± 0.001	35	7,242
NK14/Df(2R)en-A		0.074	± 0.054	0.926	± 0.054	0.000		12	594
NK14/CyO		0.897	± 0.038	0.103	± 0.038	0.000		12	636
Oregon R		0.971	± 0.026	0.029	± 0.026	0.000		6	176

S.D.: Standard deviation.

Functional analysis of spermathecae in NK14

To investigate the abilities of sperm storage and usage in NK14, females were individually crossed with a male of ProtB–GFP, whose sperm head expresses GFP (Fig. 2A–2D). After a 15-min mating, the female was separated from the male and the sperms in the female organs were counted. There were fewer sperms in each spermatheca in NK14 than in wild type at 1 h after copulation (t = 2.82, P = 0.007), but there was no significant difference between the lines at the other time points examined (P > 0.05) (Fig. 2E). Number of sperms in the ventral receptacle was not markedly different between lines, except for a weak significance of more sperms in NK14 at 7 days after copulation (Fig. 2F). A considerable number of sperms existed in spermathecae even after 15 days, but almost none remained in the ventral receptacle in both lines. NK14 females stored more sperm in total than wild type from 5 h to 7 days after copulation, although the difference was not significant at 1 day after copulation and significance in the difference was lost by 10 days (Fig. 2G). Total numbers of sperms in the ventral receptacle and spermathecae were maximal at 5 h after copulation in both lines, 626.2 ± 108 for NK14 and 455.1 ± 71.6 for Oregon R, and implied that the NK14 female is capable of storing a larger number of inseminated sperms in her sperm storage organs.

Fig. 2.

Sperm storage and usage in females. (A, B) Sperms in the spermathecae and (C, D) in the ventral receptacles. Females of en^NK14 (A, C) and Oregon R (B, D) were used 10 days after copulation with males of ProtB–GFP. Bars are 50 μm. Changes in the number of sperms per spermatheca (spt) (E), ventral receptacle (vr) (F) and female (G) (n = 5–15). The number of sperms in each female is the total in three spermathecae and the ventral receptacle for NK14, and in two spermathecae and the ventral receptacle for Oregon R. Microsoft Excel 2020 was used for t-tests. * P < 0.05 and ** P < 0.01.

To determine whether the larger number of sperms stored by NK14 could lead to increased fecundity we compared the number of F1 progeny between NK14 females and wild type females. NK14 females produced fewer F1 progeny than Oregon R females, both in the intra-crosses (t = –7.545, P = 5.6 × e^–7) and in the inter-crosses between them (t = –11.82, P = 6.5 × e^–10) (Fig. 3A). Numbers of eggs laid by NK14 females were significantly lower than those by Oregon R females during the first 12 days after copulation (Fig. 3C), and the averages of the total number of eggs were 93.8 ± 21.0 for NK14 and 215.8 ± 27.1 for Oregon R. Almost all eggs hatched and developed to pupae in both lines (Fig. 3D) and all pupae successfully emerged (data not shown). On the other hand, no difference was found in the number of ovarioles between NK14 and wild type females (t = 1.64, P = 0.164) (Fig. 3B). The lower progeny number of NK14 females appears to be explained by reduced oviposition.

Fig. 3.

Relationship between numbers of progeny and eggs laid. (A) Total number of adult flies arising from a female during 21 days after copulation (n = 10 for each cross). (B) Number of ovarioles in each female (n = 25). (C) Number of eggs laid every three days by individual females. The total number of eggs was also counted: 93.8 ± 21.0 for NK14 (n = 10) and 215.8 ± 27.1 for Oregon R (n = 12). (D) Frequencies of pupariation of the eggs laid in (C), every three days after copulation. Pupae were counted at 10 days after oviposition. Microsoft Excel 2020 was used for t-tests. ** P < 0.01.

Mapping the gene responsible for the NK14 mutation

For mapping the gene responsible, we made isochromosomal sublines for each of the X, II and III chromosomes of NK14 (Supplementary Fig. S2 and S3). Among them, the iso II sublines showed the highest frequency, 0.399, of females having three spermathecae (Table 1). Females of the iso X sublines had supernumerary spermathecae only 12.5% of the time, and those of the iso III sublines had none. However, when the II and III chromosomes were re-combined in the iso II & III sublines (Supplementary Fig. S4), the frequency of three spermathecae was significantly higher, 0.602, than that in the iso II sublines (t = –4.854, P = 2.0 × e^–10), but not equal to the level in the original NK14. Such a partial synergistic effect suggests that the major gene(s) responsible exist on the II chromosome and some minor or modifier genes on other chromosomes. As one of the probable candidates on the II chromosome, en was highlighted, because en^spt, an allele of en, is known for giving supernumerary spermathecae (Chase and Baker, 1995).

To examine the above assumption, we crossed NK14 and Df(2R)en-A/CyO, in which the chromosome Df(2R)en-A has no en due to a deletion (Table 1). The F1 female progeny NK14/Df(2R)en-A had three spermathecae as frequently as NK14 itself, while most of the NK14/CyO control females have only two spermathecae, implying that the chromosome CyO rescued the mutant phenotype of NK14, but that Df(2R)en-A did not. For further testing, we next mated NK14 with en^spt for a complementation test. The number of spermathecae in NK14 females was constantly three irrespective of the temperature, whereas the number in en^spt varied, consistent with a previous report: mainly three at 18 ℃, one at 25 ℃ and two at 28 ℃ (Hadorn and Graber, 1946) (Table 2). Among the F1 progeny of NK14 and en^spt, all females had two or three spermathecae at 25 ℃, but none had only one spermatheca. At 18 ℃, about 18–20% of F1 females had three spermathecae, thus indicating a partial complementation. These results were similar in both directions of the cross mating. Therefore, the allele responsible for the NK14 mutation is probably en, which differs from en^spt at least in temperature sensitivity.

Table 2. Temperature effects on numerical variation in spermathecae

Line	Temp. (℃)	Frequency (S.D.)						Number of tests	Number of females dissected
		one		two		three
NK14 (enNK14)	18	0.000		0.120	± 0.040	0.880	± 0.040	4	152
	25	0.000		0.010	± 0.014	0.990	± 0.014	2	99
	28	0.000		0.099	± 0.017	0.901	± 0.017	2	41
enspt	18	0.030	± 0.043	0.297	± 0.110	0.672	± 0.067	2	106
	25	0.960	± 0.056	0.040	± 0.056	0.000		2	90
	28	0.342	± 0.012	0.658	± 0.012	0.000		2	23
NK14 × enspt	18	0.000		0.804	± 0.012	0.196	± 0.012	2	216
	25	0.000		0.725	± 0.194	0.275	± 0.194	2	179
enspt × NK14	18	0.000		0.823	± 0.041	0.177	± 0.041	2	231
	25	0.000		0.867	± 0.024	0.133	± 0.024	2	170

S.D.: Standard deviation.

Structures of gene and transcripts of en^NK14 and en^spt

To understand the relationship between the structure of the en gene and the supernumerary spermathecae, we compared the nucleotide sequences of en^NK14 and en^spt with wild type en⁺ (Fig. 4). The nucleotide sequence was determined for 4,192 bp in NK14. Compared with the en⁺ sequence, NK14 had 23 nucleotide substitutions and 31 indels, of which 19 substitutions and eight indels were in en exons. A deletion of 15 nt in the first exon was deduced to replace six contiguous amino acid residues, Ser340–Ala345, with one Thr in En^NK14 (Fig. 4C). The other nucleotide changes do not alter the amino acid sequence of En^NK14. The nucleotide sequence of en^spt, 4,212 bp, carried 37 nucleotide substitutions and 21 indels, of which 16 substitutions and seven indels were in exons. Among these, only one nucleotide substitution, from T to C in the first exon, causes an amino acid change, from Ser325 to Cys in En^spt (Fig. 4C). To reveal the transcriptional pattern of en, RT-PCR was carried out with two sets of primers. As a result, the second and third introns were similarly spliced out in embryos, larvae, pupae and adult females in NK14 and wild type (Fig. 4B).

Fig. 4.

Structure of en, its transcripts and En. (A) Structure of the en gene. The arrow indicates the start position of the first exon of en. Triangles depict the approximate positions and orientations of the primers for PCR (white) or RT-PCR (gray): a, enF7411355; b, en-f3; c, en-f4; d, en-r14; e, en-r17; and f, enR7415907. (B) RT-PCR of Canton S (CS) and NK14. The en mRNAs were detected independently: with primers b and e in (A) (lanes b), with primers c and d (lanes c), and a mRNA fragment of Rp49 (lanes r). (C) Deduced amino acid sequences of the serine-rich region (S-st; 320–368) in En proteins. Dots below the map depict the Ser residues targeted by Casein Kinase II in the E/D/S/T-rich region (Bourbon et al., 1995). EH1–EH5: En homology regions (Logan et al., 1992; Gibert, 2002; Morgan, 2006; Wizenmann et al., 2015).

The amino acid changes seen in the two mutants, En^NK14 and En^spt, were located close to each other, but did not map to any known functional domains (Fig. 4C). Instead, they were located in a serine-rich region, which we call the Ser-stretch, where 55% (27/49 aa) of the residues are Ser. To address the functionality of this region, we searched for possible phosphorylation sites by an in silico analysis. NetPhos 3.1 (Blom et al., 2004) deduced probable target sites of PKC, DNAPK and CDC2 at the mutated amino acids in En^NK14 and En^spt (Table 3). PKC was highly nominated as the functional kinase at Ser325, Ser340 and Thr341. Among the Drosophila species examined, the amino acid sequence of this Ser-stretch is shown to have a considerably higher conservation than other parts of En, except for D. sechellia and D. erecta, and the three putative target sites of PKC were perfectly conserved in the seven species of the D. melanogaster species subgroup (Supplementary Fig. S5).

Table 3. Possible targets of phosphorylation

Residue (position)	Kinase	Score
Ser (325)*	PKC	0.723
	DNAPK	0.503
Ser (340)**	PKC	0.512
Thr (341)	PKC	0.601
	CDC2	0.559
Ser (343)	PKC	0.707
Ser (394)***	CK2	0.372
Ser (397)***	CK2	0.498
Ser (401)***	CK2	0.604
Ser (402)***	CK2	0.628

*  Substituted by Cys in En^spt.

**  Lost in En^NK14.

***  Experimentally shown as a target of CK2 by Bourbon et al. (1995).

DISCUSSION

Functional aspects of the spermathecae of en^NK14 females

In the mutation en^NK14, more than 98% of females have three spermathecae, while males seem morphologically normal (Fig. 1). Each spermatheca of an NK14 female accepted a similar number of inseminated sperms, although there was a delay of at least one hour in the initial entry of sperm compared with wild type (Fig. 2). Beyond 5 h after copulation, more sperms tended to be observed, both in the ventral receptacle and in total, in NK14 than in wild type. The time courses of sperm usage were thoroughly similar between NK14 and wild type, suggesting that a larger number of sperms were released from the storage tracts to the uterus in NK14 than in wild type (Fig. 2). Nevertheless, the number of offspring from NK14 females was less than half that of wild type. This poor fecundity was consistent with their laying fewer eggs, but not with the ratio of fertilization of the eggs, the viability from embryos through to pupation or the number of ovarioles (Fig. 3). Interestingly, similar phenotypes were reported in a mutant line, SP3, whose females have three spermathecae but produce fewer offspring than wild type females with two spermathecae (Bangham et al., 2003).

Both initial sperm entry and egg laying, as well as sperm survival, activation and selection in the genital tracts, are known to be regulated by secreted products from secretory glands of the spermathecae and glandular parovaria in adult females (Sun and Spradling, 2012, 2013; Mayhew and Merritt, 2013). Defects of the secretory cells induced a slowing down of sperm entry to the storage organs (Schnakenberg et al., 2011). When the number of secretory cells decreased, by means of knockdown of hr39, the rate of ovulation, and thus the egg laying, of females decreased in proportion to the cell number (Sun and Spradling, 2013). It is therefore possible that the secretory cells in NK14 carry some defects. For instance, disability or deficiency of an attractant for sperms would weaken sperm movement into the spermathecae and could result in an increase of sperms in the ventral receptacle. Similarly, defects of regulator(s) for ovulation are assumed to diminish egg laying. However, specific factor(s) are yet unidentified for each characteristic among the secretory products (Sun and Spradling, 2013). Physiological and histological details of spermathecae and biochemical assay of the secretome of NK14 will be needed to clarify the functional aspects. We cannot completely exclude the possibility that the delayed sperm entry and reduced egg laying of NK14 are unrelated to en^NK14, but dependent upon other gene(s).

There are some previous reports of unusual structures in the vicinity of the spermathecae. Wexelsen (1928) showed that females with the mutation degenerated spermatheca (dg-a) had triple spermathecae accompanied by dark brown pigments or brown granules in the epithelial cells of the capsule. Another anomalous structure, SDP (spermathecal duct presence), was recently found inside the duct of the spermatheca in Drosophila females (Hopkins et al., 2020). SDPs have a similar color to the capsule of the spermatheca and may influence the outcome of sperm competition by decreasing the entry of a second male sperm (Hopkins et al., 2020). Because of the difference in localization, the mass found in en^NK14 (Fig. 1, Supplementary Fig. S1) is unlikely to be either the dark brown pigments of Wexelsen or the SDP. The amorphous shape of the mass also supports this interpretation, because the shape of the SDP resembles a column or cylinder, like a plug (Hopkins et al., 2020). Interestingly, Kosuda (1992, 1996) reported a spermatheca-specific melanotic tumor that was visible as an “irregular shaped dense black mass” which could surround the whole of the capsule of the spermatheca when it developed. Coincidences in coloration, location and appearance let us speculate that the mass in en^NK14 would also be associated with a tumor. Most brown masses were, however, too small to surround the capsule in en^NK14 (Fig. 1, Supplementary Fig. S1). Their size variation may reflect differences in the development of such a tumor, although the origin of the mass is unknown.

The gene responsible for supernumerary spermathecae of NK14 is en^NK14

Our present results consistently suggested that the supernumerary spermathecae in NK14 are the consequence of the nucleotide deletion in en. First, chromosomal mapping by means of the isochromosomal sublines suggested that the major gene(s) responsible is located on the II chromosome. Second, Df(2R)en–A, in which en was lost, could not rescue the supernumerary spermathecae phenotype of NK14. Third, when NK14 was crossed to en^spt at 18 ℃, about 20% of F1 female progeny had three spermathecae, and thus a partial complementation between the two mutations. Furthermore, nucleotide sequences of en^NK14 and en^spt revealed non-synonymous nucleotide substitutions in the first exon of en in both mutations, which result in a replacement of six amino acids (340–345) by a Thr in En^NK14 and a replacement of Ser325 by Cys in En^spt. These results also support the previous report that en^spt, a hypomorph of en, can affect the number of spermathecae (Chase and Baker, 1995). Accordingly, we designate the new mutation en^NK14.

The results of chromosomal mapping also implied that other genes can modulate the en^NK14 phenotype. Many genes have been reported to affect the number of spermathecae. Females of loss-of-function mutants of Gef26 (guanine nucleotide exchange factor for Rap GTPase) possess three spermathecae, and the phenotype was enhanced by Rap (Ras-associated protein) mutations and could be rescued by overexpression of E-cadherin (Singh et al., 2006). Expression of lz (lozenge), encoding a Runt-domain transcription factor, is essential for spermatheca formation in the female genital disc (Oliver and Green, 1944), and hh (hedgehog) and dpp (decapentaplegic) are necessary for regulation of lz (Chatterjee et al., 2011). Overexpression of Hr39, a mammalian steroidogenic factor 1 (Sf1)-related nuclear hormone receptor, resulted in three spermathecae (Allen and Spradling, 2008). Development of the spermathecal duct requires expression of dac (dachshund) (Keisman et al., 2001) and may be influenced by invected (inv), which is located close to en and functions similarly to en (Poole et al., 1985; Coleman et al., 1987; Tabata et al., 1995). Recently, females having three spermathecae were found in a P element-induced mutation line, in which the P element is inserted in CG7956 (now renamed sp3) on III chromosome (Dhillon et al., 2020). In another lacW insertion line, FBst10175, females had three spermathecae, although the P element insertion was not the direct cause of the mutation (Bangham et al., 2003). The wide variety of genetic loci implicated supports the possibility that multiple genes cooperate for spermatheca development: lz on X, Gef26, dpp and hr39 on another arm of II, and hh and sp3 on III chromosome.

As previously mentioned, Wexelsen (1928) reported a mutation of females with an extra spermatheca accompanied by dark brown granules. He mapped one of the genes responsible, dg-a, near the two marker genes cn (cinnabar) and c (curved) on II chromosome; en is located between them. We speculate that dg-a was another allele of en having a similar molecular basis to en^NK14. Unfortunately, no stock of dg-a is currently available as far we know.

Possible molecular basis of the supernumerary spermathecae in en^NK14

All amino acid residues changed in En^NK14 and En^spt are located in a Ser-stretch. Among them, three amino acids, Ser325, Ser340 and Thr343, were predicted as PKC (Protein Kinase C) targets by an in silico analysis. PKC is one of the major protein kinases that is activated by Ca²⁺ and/or diacylglycerol and plays important roles in many signal transduction pathways from insect to human (reviewed by Steinberg, 2008; Saxena et al., 2017; Newton, 2018; Kikkawa, 2019). It is noteworthy that the three residues Ser325, Ser340 and Thr341 showed higher scores comparable to the four Ser residues targeted by CK2 (Casein Kinase II) that were also identified by experiments (discussed more later). These three amino acids were also highly conserved among the closely related species (Supplementary Fig. S5), suggesting that they have an important function.

The pleiotropy of En (McGrath et al., 2013; Wizenmann et al., 2015; Punia et al., 2019; Cao et al., 2020) gives rise to a wide variety of mutation phenotypes of en, such as scutellar cleft, malformation of the wing, an extra sex comb and lethality in D. melanogaster (Lindsley and Zimm, 1992). Two spermathecae arise from the anterior compartment of female genital primordia of the A8 segment in the wild type female genital disc during the pupal stage. When cAMP-dependent Protein Kinase (PKA) is inactive, some females have three (ca. 14%) or four (ca. 4%) spermathecae, because PKA modulates ptc, hh and wg (wingless) in the anterior compartment and En suppresses dpp and hh in the posterior compartment, forming the normal pattern of genital discs (Chen and Baker, 1997).

There are some more lines of evidence supporting a relationship between phosphorylation and En. The four Ser residues (394, 397, 401 and 402) in the E/D/S/T-rich region of Drosophila En are targets of CK2 and the DNA binding affinity of the En homeodomain is weakened by phosphorylation (Bourbon et al., 1995). CK2 is expressed ubiquitously and phosphorylates Ser/Thr and Tyr of various substrates related to growth and development in many animals, and thus is suggested to be one of the essential kinases in kinomes (reviewed by Franchin et al., 2017; Götz and Montenarh, 2017). Furthermore, sp3, the gene responsible for a recently identified supernumerary spermathecal mutation, encodes a phosphatase that is thought to hydrolyze phosphate from inositol, although the substrate is not yet identified (Dhillon et al., 2020). It is therefore conceivable that En plays an important role via phosphorylation in the development of spermathecae and that Ser325 and Ser340 of En are the targets of kinases.

In summary, we identified a new allele of en, en^NK14, that frequently causes supernumerary spermathecae in females. The phenotype appears to be associated with one of the pleiotropic effects of en. Phosphorylation by kinases is likely to play important roles in regulating the number of spermathecae and En may be one of their substrates. We believe that the supernumerary spermathecal mutants characterized here will provide valuable information for research on the biological importance and developmental control of the sperm storage organs. Their female-specific mutation phenotype also presents an exciting challenge for future work.

MATERIALS AND METHODS

Fly lines

NK14 is an isofemale line established from wild females caught in a grape yard in Nankoku, Kochi, Japan, where D. melanogaster flies were collected by net-sweeping over a heap of damaged grapes with permission of the owner in September, 2001. After collection, females were kept individually in vials to establish isofemale lines. The subline NK14-G6-8-4 was used in this study as en^NK14. en^spt is a mutant having supernumerary spermathecae (Hadorn and Graber, 1946). w; P{dj–GFP.S} AS1/CyO, P{savRas1.v12} FK1 (dj–GFP) expresses GFP in the tail of sperm and w; P{w^mW.hs=ProtB-GFP1}75 (ProtB–GFP) expresses GFP in the head of sperm. Df(2R)en–A/CyO is hemizygous for en due to a chromosomal deletion, 47D7–48B2, in Df(2R)en–A. Oregon R and Canton S were used as standard lines of wild type of D. melanogaster.

The stocks of en^spt (Ky103486), w; P{dj–GFP.S} AS1/CyO, P{savRas1.v12} FK1 (Ky108217), w; P{w^mW.hs=ProtB-GFP1}75 (Ky1808217) and Df(2R)en–A/CyO (Ky108995) were provided by Kyoto Stock Center. Flies were maintained on standard food medium at 24 ℃ except for specific tests (see below).

Cross mating

For establishing homozygous sublines of the X chromosome (iso X sublines) of NK14, FM7c, an X chromosomal balancer line, was used (Supplementary Fig. S2). For establishing homozygous sublines of the II and III chromosomes (iso II and iso III sublines) of NK14, second and third chromosomal double-balancer lines, yw; CyO/Sp; TM3 Sb Ser/Dr, were used (Supplementary Fig. S3). The established iso II and III sublines were then crossed to each other to re-combine the II and III chromosomes (iso II & III) (Supplementary Fig. S4). Females of NK14 were mated with males of Df(2R)en-A/CyO for a complementation test, where the genotype of F1 females having curly wings should be II_NK14/CyO and that of females having normal wings should be II_NK14/Df(2R)en-A. Ten females and ten males of NK14 and en^spt were mated in both directions for the complementation test at 18 ℃ or 25 ℃.

Observation of reproductive organs and sperms

Females were dissected in 70% ethanol under a stereo microscope for evaluation. The separated reproductive organs were mounted in 2% agarose in a concave slide glass for imaging (González-Morales et al., 2015). For fine observation, females were mated with males of dj−GFP or ProtB−GFP. The male was separated from the female after 15 min copulation. The females were dissected as above and the reproductive organs were observed in VECTASHIELD Mounting Medium H-1000 (Vector Laboratories) under a confocal laser scanning microscope, FluoView FV10i (Olympus), or a Stemi stereo microscope (Zeiss). The females mated with dj−GFP were dissected and observed just after mating. The females mated with ProtB−GFP were dissected 1 h, 5 h, 24 h, 5 days, 7 days, 10 days and 15 days after copulation and the organs were fixed in 99.5% ethanol for 15 min and rinsed with 0.05% Tween 20/PBS before observation. Sperms in each spermatheca and ventral receptacle were counted in five females or more for each time point. The number of ovarioles for each ovary was counted in 25 adult females five days after emergence.

Counting the numbers of eggs and progeny

A virgin female and male, both 3–5 days old, were mated in a vial. The male was separated from the female by hand after 15 min of copulation. The female freely laid eggs on a normal medium in a plastic dish. The dishes were renewed every three days. Numbers of eggs laid and hatched in each dish were counted. Number of pupae was counted about 10 days after oviposition, and number of adult flies was counted later.

Nucleotide sequence determination

Genomic DNA was extracted from adult flies using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich). The en gene was amplified with KOD -Plus- Neo (Toyobo) using the primer pair enF7411355 (5′-AGCCTTTGATGTGACAGATG-3′) and enR7415907 (5′-CAGAGGGAGTGAACAGGTG-3′). The PCR conditions were as follows: 94 ℃ for 2 min followed by 30 cycles of 98 ℃ for 10 s, 50 ℃ for 30 s and 68 ℃ for 3 min. After electrophoresis in a 0.7% agarose gel (Seakem GTG Agarose, Lonza), DNA fragments were separately extracted and purified using the QIAquick Gel Extraction Kit (Qiagen). BigDye Terminator v3.1 (Thermo Fisher Scientific) and Genetic Analyzer 3100 or 3500 (Thermo Fisher Scientific) were used for DNA sequencing. The nucleotide sequences determined in this study are deposited in DDBJ under the accession numbers AB807860 for en^NK14, AB808580 for en^spt and AB808579 for en⁺ in Canton S.

RT-PCR

Total RNA was extracted from embryos (0–24 h old), third instar larvae, pupae or adult females with the Aurum Total RNA Mini Kit (Bio-Rad). cDNA was synthesized by ReverTra Ace (Toyobo) using total RNA (450–720 ng) with the supplied oligo-dT primer. Amplification conditions of cDNA were as follows: 94 ℃ for 1 min followed by 30 cycles of 98 ℃ for 10 s, 51 ℃ for 30 s and 72 ℃ for 3 min for en with two primer pairs, en-f3 (5′-AGTGACCCAGTGACAAGTG-3′) and en-r17 (5′-TACGGATGGGTCTTACTCT-3′) and en-f4 (5′-CAACAGCAGCAGCAAATG-3′) and en-r14 (5′-GCTTCTCGTCGTTGGTCTT-3′). The former pair should amplify a 1,896-bp DNA containing all three exons and the latter a 1,274-bp fragment containing exons 1 and 2. The rp49 gene was chosen as an endogenous expression reference, for which PCR conditions were as follows: 94 ℃ for 1 min followed by 30 cycles of 98 ℃ for 10 s, 62 ℃ for 30 s and 72 ℃ for 1.5 min, with the primers 5′-GCTTCTGGTTTCCGGCAAGCTTCAAG-3′ and 5′-GACCTCCAGCTCGCGCACGTTGTGCACCAGGAAC-3′ (Trong-Tue et al., 2010).

Bioinformatic analyses and statistical tests

Putative phosphorylation sites of En proteins were searched for using NetPhos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) (Blom et al., 2004). Phylogenetic analyses were conducted in MEGA X (Kumar et al., 2018; Stecher et al., 2020). Evolutionary history of En proteins was inferred using the maximum likelihood method and JTT matrix-based model (Jones et al., 1992) and the tree with the highest log likelihood is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with superior log likelihood value. All positions containing gaps and missing data were eliminated (complete deletion option). Accession numbers of En in Drosophila species are: D. melanogaster NP_523700, D. simulans XP_002081053, D. mauritiana XP_033154367, D. sechellia XP_002033411, D. yakuba XP_002091199, D. santomea XP_039483386, D. erecta XP_001976053, D. suzukii XP_016927909, D. ananassae XP_001958778, D. pseudoobscura XP_001360552, D. willistoni XP_002066183 and D. virilis XP_032292262. Microsoft Excel 2020 was used for statistical tests.

ACKNOWLEDGMENTS

We thank T. Ohsako for his technical advice for counting sperms in the female organs. We thank I. A. Boussy for his valuable comments for improving the manuscript. This work was partly supported by the Japan Society for the Promotion of Science KAKENHI Grant Number 19K06071 to Y. K. and M. I. We thank Kyoto Stock Center for supplying fly stocks. One of the authors, T. U., who established the line of NK14 and found its supernumerary spermathecae, passed on in 2006.

REFERENCES

•  Allen,  A. K., and  Spradling,  A. C. (2008) The Sf1-related nuclear hormone receptor Hr39 regulates Drosophila female reproductive tract development and function. Development 135, 311–321.
•  Bangham,  J.,  Chapman,  T.,  Smith,  H. K., and  Partridge,  L. (2003) Influence of female reproductive anatomy on the outcome of sperm competition in Drosophila melanogaster. Proc. Biol. Sci. 270, 523–530.
•  Blom,  N.,  Sicheritz-Pontén,  T.,  Gupta,  R.,  Gammeltoft,  S., and  Brunak,  S. (2004) Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Proteomics 4, 1633–1649.
•  Bourbon,  H. M.,  Martin-Blanco,  E.,  Rosen,  D., and  Kornberg,  T. B. (1995) Phosphorylation of the Drosophila engrailed protein at a site outside its homeodomain enhances DNA binding. J. Biol. Chem. 270, 11130–11139.
•  Cao,  Y.,  Wang,  X.,  Tang,  L.,  Li,  Y.,  Song,  X.,  Liu,  X.,  Li,  M.,  Chen,  F., and  Wan,  H. (2020) Engrailed-2 promotes a malignant phenotype of esophageal squamous cell carcinoma through upregulating the expression of pro-oncogenic genes. PeerJ 8, e8662.
•  Chase,  B. A., and  Baker,  B. S. (1995) A genetic analysis of intersex, a gene regulating sexual differentiation in Drosophila melanogaster females. Genetics 139, 1649–1661.
•  Chatterjee,  S. S.,  Uppendahl,  L. D.,  Chowdhury,  M. A.,  Ip,  P.-L., and  Siegal,  M. L. (2011) The female-specific doublesex isoform regulates pleiotropic transcription factors to pattern genital development in Drosophila. Development 138, 1099–1109.
•  Chen,  E. H., and  Baker,  B. S. (1997) Compartmental organization of the Drosophila genital imaginal discs. Development 124, 205–218.
•  Coleman,  K. G.,  Poole,  S. J.,  Weir,  M. P.,  Soeller,  W. C., and  Kornberg,  T. (1987) The invected gene of Drosophila: sequence analysis and expression studies reveal a close kinship to the engrailed gene. Genes Dev. 1, 19–28.
•  Dhillon,  A.,  Chowdhury,  T.,  Morbey,  Y. E., and  Moehring,  A. J. (2020) Reproductive consequences of an extra long-term sperm storage organ. BMC Evol. Biol. 20, 159.
•  Epper,  F., and  Sánchez,  L. (1983) Effects of engrailed in the genital disc of Drosophila melanogaster. Dev. Biol. 100, 387–398.
•  Estrada,  B.,  Casares,  F., and  Sánchez-Herrero,  E. (2003) Development of the genitalia in Drosophila melanogaster. Differentiation 71, 299–310.
•  Fowler,  G. (1973) Some aspects of the reproductive biology of Drosophila: sperm transfer, sperm storage, and sperm utilization. Adv. Genet. 17, 293–360.
•  Franchin,  C.,  Borgo,  C.,  Zaramella,  S.,  Cesaro,  L.,  Arrigoni,  G.,  Salvi,  M., and  Pinna,  L. A. (2017) Exploring the CK2 paradox: restless, dangerous, dispensable. Pharmaceuticals 10, 11.
•  Gibert,  J.-M. (2002) The evolution of engrailed genes after duplication and speciation events. Dev. Genes Evol. 212, 307–318.
•  González-Morales,  N.,  Géminard,  C.,  Lebreton,  G.,  Cerezo,  D.,  Coutelis,  J.-B., and  Noselli,  S. (2015) The atypical cadherin dachsous controls left-right asymmetry in Drosophila. Dev. Cell 33, 675–689.
•  Götz,  C., and  Montenarh,  M. (2017) Protein kinase CK2 in development and differentiation. Biomed. Rep. 6, 127–133.
•  Hadorn,  E., and  Graber,  H. (1946) New mutant reports. Dros. Inform. Serv. 20, 65.
•  Hopkins,  B. R.,  Sepil,  I., and  Wigby,  S. (2020) Structural variation in Drosophila melanogaster spermathecal ducts and its association with sperm competition dynamics. R. Soc. Open Sci. 7, 200130.
•  Jones,  D. T.,  Taylor,  W. R., and  Thornton,  J. M. (1992) The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8, 275–282.
•  Keisman,  E. L.,  Christiansen,  A. E., and  Baker,  B. S. (2001) The sex determination gene doublesex regulates the A/P organizer to direct sex-specific patterns of growth in the Drosophila genital imaginal disc. Dev. Cell 1, 215–225.
•  Kikkawa,  U. (2019) The story of PKC: a discovery marked by unexpected twists and turns. IUBMB Life 71, 697–705.
•  Kornberg,  T. (1981) engrailed: a gene controlling compartment and segment formation in Drosophila. Proc. Natl. Acad. Sci. USA 78, 1095–1099.
•  Kornberg,  T.,  Sidén,  I.,  O’Farrell,  P., and  Simon,  M. (1985) The engrailed locus of drosophila: in situ localization of transcripts reveals compartment-specific expression. Cell 40, 45–53.
•  Kosuda,  K. (1992) Chromosomal assignment of the genetic factor, tu-91k, responsible for a malanotic tumour in the Drosophila melanogaster adult female. Genet. Sel. Evol. 24, 561–565.
•  Kosuda,  K. (1996) Deleterious effect of the melanotic tumour on the survival rate of Drosophila melanogaster female flies. Zoolog. Sci. 13, 821–824.
•  Kumar,  S.,  Stecher,  G.,  Li,  M.,  Knyaz,  C., and  Tamura,  K. (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549.
•  Lindsley,  D., and  Zimm,  G. (1992) The genome of Drosophila melanogaster. Academic Press, San Diego.
•  Logan,  C.,  Hanks,  M. C.,  Noble-Topham,  S.,  Nallainathan,  D.,  Provart,  N. J., and  Joyner,  A. L. (1992) Cloning and sequence comparison of the mouse, human, and chicken engrailed genes reveal potential functional domains and regulatory regions. Dev. Genet. 13, 345–358.
•  Mayhew,  M. L., and  Merritt,  D. J. (2013) The morphogenesis of spermathecae and spermathecal glands in Drosophila melanogaster. Arthropod. Struct. Dev. 42, 385–393.
•  McGrath,  S. E.,  Michael,  A.,  Pandha,  H., and  Morgan,  R. (2013) Engrailed homeobox transcription factors as potential markers and targets in cancer. FEBS Lett. 587, 549–554.
•  Morgan,  R. (2006) Engrailed: complexity and economy of a multi-functional transcription factor. FEBS Lett. 580, 2531–2533.
•  Neubaum,  D. M., and  Wolfner,  M. F. (1998) Wise, winsome, or weird? Mechanisms of sperm storage in female animals. Curr. Top. Dev. Biol. 41, 67–97.
•  Newton,  A. C. (2018) Protein kinase C: perfectly balanced. Crit. Rev. Biochem. Mol. Biol. 53, 208–230.
•  Oliver,  C. P., and  Green,  M. M. (1944) Heterosis in compounds of lozenge alleles in Drosophila melanogaster. Genetics 29, 331–347.
•  Pitnick,  S.,  Marrow,  T., and  Spicer,  G. S. (1999) Evolution of multiple kinds of female sperm-storage organs in Drosophila. Evolution 53, 1804–1822.
•  Poole,  S. J.,  Kauvar,  L. M.,  Drees,  B., and  Kornberg,  T. (1985) The engrailed locus of drosophila: structural analysis of an embryonic transcript. Cell 40, 37–43.
•  Prokupek,  A.,  Hoffmann,  F.,  Eyun,  S. I.,  Moriyama,  E.,  Zhou,  M., and  Harshman,  L. (2008) An evolutionary expressed sequence tag analysis of Drosophila spermatheca genes. Evolution 62, 2936–2947.
•  Punia,  N.,  Primon,  M.,  Simpson,  G. R.,  Pandha,  H. S., and  Morgan,  R. (2019) Membrane insertion and secretion of the Engrailed-2 (EN2) transcription factor by prostate cancer cells may induce antiviral activity in the stroma. Sci. Rep. 9, 5183.
•  Sánchez,  L.,  Casares,  F.,  Gorfinkiel,  N., and  Guerrero,  I. (1997) The genital disc of Drosophila melanogaster. Dev. Genes Evol. 207, 229–241.
•  Sánchez,  L., and  Guerrero,  I. (2001) The development of the Drosophila genital disc. Bioessays 23, 698–707.
•  Saxena,  A.,  Scaini,  G.,  Bavaresco,  D. V.,  Leite,  C.,  Valvassoria,  S. S.,  Carvalho,  A. F., and  Quevedo,  J. (2017) Role of protein kinase C in bipolar disorder: a review of the current literature. Mol. Neuropsychiatry 3, 108–124.
•  Schnakenberg,  S. L.,  Matias,  W. R., and  Siegal,  M. L. (2011) Sperm-storage defects and live birth in Drosophila females lacking spermathecal secretory cells. PLoS Biol. 9, e1001192.
•  Schnakenberg,  S. L.,  Siegal,  M. L., and  Bloch Qazi,  M. C. (2012) Oh, the places they’ll go: female sperm storage and sperm precedence in Drosophila melanogaster. Spermatogenesis 2, 224–235.
•  Singh,  S. R.,  Oh,  S.-W.,  Liu,  W.,  Chen,  X.,  Zheng,  Z., and  Hou,  S. X. (2006) Rap-GEF/Rap signaling restricts the formation of supernumerary spermathecae in Drosophila melanogaster. Dev. Growth Differ. 48, 169–175.
•  Snodgrass,  R. E. (1993) Principles of Insect Morphology, with a new foreword by George C. Eickwort. Cornell University Press, Ithaca, NY.
•  Stecher,  G.,  Tamura,  K., and  Kumar,  S. (2020) Molecular evolutionary genetics analysis (MEGA) for macOS. Mol. Biol. Evol. 37, 1237–1239.
•  Steinberg,  S. F. (2008) Structural basis of protein kinase C isoform function. Physiol. Rev. 88, 1341–1378.
•  Sturtevant,  A. (1925) The seminal receptacles and accessory glands of the Diptera, with special reference to the Acalypterae. Journal of the New York Entomological Society 33, 195–215.
•  Sun,  J., and  Spradling,  A. C. (2012) NR5A nuclear receptor Hr39 controls three-cell secretory unit formation in Drosophila female reproductive glands. Curr. Biol. 22, 862–871.
•  Sun,  J., and  Spradling,  A. C. (2013) Ovulation in Drosophila is controlled by secretory cells of the female reproductive tract. Elife 2, e00415.
•  Tabata,  T.,  Schwartz,  C.,  Gustavson,  E.,  Ali,  Z., and  Kornberg,  T. B. (1995) Creating a Drosophila wing de novo, the role of engrailed, and the compartment border hypothesis. Development 121, 3359–3369.
•  Trong-Tue,  N.,  Thao,  D. T. P., and  Yamaguchi,  M. (2010) Role of DREF in transcriptional regulation of the Drosophila p53 gene. Oncogene 29, 2060–2069.
•  Wexelsen,  H. (1928) Two new mutant characters on the spermathecae of the females of Drosophila melanogaster. Cell-degenaration and supernumerary spemathecae. Genetics 13, 389–400.
•  Wizenmann,  A.,  Stettler,  O., and  Moya,  K. L. (2015) Engrailed homeoproteins in visual system development. Cell. Mol. Life Sci. 72, 1433–1445.