RESEARCH ARTICLE Cytoskeleton, April 2016 73:209–218 (doi: 10.1002/cm.21291) C V 2016 Wiley Periodicals, Inc. The Evolution of Sperm Axoneme Structure and the Dynein Heavy Chain Complement in Cecidomid Insects S. Ciolfi, C. Mencarelli, and R. Dallai* Department of Life Sciences, University of Siena, Siena, Italy Received 25 September 2015; Revised 10 February 2016; Accepted 1 March 2016 Monitoring Editor: Ritsu Kamiya The 9 1 2 axoneme of cilia and flagella is specialized machinery aimed at the production of efficient, finely tuned motility, and it has been evolutionarily conserved from protists to mammals. However, the sperm cells of several insects express unconventional axonemes, which represent unique models for studying the structural– functional relationships underlying axonemal function and evolution. Cecidomids comprise a group of dipterans characterized by an overall tendency to deviate from the standard axonemal pattern. In particular, the subfamily Cecidomyiinae shows a series of progressive modifications of the sperm axoneme. We previously analyzed the unusual sperm axonemes of Asphondylia ruebsaameni (Asphondyliidi) and Monarthropalpus buxi (Cecidomyiidi), which are characterized by the absence of any structure related to the control of motility (that is, the central pair complex, radial spokes and inner dynein arms); however, these sperm are motile, and motility is driven by the outer dynein arms only. This simplification of the motility machinery is accompanied by a parallel reduction in the dynein isoform complement. Here, we complete our survey of the axonemal organization and the parallel evolution of sperm dynein complement in cecidomids with the characterization of both the sperm ultrastructure and the dynein genes in Dryomyia lichtensteini, a representative of Lasiopteridi, the cecidomid taxon with aberrant and immotile sperm cells. On the basis of the whole set of our data, we discuss the potential molecular mechanism(s) underlying the progressive modification of axoneme in cecidomids, leading first to a reduction of dynein genes and eventually to the complete loss of motility. V 2016 Wiley Periodicals, Inc. C Key Words: sperm axoneme; dynein; insect; cecidomid *Address correspondence to: R. Dallai, Department of Life Sciences, University of Siena, Siena, Italy. E-mail: dallai@unisi.it Published online 3 March 2016 in Wiley Online Library (wileyonlinelibrary.com). Introduction T he 9 1 2 axoneme of cilia and flagella is a specialized and strictly integrated supramolecular protein machinery that has been achieved during eukaryotic evolution to provide cells with an efficient and finely tuned motility. As a consequence, both the architecture of this organelle and the basic molecular mechanisms underlying motility generation and control have been generally conserved from unicellular protists to mammals. Notwithstanding, unconventional axonemal organizations have been observed in several species scattered among evolutionarily distant taxa [e.g., Schrevel and Besse, 1975; Prensier et al., 1980; Gibbons et al., 1983; Perkins, 1991; Justine, 1998; Dallai et al., 2006, 2014; B^a et al., 2007; Mencarelli et al., 2008; Bru nanska et al., 2014; Diagne et al., 2015; Kacem et al., 2015]. These peculiar axonemes are generally endowed with a reduced or even absent motility which appears, however, to be fully compatible with species viability. In particular, insects provide an unusually high number of unconventional axonemal models expressed in the sperm cells of different species [Dallai, 2014]. Such variability in sperm architecture can reasonably be related to the widespread radiation and the high number of species in this animal group that—due to a short generation lifespan and a consequent high speciation rate, as well as to their capability of tolerating a variety of environments [Mayhew, 2007]—is the largest and most diversified among animal taxa. Starting from the basic 9 1 2 model that is still expressed in the basal collembolans, insects have achieved the 9 1 9 1 2 model, so-called for the presence of an important apomorphic feature, that is, a crown of nine accessory microtubules that surrounds the central axoneme. Such a feature is also present in Diplura, which thus can be considered the sister group of insects sensu strictu [Dallai et al., 2011; Misof et al., 2014]. In several insect taxa, however, this general organization displays a number of modifications, and several aberrant and even bizarre axonemes have 209 䊏 been described in many insect species [reviewed by Dallai et al., 2006; Mencarelli et al., 2008]. Consequently, the architecture of the insect sperm axoneme is generally recognized as a useful feature for the analysis of the phylogenetic relationships existing among different taxa [Dallai, 2014]. A puzzling question concerning the great variability of sperm axoneme organization in insects is how these peculiar models might have been accepted during the evolution of insect lineages. Besides providing phylogenetic information, the study of such modified insect axonemes, which may be considered as naturally occurring mutants, is also of interest in light of the potential contributions they may provide towards comprehending the structural–functional relationships underlying axoneme function and evolution. Cecidomyiidae, a family of the dipteran suborder Nematocera, comprise a group of insect species that are characterized by an overall tendency to deviate from the standard 9 1 9 1 2 axonemal pattern [Baccetti and Dallai, 1976; reviewed by Dallai et al., 2006; Dallai, 2014]. This trend has been particularly well analyzed in the subfamily Cecidomyiinae, in which an interesting series of progressive structural modifications of the sperm axoneme have been described [Dallai et al., 1996 a,b; Dallai, 2014]. In this group a strong differentiation has been accomplished between the axoneme organization expressed in the supertribes Asphondyliidi and Cecidomyiidi, on one side, and the supertribe Lasiopteridi on the other side; while the former are characterized by a highly aberrant axonemal organization which is, however, still able to guarantee a certain degree of motility, the latter possess immotile sperm cells that are endowed with just a residual rudimentary axoneme [Dallai, 1988, 2014; Dallai and Mazzini, 1989; Dallai et al., 1996a]. We previously focused our interest on the unusual sperm axonemes of the two species, Asphondylia ruebsaameni (Asphondyliidi) and Monarthropalpus buxi (Cecidomyiidi) [Lupetti et al., 1998; Mencarelli et al., 2000, 2001]. The high peculiarity of these models resides in the absence of any structure related to the control of motility (that is, the central pair complex, radial spokes and inner dynein arms). Notwithstanding, these sperm cells are motile, and motility is essentially driven by outer dynein arms only. This simplification of the motility machinery is accompanied by a parallel reduction in the gene complement coding for axonemal dyneins. In fact, dynein arms usually comprise at least seven different isoforms, each one with a distinct catalytic heavy subunit [Kamiya and Yagi, 2014]; accordingly, a comparative genomic analysis carried out on 24 different eukaryotic organisms identified seven gene families coding for axonemal dynein heavy chains [Wickstead and Gull, 2007]. In contrast, only one or two dynein heavy chain gene(s) could be found in Monarthropalpus [Lupetti et al., 1998] and in Asphondylia [Mencarelli et al., 2001], respectively, suggesting the occurrence of a progressive loss of dynein genes during the cecidomid lineage. 䊏 210 Ciolfi et al. Here, we complete our survey of the axonemal organization and the parallel evolution of the dynein complement in cecidomiids with the characterization of both the ultrastructure of the sperm cell and the dynein genes in Dryomyia lichtensteini, a representative of Lasiopteridi, the cecidomyiid taxon expressing aberrant and immotile sperm cells. On the basis of the whole set of our data, we discuss the possible molecular mechanism(s) that underlaid the progressive modification of the axoneme that occurred in this insect group, eventually leading to the complete loss of motility. Materials and Methods Origin and Collection of Samples Mature individuals, larvae and pupae of Dryomyia lichtensteinii, living in gall-midges on the leaves of Quercus ilex, were collected from the neighbourhood of Siena. Testes and deferent ducts were dissected in 0.1M phosphate buffer (PB), pH 7.2, for electron microscopic analyses. Transmission Electron Microscopy For thin-sectioning, the material was fixed for 1.5 h at 48C in 2.5% glutaraldehyde diluted in PB, rinsed in buffer overnight, and then postfixed in 1% osmium tetroxide for 1 h at 48C. The material was then dehydrated with a graded series of ethanol and embedded in Epon-Araldite. Before observation by TEM, thin sections were routinely stained with uranyl acetate and lead citrate. Part of the material was prepared according to Dallai and Afzelius [1990], using tannic acid impregnation. RNA Isolation and Reverse-transcription Polymerase Chain Reaction (RT-PCR) Analysis Dryomyia larvae or young pupae were frozen in liquid nitrogen and homogenized by Polytron homogenizer (Kinematica AG); total RNA was then extracted using the protocol described by Chomczynski and Sacchi [1987]. Total RNA was treated with DNaseI (1 U mg21 RNA; Promega) and then reverse transcribed using SuperScriptTM II Reverse Transcriptase (Invitrogen) and oligo-dT12–18 (Invitrogen), CDSIII/SMARTIII (Clontech) or random nonamers (Takara) in parallel experiments according to the manufacturer’s instructions. The resulting cDNAs were used in PCR reactions and amplified with 200 pmol of the degenerated primers—S2: 50 -CCCCGGATCCTGCTGG(GATC)AC(GATC)GG(GATC)AA(AG)AC-30 , and A2: 50 -GG GCGAATTC(TC)(AG)TT(AG)AA(TC)TC(AG)TC(AG) AA(AG)CA-30 —in a reaction mixture containing: 1.5 mM MgCl2, 0.5 mM each dNTP and 2,5U GotaqV DNA Polymerase (Promega). PCRs were performed using the following profile: 5 cycles of 948C for 1 min, 508C for 2 min and 728C for 2 min; 40 cycles of 948C for 1 min, 358C for 2 min and 728C for 2 min and an extension step R CYTOSKELETON at 728C for 10 min at the end. The amplification products were run on a 3% agarose gel and purified by the WizardV SV Gel and PCR Clean-Up System (Promega), then subcloned in pGEMV-T Easy Vector (Promega) and sequenced. The results we obtained were independent of the developmental stage and of the primers used to obtain cDNA. The above described experimental approach was previously used extensively to analyze the dynein heavy chain complement in several eukaryotic organisms [Asai et al., 1994; Gibbons et al., 1994; Rasmusson et al., 1994; Tanaka et al., 1995; Andrews et al., 1996; Porter et al., 1996; Lupetti et al., 1998; Mencarelli et al., 2001]. The two degenerate primers S2 and A2 were designed on strictly conserved sequences that are located around the catalytic domain of the dynein heavy chain genes [Asai and Criswell, 1995]. The primer A2 matches the sequence PAGTGKT of the P1-loop, which is the most highly conserved region of the dynein heavy subunit [Mikami et al., 1993], while S2, located 40 amino acids downstream, corresponds to the sequence CFDEFNR. The use of these primers results in the amplification of the nucleotide-binding region of both cytoplasmic and axonemal dyneins; however, two distinct consensus sequence motifs mark the two classes of dynein motors [Asai and Brokaw, 1993; Asai et al., 1994]. The two motifs are very similar to one another, but they contain a few notable differences that allow assigning any new sequence to either the cytoplasmic or the axonemal dynein family. In particular, the so-called motif A is common to all known axonemal dynein heavy chains, while motif B is an established hallmark of cytoplasmic dyneins. Both motifs have been evolutionarily conserved. R A total of 5 mg of genomic DNA was digested with the appropriate restriction enzyme and used for Southern blot analysis as described in Mencarelli et al. [2001]. R Specific PCR for Axonemal Dynein Gene Specific primers were designed on the genomic axonemal dynein gene and used in PCR reactions on cDNAs to prove the absence of gene expression. Sequence of specific primers: Dryax_for: 50 -GAATTCTGAAACTACAAAGAG TG-30 ; Dryax_rev: 50 -GGATCCGCCCCAAGATCCAGT TTG-30 . The PCR reaction mixture was the same described above for degenerate primers, while the amplification profile was: 958C for 10 min, followed by 40 cycles of: 958C for 1 min, 558C for 1 min, 728C for 2 min, and a final elongation step for 10 min at 728C. The PCR was performed in parallel on both Dryomyia total cDNA and genomic DNA, but we obtained only an amplification product of about 300bp in the genomic DNA. The PCR products were run on a 3% agarose gel and sequenced. DNA Isolation and Analysis Genomic DNA was extracted according to the method of Sambrook et al. [1989]. PCR amplifications on genomic DNA, as well as cloning and sequencing, were performed under the same conditions described previously for cDNA. CYTOSKELETON Sequencing Nucleotide sequences were obtained by Biofab Sequencing Center (Roma). Sequences were then analyzed by using the Basic Logical Aligment Search Tool (Blast) algorithm [Altschul et al., 1990]. Nucleotide sequences of axonemal and cytoplasmic dynein genes from D. lichtesteini genomic DNA were deposited in the GenBank/EMBL/DDBJ nucleotide sequence database under the accession numbers KT162141 and KT162142, respectively. Results The Structural Organization of the Sperm Cell in Dryomyia Consistent with all Lasiopterid species analyzed to date, D. lichtesteini possesses elongated, ear-like, immotile sperm cells that have completely lost the 9 1 9 1 2 organization characterizing many insect orders (Fig. 1). Typical features of these highly aberrant cells are the lack of an acrosome, the occurrence of a centrally located, irregularly elliptical nucleus with non-homogeneously condensed chromatin, and an abundance of singlet cytoplasmic microtubules, which are most often associated with the plasma membrane. Singlet microtubules also appear to sustain peculiar ruffle-like structures that project out of the cell (arrows in Figs. 1A and 1B). At the posterior level, no sperm tail endowed with a structured axoneme is present. However, microtubular doublets are evident in cross-sections of the sperm cell, where they appear to be quite variable, both in number and disposition, depending on the section level (Figs. 2B–2F). Nine doublets arranged in a regular circular organization can be observed only for an extremely short tract and in a region very close to the nucleus (Figs. 2A and 2B). In other sections, in which the doublets appear to be less strictly associated to the nucleus, they appear to be variably scattered in the cytoplasm (Figs. 2C–2F). These doublets do not carry any evident dynein projection, and appear to originate from a centriolar structure that is closely apposed to the nuclear envelope (Fig. 2A). The overall organization of Dryomyia sperm cells is schematized in Fig. 3. The whole set of observations suggests that the assembly of a 9 1 0 axoneme is indeed initiated in Dryomyia sperm cells, but also that, as long as the assembly proceeds, the axoneme very rapidly loses its integrity and doublets become randomly distributed in the cytoplasm. Furthermore, the occurrence of a variable number of doublets indicates that they may possess variable individual lengths, and that the ectopic assembly of microtubular doublets may sometimes occur (see Fig. 2D). The latter process has also Cecidomid Axonemal Dynein 211 䊏 Fig. 1. Longitudinal (A) and transversal (B) section of mature Dryomyia sperm cells. The cytoplasm is characterized by the occurrence of a large number of singlet microtubules, many of which are regularly aligned below the plasma membrane and engaged in the formation of long and thin extensions of the cytoplasm (arrows). The occurrence of an organized array of axonemal doublets is not appreciable. been previously documented in the sperm cells of the lasiopterid Mayetiola (Dallai and Mazzini, 1980). Molecular Analysis of Axonemal Dynein Gene(s) To establish whether the absence of dynein arms on microtubular doublets of Dryomyia sperm cells is due to a defect in the process of dynein arm assembly or binding onto the microtubular surface or, rather, to the loss of dynein gene expression in such an aberrant axonemal model, we employed the same approach we previously applied for the analysis of dynein genes in the cecidomid species Asphondylia and Monarthropalpus [Lupetti et al., 1998; Mencarelli et al., 2001]. Thus, we used degenerate primers designed for two strictly conserved sequences located around the catalytic domain (see Materials and Methods) to amplify the dynein heavy chain-related sequences from both transcripts and genomic DNA of whole Dryomyia pupae. Amplification products were cloned, sequenced and analyzed by the BLAST algorithm [Altschul et al., 1990]. All the deduced amino acidic sequences, either from genomic DNA or cDNA, were then checked for the presence of consensus motif A or B [Asai et al., 1994; see Materials and Methods]. We first analyzed the sequences obtained from transcript amplification by RT-PCR. Five PCR reactions were carried out on the cDNA obtained from five different Dryomyia samples, and each time 15–20 clones were analyzed randomly. All the nucleotide and the deduced amino acid sequences we obtained were submitted to the BLAST program and always revealed the presence of only one type of transcript that clearly corresponds to a fragment of cytoplasmic dynein (Dry-cyt) (Fig. 4A). In fact, it shows a nucleotide identity of 82% with the cytoplasmic dynein heavy chain from several species, including the two insect 䊏 212 Ciolfi et al. species Acromyrmex echinatior and Apis dorsata (GenBank: XM_011059491 and XM_006622875) and an amino acid identity of 96% with cytoplasmic dynein heavy chains from several other species, among which are both Homo sapiens and the insects Apis mellifera and Ixodes scapularis (GenBank AAA16065; XP_006570926; XP_002411858) (not shown). When the Dryomyia fragment is compared with the other known dynein heavy chain sequences from Cecidomyiidae species that we have previously studied, we found a nucleotide identity of 77 and 72%, respectively, with the two Asphondylia cytoplasmic sequences Asph-cyt1 and Asph-cyt2 (GenBank: AJ309816 and AJ309817), and of 79% with Mon-cyt, the single cytoplasmic dynein reported for Monarthropalpus flavus (GenBank: Y16229) [Lupetti et al., 1998; Mencarelli et al., 2001] (not shown). At the primary structure level, we observed an amino acid identity of 93% with Asph-cyt1, 81% with Asph-cyt2 (GenBank CAC32446 and CAC32447) and of 98% with Mon-cyt (GenBank CAA76126); the occurrence of motif B in all these sequences is evidenced in Fig. 4A. As far as it can be inferred from the partial sequence we have amplified and sequenced, the Dryomyia cytoplasmic fragment is more closely related to cytoplasmic dynein-1 than to dynein-2 (not shown). We underline that, notwithstanding the great number of clones sequenced, we were not able to identify any transcript coding for an axonemal dynein heavy chain. On the contrary, we could obtain evidence for both a cytoplasmic-type and an axonemal-type of dynein heavy chain sequence when the same degenerate primers were used to amplify genomic DNA. Two PCR fragments were amplified, one of 321 bp and the other of 237 bp; these were cloned, sequenced and the corresponding nucleotide and deduced amino acid sequences were analyzed by BLAST. The 321 bp and the 237 bp fragments were found to contain, respectively, the axonemal consensus motif A and the cytoplasmic motif B (Figs. 4A and 4B) and have CYTOSKELETON Fig. 2. A 9 1 0 axoneme is assembled close to the nuclear envelope in Dryomyia sperm cells (A–B); the original geometry is progressively lost, and microtubular doublets become scattered in the cytoplasm (C–F). N 5 nucleus; BB 5 basal body. thus been considered as fragments of an axonemal (Dry-ax) and a cytoplasmic (Dry-cyt) dynein heavy chain. The genomic cytoplasmic dynein sequence is identical to the sequence obtained by RT-PCR on Dryomyia cDNA, and presents an intron of 110 bp (not shown). The Dry-ax genomic sequence shares a 100 and 86% amino acid identity, respectively, with the two axonemal heavy chain fragments previously identified in Asphondylia ruebsaameni, Asph-ax1 and Asph-ax2 (accession numbers AJ309818 and AJ309819, respectively), and a 67% identity with Mon-ax (accession number Y16252), the only axoneCYTOSKELETON mal heavy chain expressed in Monarthropalpus [Lupetti et al., 1998; Mencarelli et al., 2001]. Also, the Dry-ax sequence revealed the presence of two introns: the first one of 80 bp and the other of 115 bp. Interestingly, the second intron occurs in the same position as the intron previously found in Asph-ax1 (Fig. 4B). This position corresponds exactly to the site of a 7 nucleotide deletion occurring in a putative dynein pseudogene of Monarthropalpus [Lupetti et al., 1998]. A Blast search using the axonemal and the cytoplasmic nucleotide sequences of Dryomyia dynein genes against the Cecidomid Axonemal Dynein 213 䊏 the basis of the genomic sequence coding for the axonemal dynein gene and used to test all the cDNAs and genomic DNAs used in this study by PCR. Once again, we could not amplify any sequence on the cDNAs, while we had a specific band of 321 bp on the genomic DNA. This band was sequenced and confirmed the presence of one single form for the axonemal dynein gene. When the motif A-containing genomic sequence Dry-ax was used to probe a Southern blot of Dryomyia genomic DNA, we could evidence only a single band for each one of the three restriction enzymes we used (EcoRI, EcoRV and HindIII) (Fig. 5); in this type of experiment, hybridization was performed under high stringency conditions to avoid any possible cross-reaction with the cytoplasmic dynein sequence. As a whole, these results indicate that only a single gene related to the axonemal type of dynein heavy chain sequences occurs in the Dryomyia genome, and that this gene is not transcribed during spermatogenesis. Discussion Fig. 3. Schematic diagram of the sperm cell organization in Dryomyia. Axonemal doublets (ad) and the basal body (bb) are in red, the Golgi apparatus (G) is in blue. N: nucleus; m: mitochondria, mt: cytoplasmic singlet microtubules. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] complete genome of the lasiopterid Mayetiola destructor, allowed the identification of two genomic fragments, a first one of 5089 bp (GeneBank: JXPD01008499) corresponding to the cytoplasmic dynein heavy chain gene, and a second one of 3988 bp (GeneBank: JXPD01020318) corresponding to the axonemal genomic dynein gene. The two fragments share the 84 and 70%, respectively, of nucleotide sequence with the genes from D. lichtesteini. Interestingly, the deduced amino acidic sequence of the axonemal dynein P1-loop from M. destructor, suggests the presence of two introns that are in the same position and of similar length (the first one is of 63 bp and the second is of 123 bp) as those identified in D. lichtesteini (Fig. 4B). The cytoplasmic genomic sequence does not show any intron in the same part of the catalytic domain of the gene. To confirm that the Dryomyia axonemal dynein heavy chain is not expressed, specific primers were designed on 䊏 214 Ciolfi et al. The motile 9 1 2 axoneme is an efficient molecular machinery whose function essentially relies on the activity of inner and outer dynein arms—which carry out the sliding process between adjacent microtubular doublets—and on the presence of a highly integrated regulatory system that locally governs their activation. The molecular pathway controlling dynein activity is not yet completely clarified, but it has been clearly established that it involves signals from both the central pair complex and the radial spokes [reviewed by Smith and Yang, 2004; Wirschell et al., 2011; King, 2012]. Different functional requirements are likely to be imposed on dynein activity during the different phases of the motility process, e.g. in the initiation of motility in the proximal part of the axoneme or in the transmission of motility along the axoneme. Such functional specialization is likely to underlie the molecular differentiation of axonemal dyneins that has been observed in most eukaryotic organisms, from protists to humans [Wickstead and Gull, 2007]. Axonemal dyneins share a common molecular organization, consisting of the so-called heavy, intermediate and light chain subunits, the former carrying the catalytic ATPase site and the latter being involved in regulatory/ structural roles [Kamiya and Yagi, 2014]. According to the so-called “multi-dynein hypothesis” [Asai, 1995], different heavy chain complements provide dynein arms with specific functional properties, as is also indicated by the differential localization of distinct inner arm dyneins along the Chlamydomonas flagella [Piperno and Ramanis, 1991; Gardner et al., 1994; Bui et al., 2012]. Based on the different features occurring in the heavy chain motor domain sequence, nine functional classes of dyneins have been identified, which includes two classes of cytoplasmic dyneins, two classes of outer arm dyneins, and five classes of inner arm CYTOSKELETON Fig. 4. Alignment of the hypothetical translational product of Dryomyia cytoplasmic (A) or axonemal (B) dynein heavy chain fragment (accession numbers KT162142 and KT162141, respectively) obtained from genomic DNA, with the corresponding cytoplasmic or axonemal dynein heavy chain fragments obtained from other cecidomid species. Asph: Asphondylia ruebsaameni; Mon: Monarthropalpus flavus; May: Mayetiola destructor. Motif A- and motif B-related sequences are underlined, and residues conserved in at least one other species are indicated in bold. Arrowheads mark the location of introns in Dry-ax, Asph-ax1 and May-ax sequence fragments. dyneins [Wickstead and Gull, 2007; Wilkes et al, 2008]. Such a clustering of dynein heavy chain sequences into nine branches has been evolutionarily conserved among eukaryotic organisms and occurs in all metazoans expressing motile 9 1 2 cilia or flagella, from simple protists up to humans. Thus, it appears that the expression of multiple forms of dynein is a common feature of axonemes capable of coordinated motility. In particular, inner dynein arms, which are strictly connected with the controlled generation of motility, appear to be indispensable for motility. Mutant Chlamydomonas strains with large defects in the inner dynein arms are immotile [Kurimoto and Kamiya, 1991]. Moreover, while axonemes endowed with only the inner dynein arms are expressed in diverse organisms [Gibbons et al., 1983; Hyams and Campbell, 1985; Dallai et al., 1992; Dallai and Afzelius, 1994; Woolley, 1997; Lupetti et al., 2011; Dallai, 2014; Mencarelli et al., 2014;] and are still motile, an extremely limited number of protozoan and metazoan species are known which express axonemes whose motility is based only on the activity of outer dynein arms [Dallai, 1988; Dallai et al., 1996a,; Wickstead and Gull, 2007; Mencarelli et al., 2008]. Interestingly, all the metazoan species are comprised in the same group of insects, the dipteran family Cecidomyiidae [Dallai, 1988; Dallai et al., 1996a,; Mencarelli et al., 2008]. Two distinct evolutionary trends have occurred within this group, one comprising the subfamilies Lestremiinae and Cecidomyiinae, and the other leading to Lasiopteridae [Dallai and Mazzini, 1989; Dallai et al., 1996a]. The first trend still retains a geometric— CYTOSKELETON though deeply altered—organization of the axoneme, which is characterized by the absence of the central complex (substituted for by elongated, axial mitochondria), by a Fig. 5. Southern blot analysis of Dry-Ax axonemal dynein heavy chain genes in genomic DNA from Dryomyia digested with EcoRI (E), HindIII (H), EcoRV (V) and double digestions with EcoRI/HindIII (E/H), EcoRI/EcoRV(E/V) and EcoRV/HindIII (V/H). Molecular sizes of the bands are expressed in kilobases (kb). Cecidomid Axonemal Dynein 215 䊏 progressively increasing number of doublets (up to 2500 in Asphondylia), and by the loss of the inner dynein arms, which are only present in the less evolved Lestremiinae [Dallai and Mazzini, 1983; Dallai et al., 1996b]. However, and albeit only under particular conditions, these sperm cells are motile [Lupetti et al., 1998; Mencarelli et al., 2001]. The high ultrastructural complexity of their axonemes is only apparent, being accompanied by the molecular simplification of the motility apparatus. In fact, we have reported that the loss of the central pair/radial spoke complex is not only followed by the loss of the inner dynein arms, but also results in a reduced complement of heavy chains in the outer dynein arms [Lupetti et al., 1998; Mencarelli et al., 2001]. The sperm cells of the species belonging to the other trend, on the contrary, assemble 9 1 0 axonemes that are able to maintain their ultrastructural integrity only in their very proximal part, after which doublets appear scattered in the cytoplasm [this article; Dallai and Mazzini, 1989; Dallai et al, 1996b]. Both dynein arms are lost in this evolutionary lineage, and sperm cells are immotile. Our present data indicate that, in Dryomyia, only a single gene coding for an axonemal dynein heavy chain occurs. However, this gene does not appear to be transcribed in sperm cells, as the inability to obtain amplification products in RT-PCR experiments using degenerate primers, as well as primers specific for the genomic sequence, strongly suggests. Such an absence of axonemal transcripts may be indicative of a defect in the upstream regulatory process controlling the expression of this gene, or, alternatively, the gene might be a pseudogene, as has been reported in the cecidomid Monarthropalpus (Lupetti et al., 1998). In the first case, the defect might be restricted to the germ line, and the gene might be actively expressed in the dendritic cilia of chordotonal organs; our inability to detect by RTPCR its transcript in the larval or pupal cDNA could be attributable to its extremely low abundance. However, we note that, though sensory cilia have been shown in Drosophila to be endowed with dynein arms [Kavlie et al., 2010; Karak et al., 2015], most of the information that is currently available is limited to this species, and no observations have been reported on the sensory organs in cecidomids. It is thus possible that, similar to sperm cells, cecidomid sensory organs have realized structural adaptations allowing them to accomplish their function in the absence of dynein arms. Such a possibility might be coherent with our preliminary finding suggesting that in Dryomyia cytoplasmic dynein-2 might also be absent; in fact, in Drosophila, this dynein has been demonstrated to be involved in ciliogenesis, but not in spermatogenesis [Han et al., 2003; Sarpal et al., 2003]. Further studies are required to clarify the functional morphology of chordotonal organs in cecidomids. In both the two cecidomid evolutionary lineages, the loss of one or both types of dynein arms is preceded by dramatic 䊏 216 Ciolfi et al. alterations of the axoneme geometry. In this regard, we note that the basic axonemal model common to both lineages is likely to have been a 9 1 0 model, similar to that occurring in the porrycondylid group of Cecidomyiidae [Dallai et al., 1996c]. In other words, the loss of the central pair complex seems to be the first evolutionary event that apparently opens the door to successive ultrastructural modifications. In the 9 1 2 axoneme, this axonemal structure plays a crucial functional role, is assembled through a route that is distinct from that of microtubular doublets [McKean et al., 2003], and its assembly poses specific molecular requirements [Nielsen et al., 2001]. The central pair complex has been proposed to act as a major constraint on the evolution of the axoneme, since it provides motility with a greater coordination and hence a greater efficiency [Mitchell, 2004]. Indeed, the strict evolutionary conservation of specific sequence features in the carboxyterminal domain of beta tubulin has been shown to result from specific structural requirements imposed by the assembly of central pair [Nielsen and Raff, 2002]; as a consequence, their removal by site-directed mutagenesis results in the assembly of 9 1 0 axonemes [Nielsen et al., 2001]. In this context, it is interesting to note that, in Drosophila, removal of the isotype-defining 15 carboxyterminal residues from the testis-specific b2 tubulin results not only in the loss of the central pair complex, but also in a strongly diminished stability of the axoneme [Fackenthal et al., 1993; Nielsen et al., 2001]. In fact, in these mutants the assembly of the sperm axoneme starts correctly at the basal body, but then the axoneme maintains its circumferential arrangement only over an extremely short tract of its length, and distally loses its coherent organization [Nielsen et al., 2001]. Also, such disorganized doublets do not carry any type of projections, including dynein arms and an ectopic tenth doublet can sometimes be observed. Similarly, we have shown here that Dryomyia sperm cells start to assemble a circular 9 1 0 axoneme close to the nuclear envelope, but then doublets— deprived of projections—soon become disorganized, and ectopic doublets may occur. No information is currently available on the axonemal tubulin sequence in any cecidomid species, but we note that the beta tubulin specialized sequence features that are required for central pair assembly overlap with the polyglycylation sites [Xia et al., 2000; Nielsen et al., 2001; Hoyle et al., 2008]; this post-translational modification is evolutionarily conserved and is required for axoneme assembly [Thazhath et al., 2002, 2004], but is strongly reduced in cecidomid sperm flagella [Mencarelli et al., 2004, 2005]. It seems therefore likely that some alterations in the tubulin properties may have occurred during cecidomid evolution. Our present data complete the previous studies we carried out on the organization of cecidomid sperm flagella and the composition of their dynein isoforms [Lupetti et al., 1998; Mencarelli et al., 2001]. As a whole, our data sustain the hypothesis of a crucial role of the central pair CYTOSKELETON complex in limiting the evolutionary variability of the 9 1 2 axoneme and its molecular components. Once the structures responsible for coordinated control of motility are lost, the expression of multiple dynein isoforms becomes dispensable; as a consequence, mutations are allowed to accumulate in dynein genes and/or in their regulatory sequences, leading to a progressive reduction of the expressed dynein heavy chain complement. Such a trend is exemplified by the dynein heavy chain complement in the three cecidomid species we have analysed: the outer dynein arm comprise in fact a heterodimer of two distinct heavy subunits in Asphondylia [Mencarelli et al., 2001] but is a homodimer in Monarthropalpus [Lupetti et al., 1998], while no heavy chain seems to be expressed in Driomyia (this article). 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