BACKGROUND
to execute a complex behavior, an animal must perceive and integrate information from various sources, process this information and perform the appropriate task.
in many animals, one such complex behavior is mating, as different aspects of this behavior must be coordinated to ensure sexual reproduction.
male mating behavior in c. elegans affords the opportunity to study a complex multi-step behavior in an organism with a relatively simple nervous system.
when mating, c. elegans males exhibit a highly reproducible series of behavioral steps, culminating in the fertilization of hermaphrodite eggs  <cit> .
briefly, when the male comes in contact with a hermaphrodite, the male responds by placing the ventral side of his tail against the hermaphrodite, and moves backward along the body, scanning for the vulva.
if the male's tail reaches the end of the hermaphrodite before encountering the vulva, the male turns and continues scanning the other side.
when the vulva is located, the male ceases backward motion, prods with his copulatory spicules, inserts them into the vulva, and transfers sperm into the uterus  <cit> .
although the steps of reproductive behavior have been described in many organisms, relatively little is known about the genes that specifically control these behaviors  <cit> .
we and others have begun molecular genetic analyses of the c. elegans male response  <cit> , turning  <cit> , vulval location  <cit> , spicule insertion  <cit> , and most recently, sperm-transfer  <cit>  behaviors.
by dissecting each step, their eventual integration as a complex series of overlapping sub-behaviors can be understood, and provide insight into nervous system function.
in addition, the study of each individual step, each with its own unique aspects, may provide knowledge about the regulation of behavior not gotten from studying the other steps of mating.
for example, the sperm-transfer sub-behavior in c. elegans is slower than any of the other mating steps, and does not occur instantaneously upon spicule insertion; therefore, its analysis will further our understanding of behavior.
to generate a behavior, an animal must employ its nervous system to perceive and integrate stimuli, and often coordinate this information with non-neuronal targets necessary for its proper execution.
what is known about the neuronal contribution to sperm-transfer behavior comes from systematic ablation of the sexually dimorphic neurons in the male to assign them function during mating behavior  <cit> .
each of the two male spicules contains three neurons: two sensory neurons, designated spv and spd, and the motor neuron spc  <cit> .
ablation of the spv spicule neurons leads to premature sperm transfer at the vulva; therefore, the spv neurons regulate the timing of sperm transfer by acting as a negative regulator  <cit> .
less is known about non-neuronal contribution to sperm-transfer behavior.
gower et al have recently shown that signaling mediated by inositol  <dig> ,5-triphosphate is required for efficient sperm transfer and propose that the site of action for this signaling is the male somatic gonad  <cit> .
here we present an analysis of the sperm-transfer step of male mating behavior.
direct observation suggests that sperm transfer comprises four sub-steps: initiation, release, continued transfer, and cessation.
in a genetic screen for mutations affecting successful copulation, we isolated mutants in the sperm transfer process.
of the mutants we isolated, unc- <dig>  the focus of this work, has defects in two distinct sub-steps of sperm transfer: initiation and continued transfer.
unc- <dig> is a member of the sm family of highly conserved proteins involved in membrane-trafficking pathways, particularly in regulated exocytosis  <cit> .
the mammalian unc- <dig> homologue  has been reported to be expressed in neurons, as well as in non-neuronal cell types  <cit> .
in c. elegans, unc- <dig> expression and function has been previously reported only in neuronal cells  <cit> .
here we show that, while the unc- <dig> site of action for initiation involves neuronal expression, the continuation of transfer involves non-neuronal expression of unc- <dig> in the male gonad.
RESULTS
steps of transfer and timing
as a first step toward understanding the molecular genetic processes underpinning sperm-transfer behavior and its regulation in c. elegans, and to establish a context to characterize sperm transfer mutants, we further defined this behavior.
our observation of wild-type sperm transfer and subsequent genetic screening  has defined at least four steps in the transfer process: initiation, release, continued transfer, and cessation .
once a male has inserted his spicules into the hermaphrodite's vulva, there is movement of sperm out of the seminal vesicle, which we call 'initiation'.
the initiation step, from tonic insertion of spicules to sperm exit from the seminal vesicle, takes  <dig>  ±  <dig>  seconds  .
no gross changes in morphology were observed, except for the exit of sperm from the seminal vesicle, through the 'valve region', and into the tube formed by the vas deferens.
the sperm leave the seminal vesicle, travel through the vas deferens, and are released from the animal through the cloaca, a sub-step termed 'release' .
the anal sphincter is contracted at this time, presumably to open the cloaca  <cit> .
release of sperm takes  <dig>  ±  <dig>  seconds  from the time of insertion, and failure at this step causes sperm to collect in the vas deferens.
failure to release is rarely observed in wild-type animals; however, if a male's spicules fall out of the vulva while transferring sperm, sperm will sometimes collect in the vas deferens.
sperm continue to exit the seminal vesicle in a genetically separable step  that we call 'continued transfer' .
when all sperm that exit the seminal vesicle have been released, the spicules retract into the male.
this is the 'cessation' sub-step and the termination of sperm transfer .
the entire process from spicule insertion to cessation takes  <dig>  ±  <dig> seconds .
liu and sternberg reported that it took approximately  <dig> seconds, compared to our reported  <dig> seconds, to transfer sperm  <cit> .
we attribute the discrepancy between the results to the microscope used for behavioral analysis .
because the majority of sperm to be transferred are ejaculated in the beginning of the transfer process, it is difficult to observe without the use of a high-magnification dissecting microscope that as the behavior progresses, the male still continues to intermittently transfer a few sperm.
screen for mating behavior mutants
to begin to answer fundamental questions about how the sub-steps of sperm transfer are regulated, we performed a genetic screen designed to isolate males defective for this behavior.
briefly, plg- <dig> males will extrude a gelatinous substance that puts a 'plug' over the hermaphrodite vulva after sperm transfer  <cit> .
the plg- <dig> allele is a dominant allele derived from a wild c. elegans isolate that was introgressed into the n <dig> wild-type strain.
by screening for the lack of copulatory plugs in a clonal population of ems mutagenized worms, we were able to isolate mutants defective in all steps of mating .
secondary screening of putative mutants by observation of mating behavior allowed us to determine those mutants with defects in sperm transfer.
to isolate only those with non-developmental mating defects, we screened through the mutants we isolated for defects in morphology, focusing on those with wild-type anatomy.
one recessive mutant isolated, sy <dig>  is defective in the initiation step of sperm transfer.
while sy <dig> mutant males will insert their spicules into the hermaphrodite vulva and remain tonically inserted for  <dig>  ±  <dig> seconds   from wild type), they rarely initiate sperm transfer .
a second recessive mutant isolated, sy <dig>  allows initiation and release, but is defective in the continuation of sperm transfer.
sy <dig> males transfer very few sperm, although sperm are clearly visible in the seminal vesicle, and sy <dig> males keep their spicules tonically inserted during the transfer behavior for  <dig>  ±  <dig> seconds .
although the duration that sy <dig> males remain with their spicules inserted in the hermaphrodite vulva is less than wild type , they should transfer significantly more sperm, as the bulk of transfer should have already occurred by this time.
the sy <dig> mutant allows us to genetically separate the continued-transfer step from initiation and release steps.
for the remainder of this study, we will focus on the analysis of sy <dig>
a for all genotypes carrying an extrachromosomal array , at least three independent lines were assayed except for unc-18; ex, for which only two lines each were tested.
these results are the combined data from individual lines.
see additional file  <dig> for results of individual lines.
b wild type is strain ps <dig>  plg-1; him- <dig>
c rescue is defined as being different from unc- <dig>
using anova with a tukey-kramer multiple comparisons test .
sy <dig> acts downstream or in parallel to spv
as mentioned above, the spv neurons regulate sperm transfer by inhibiting release until the spicules have penetrated the vulva: spv-ablated wild-type males prematurely transfer sperm before spicule insertion  <cit> .
because sy <dig> has the opposite phenotype  we ablated the spv neurons in the sy <dig> mutant to see if this could suppress the initiation defect.
in particular, we ablated the b.β cell, the precursor of both spv neurons  .
although the b.β ablation also eliminates four of  <dig> structural cells , the sheath cells are not necessary for spicule morphology, and the function of the socket cells is redundant  <cit> .
in addition, ablation of the b.β cell causes the same defect in sperm transfer as the ablation of the spv alone  <cit> .
we found that ablation of the b.β cell  in the sy <dig> mutant did not suppress the initiation defect .
this observation suggests that the defect in our mutant lies downstream or in parallel to the spv regulation in the sperm-transfer pathway.
a number of animals assayed.
b scored as animals that transfer sperm without tonic contraction of spicules in the vulva.
c percentage altered is the number of animals with altered transfer divided by the total number of animals tested.
altered transfer for wild type includes premature transfer and no transfer.
altered transfer for sy <dig> includes premature transfer and wild-type transfer.
d wild type is strain ps <dig>  plg-1; him- <dig>
molecular characterization of sy671
to understand the sperm-transfer initiation defect in sy <dig> males, we cloned this locus.
we employed single-nucleotide polymorphism  mapping  <cit>  to map sy <dig> to a 120-kb interval between the polymorphisms f26a10: <dig> and c47c12: <dig> .
injection of cosmids and pcr fragments narrowed the sy <dig> locus to a  <dig> -kb region containing f27d <dig>  .
we sequenced the predicted coding and non-coding regions of unc- <dig> from the sy <dig> mutant, and found that the sy <dig> mutant contains a g→a missense mutation in an unc- <dig> exon, which changes the amino-acid arg <dig> to his .
in c. elegans, unc- <dig>  which encodes a member of the sec1/munc- <dig> family of proteins, is required in neurons for synaptic-vesicle exocytosis  <cit> .
for exocytosis to occur, secretory vesicles must dock, be primed, and then fuse with the plasma membrane  <cit> .
unc- <dig> has been proposed to function in each of these steps of exocytosis, based on work in various systems, and may also act as a trafficking factor for other proteins necessary for exocytosis  <cit> .
although unc- <dig> has been postulated to act in various steps of the exocytosis pathway, in c. elegans it has been shown that there is a reduction of docked vesicles at the active zone in unc- <dig> mutants, indicating that unc- <dig> functions at least as a facilitator of vesicle docking in c. elegans  <cit> .
mutations in unc- <dig> cause locomotor defects and acetylcholine accumulation, and therefore unc- <dig> has been implicated in the release of acetylcholine  <cit> .
unc- <dig> interacts with syntaxin, a protein believed to mediate fusion of synaptic vesicles to the plasma membrane and hence neurotransmitter release  <cit> .
while most alleles of unc- <dig> have severe locomotor defects, sy <dig> males have only mild locomotor defects .
although the amino-acid change in sy <dig>  occurs in a residue conserved among unc- <dig> orthologs , this residue lies outside the syntaxin-binding region  <cit> .
we were intrigued by the unique position of the molecular lesion in the sy <dig> allele of unc- <dig>  all reported sequenced alleles of unc- <dig> have a molecular defect in either exon  <dig> or exon  <dig>  <cit> ; however, sy <dig> has its defect in exon  <dig> .
of the other unc- <dig> mutants, only two, md <dig> and md <dig>  move sufficiently well to allow sperm transfer to be assayed.
although their movement is indistinguishable from sy <dig>  both mutants are able to initiate transfer .
this difference among alleles leads us to speculate that the mutation in sy <dig> has some specificity for sperm transfer through a mechanism different from the function of unc- <dig> in vesicle release.
to address if a perturbation of vesicle release is the likely defect in sy <dig> males, we assayed males that were transheterozygous for sy <dig> and for a severe allele of unc- <dig>  b <dig>  the unc- <dig> allele has a single amino-acid substitution  that causes the accumulation of acetylcholine, the inability to bind syntaxin, and a severe locomotor defect, all characteristics of defective vesicle release  <cit> .
if sy <dig> does have a defect in a yet uncharacterized unc- <dig> function, we would expect that when placed in trans to an allele that perturbs vesicle release through a single amino-acid substitution, we would see compensation for the sperm-transfer defect.
however, the b <dig> allele fails to complement sy <dig> for the initiation of transfer , and therefore it seems likely that the mutation in sy <dig> also alters vesicle release.
unc- <dig> is expressed in the male gonad and nervous system
previous characterization of unc- <dig> expression using polyclonal antisera showed that unc- <dig> was present in ventral-cord motor neurons and some unidentified head neurons in the adult hermaphrodite  <cit> .
to replicate the expression in the hermaphrodite and determine expression in the male, we fused the unc- <dig> upstream sequences used in the mutant-rescue experiment to a yfp reporter.
in transgenic animals, this reporter  recapitulated the previously observed expression in the hermaphrodite.
in the male, unc-18::yfp showed the same expression in the ventral cord and head, but surprisingly also had strong expression in the gonad.
the expression was strongest in the 'valve region', a non-neuronal tissue that lies at the junction of the seminal vesicle and the vas deferens .
expression was also present in most, if not all neurons in the male tail .
site of action: initiation
to define the site of action of unc- <dig> in sperm-transfer behavior, we created a yfp-tagged version of unc- <dig> under the regulation of the unc- <dig> upstream regulatory region , which was able to rescue the sy <dig> sperm transfer initiation defect .
this construct was also able to confer the null allele, unc- <dig>  with the ability to initiate sperm transfer .
next we expressed the unc-18::yfp functional protein under different tissue-specific, transcriptional-control regions to define its site of action in sperm-transfer behavior, beginning with the two most probable candidates: the gonad and the spicule neurons.
because unc- <dig> expression in the male gonad coincides with the region where sperm exit from the seminal vesicle to the vas deferens, this seemed a likely candidate for the site of action of unc- <dig> in the initiation of sperm-transfer behavior.
an equally attractive candidate is the spv spicule neurons in the male tail, as they are known regulators of sperm transfer.
to express unc- <dig> under a gonad-specific promoter, we used a portion of the described itr- <dig> gene  <cit> .
it was previously reported that two introns and one exon of the itr- <dig> gene  gave expression in the intestine, pharyngeal isthmus, and gonad of hermaphrodites  <cit> .
through further dissection of this region, called the pc promoter, we were able to drive expression  in the seminal vesicle, the valve region, and the vas deferens of the male .
this expression coincides with that described for the entire pc promoter in the male  <cit> .
we call this element int2itr- <dig>  we then expressed unc- <dig> under the control of the itr- <dig> gonadal enhancer , but were not able to rescue the mutant phenotype in sy <dig> .
to express unc- <dig> under a spicule neuron promoter, we used  <dig> kb of 5' upstream sequence of the gpa- <dig> gene that drives expression in all three types of spicule sensory neurons, including the spv neurons  <cit>  .
the gpa-1::unc-18::yfp transgene was also unable to rescue the sperm-transfer initiation defect in sy <dig> .
after initial testing of possible gonadal and spicule neuron contributions, we chose to express unc- <dig> more broadly to see if unc- <dig> was at least acting in neurons to initiate the transfer of sperm.
we used a pan-neuronal promoter   <cit>  to drive expression of unc- <dig>  in neurons , and also used a body-wall muscle promoter  to rule out non-specific rescue effects  <cit> .
the neuronal expression of unc- <dig> was able to rescue the initiation defect .
these results suggest that although unc- <dig> is expressed in the male gonad, its expression in this tissue is not required for the initiation of sperm transfer, but it is required in neurons.
to confirm this result, we used the 5' regulatory region of the aex- <dig> gene, another pan-neuronal promoter, to express unc- <dig>   <cit> .
as expected, this transgene was able to rescue the sperm-transfer initiation defect in sy <dig> .
the negative result using gpa-1::unc-18::yfp to drive expression in the spicule neurons does not exclude an unc- <dig> contribution from these neurons; therefore, we decided to further define the site of action of unc- <dig> in this behavior using various neuronal promoters.
unc- <dig> was initially described to be expressed in all ventral-cord motor neurons  <cit> .
the ventral cord has been divided into eight classes of motor neurons: four classes that innervate ventral muscles  and four classes that innervate dorsal muscles   <cit> .
the d-type ventral cord neurons  are gamma-aminobutyric acid ergic and the others  are cholinergic  <cit> .
the vc cholinergic neurons are hermaphrodite-specific, while in males, the homologous cells divide to give the male-specific cp and ca motor neurons  <cit> .
to test whether unc- <dig> was acting exclusively in either cholinergic or gabaergic ventral cord motor neurons to initiate sperm transfer, we drove expression specifically in each type.
to express unc- <dig> in all gabaergic neurons in the sy <dig> mutant, we used the promoter region from the unc- <dig> gene to drive expression   <cit> .
expression in only the gabaergic neurons was unable to rescue our mutant .
a for all genotypes tested, at least three independent lines were assayed.
these results are the combined data from individual lines.
see additional file  <dig> for results of individual lines.
b rescue is defined as being different from unc- <dig> in table  <dig>
using anova with a tukey-kramer multiple comparisons test .
c nd, not done.
these lines were not assayed because the introduction of the transgene caused severe mating behavioral defects in steps prior to sperm transfer assay.
to express unc- <dig> in all cholinergic neurons in the sy <dig> mutant, we initially tried using promoters from both unc- <dig>  <cit>  and cho- <dig> <cit>  to drive expression.
however, the use of either of these promoters , caused penetrant locomotion and mating behavioral defects in the steps prior to sperm transfer, and therefore the worms could not be assayed for their ability to transfer sperm.
these defects, not seen in non-transgenic worms, could be caused by overexpression of unc- <dig> or possible titration of promoter regulatory proteins caused by our transgenic arrays.
next we tried the acr- <dig> promoter, which drives expression in the va, vb, da, and db cholinergic ventral-cord motor neurons  <cit> .
the acr- <dig> construct, acr-2::unc-18::yfp, when expressed in sy <dig> mutants, was able to rescue initiation .
because acr- <dig> is expressed in cholinergic neurons throughout the ventral cord and not in the male spicule sensory neurons , these ventral-cord motor neurons are strong candidates for contributing to the initiation of sperm transfer.
to further test ventral-cord input for the initiation of sperm-transfer behavior and to further pinpoint the site of action to a specific subset of cells, we used additional promoters to attempt to rescue the sy <dig> defect.
we initially tested two classes of cholinergic ventral-cord motor neurons, the a-type neurons, va and da  and the b-type neurons, vb and db   <cit> .
we used the unc- <dig> upstream sequences to drive expression in the a-type neurons  <cit>  and the acr- <dig> promoter to drive expression in b-type neurons  <cit> .
by expressing unc- <dig> under the acr- <dig> regulatory region, we were able to rescue the sy <dig> initiation defect , but were unable to do so with the unc- <dig> promoter .
this result suggests that unc- <dig> in the b-type neurons  could be necessary for initiation, again with the exception of the neurons outside the ventral cord that express acr- <dig>
we determined that acr- <dig> is expressed in the male-specific ray sensilla  using the acr- <dig> regulatory region to drive expression of dsred <dig> .
the expression in the rays appears to be in the a-type ray neurons, as acr- <dig> expression in the tail does not overlap with pkd-2::gfp, which is expressed in the b-type ray neurons   <cit> .
acr- <dig> also shows expression in a cell in the right preanal ganglion, which we have tentatively identified as pga or pvz.
however, input from the acr- <dig> expressing cells in the male tail in initiating transfer is very unlikely, as there is no overlap with acr- <dig> expression in the tail , yet both promoters driving unc- <dig> expression can rescue the initiation defect in unc- <dig> mutants.
the overlap in expression of acr- <dig> and acr- <dig> is in the ventral cord , making a strong case for it being necessary for the initiation of sperm transfer.
although these data also suggest it is specifically the b-type motor neurons in the ventral cord that are relevant for the initiation of sperm transfer, further experiments indicate that the male-specific ca ventral-cord motor neurons are stronger candidates .
in the adult male ventral cord we noticed that acr-5::yfp was expressed not only in the b-type neurons, but also in additional neurons .
because the male has additional ventral cord neurons, the cps and cas, we thought that these may account for the additional cells showing expression, and it may be these cells that are necessary for initiation of sperm transfer.
because the cp neurons are necessary for efficient turning behavior  <cit> , we wanted to test if the acr-5-expressing cells were cp neurons.
we used a strain carrying a tph-1::gfp transgene that drives expression in the cp neurons  <cit> , and crossed it into animals expressing acr-5::dsred <dig>  the additional cells in the male ventral cord that express acr-5::dsred <dig> did not overlap with the cp-expressing cells, but in many cases these cells were anterior to the cp neurons, suggesting that they could be ca neurons  <cit>  .
because there are no reported markers for the ca neurons, we tested the possible contribution of these neurons to sperm-transfer initiation by assaying lin- <dig> mutant males.
the lin- <dig> gene, which encodes a homeobox gene, is required for mid-body region-specific development in c. elegans  <cit> .
in wild-type males, p.aap divide during late l <dig> to generate male-specific motor neurons.
the posterior cells, p.aapp, are the cp neurons  and the anterior cells, p.aapa, are the ca neurons   <cit> .
in lin- <dig> mutant males, p.aap  do not divide, but instead have a compact neuron-like morphology or die  <cit> .
also affected in lin- <dig> mutant males are p.aapp , which take on a more posterior fate, as judged by the loss of serotonin staining in cp <dig> and cp <dig>  <cit> .
this observation suggests that p.a might be transformed into p <dig> a-like cells  <cit> , thus also affecting ca <dig> and ca <dig>
if the ca neurons  are necessary for initiation of sperm transfer, we would expect lin- <dig> males to be unable to initiate this behavior, as these cells have been genetically ablated or misfated.
we tested two alleles of lin- <dig>  and both were unable to initiate sperm transfer , further supporting a role for the ca neurons and confirming ventral cord involvement, limiting it to the mid-body region.
to further corroborate that the unassigned ventral cord motor neurons expressing acr- <dig> in the wild-type males could be ca neurons, we analyzed the expression of acr-5::gfp in lin- <dig> mutant males.
as expected, we saw fewer cells expressing acr-5::gfp in the ventral cord of lin- <dig> mutant males .
site of action: continued transfer
although the sy <dig> mutants can be rescued for the initiation of sperm transfer using a transgene to drive neuronal expression of unc- <dig> in a subset of ventral cord cholinergic neurons , these animals did not transfer as many sperm as wild type.
we observed that by using the unc- <dig> promoter to drive unc- <dig> expression, the animals would initiate, and usually the sperm that were released from the seminal vesicle were released from the animal, but the animals transferred much less sperm and thus appeared to be defective in the continuation of transfer.
this observation raised the possibility that unc- <dig> functions in two separate sub-steps of the sperm-transfer process , and that neuronal expression of unc- <dig> was responsible for the initiation of transfer, while the gonad expression may be necessary for continued transfer.
to formally test this possibility, we hypothesized that if we could add back both neuronal and gonadal expression of unc- <dig>  using different 5' control regions, we should restore wild-type sperm transfer to the unc- <dig> mutants.
to do this, we again used our neuronal  construct, and remade our gonadal transgene with a cfp marker  to facilitate the scoring of worms carrying both transgenes.
to quantify the amount of sperm transferred, we devised a 'continuation assay' .
this assay uses the percentage of cross progeny as a proxy for the amount of sperm transferred.
as a control for the effectiveness of our continuation assay, we tested unc- <dig> mutants carrying a rescuing transgene  to determine if they could sire a wild-type percentage of cross progeny.
not only did we observe that these transgenic animals appeared to transfer a similar amount of sperm to that of wild type, but as expected they sired an equivalent percentage of cross progeny .
this construct also was able to completely restore wild-type sperm transfer in the unc- <dig> null mutant .
in addition, as expected based on observation of sperm transfer, animals carrying the unc-119-driven transgene sired much fewer progeny than wild type in our assay , because they initiated transfer and released sperm, but did not continue to transfer.
in addition, lines carrying only gonadally expressed unc- <dig>  did not sire cross progeny , consistent with an inability to initiate transfer.
we then tested if we could restore wild-type sperm transfer by expressing unc- <dig> in both neuronal  and gonadal  tissues.
indeed, when unc- <dig> and int2itr- <dig> promoter elements are used to drive unc- <dig> expression simultaneously in the sy <dig> mutant, sperm transfer is restored completely .
these transgenes are lost at some frequency during meiotic cell divisions  <cit> , and if either transgene is lost, wild-type sperm transfer no longer occurs in that animal : if the unc-119::unc-18::yfp transgene is lost, animals do not initiate, and if the int2itr-1::unc-18::cfp transgene is lost animals will initiate, but no longer continue to transfer and hence sire fewer crossprogeny.
the results from the continuation assays support our hypothesis that unc- <dig> functions in two different tissues to mediate two sub-steps of sperm transfer.
we also assayed unc- <dig> animals carrying either the acr-2::unc-18::yfp, the acr-5::unc-18::yfp, or the aex-3::unc-18::yfp transgene using our continuation assay.
both acr- <dig> and acr-5-driven transgenes sired fewer progeny as expected, similar to our results using the unc- <dig> regulatory region, however aex-3::unc-18::yfp was able to fully restore wild-type transfer.
we then checked to see if aex-3:: unc-18::yfp was also expressed in the male gonad, which would explain the complete rescue.
aex-3:: unc-18::yfp is indeed expressed in the gonad, while acr-2::yfp and acr-5::yfp are not .
although sy <dig> mutant animals carrying the acr-5::unc-18::yfp transgene mostly transferred less sperm, occasionally an animal transferred wild-type amounts.
considering that the acr- <dig> regulatory region we used does not drive detectable expression in the gonad, expression in the gonad may not be absolutely required, but important for proper continued transfer.
one caveat for the use of the continuation assay as a proxy for sperm transfer is that the sperm being transferred must be fertile.
mature spermatozoa that are capable of fertilization develop from spermatids following ejaculation by the male  <cit> .
as it is possible to activate spermatids in vitro to test whether they are competent to mature , we isolated and activated sperm from unc- <dig> males.
spermatids from unc- <dig> males were capable of developing into mature spermatozoa and there appeared to be similar quantities of spermatids released upon dissection compared with wild-type males .
this observation suggests that there is no sperm formation or activation defect in this mutant.
although the sperm from unc- <dig> males are capable of being activated in vitro, we cannot rule out the possibility that the vas deferens in these mutant males does not release the secreted material necessary to activate the sperm in vivo.
because unc- <dig> is involved in regulated exocytosis, such a secretory function in the vas deferens could be postulated.
although this defect would account for a decrease in cross progeny, we do observe a coincident decrease in sperm being transferred.
this observation means that even if there is a defect in seminal release, there must be some feedback to the gonad to tell it to stop the continuation of transfer.
in either case, unc- <dig> is necessary for the proper execution of this step.
discussion
understanding how genes function within neural circuits and networks to coordinate a behavioral output is a central focus in neurogenetics.
we are using c. elegans mating behavior as a model to address how genes control the ability of an animal to execute a stereotyped behavior.
during c. elegans mating behavior, the male must perceive sensory cues, integrate and process information, and generate the appropriate motor responses in order to successfully mate.
here we have defined the motor outputs , one of the molecules , and parts of the circuitry  involved in c. elegans male sperm transfer, the last step in mating behavior.
our results indicate that there are at least four sub-steps in the sperm-transfer behavior in c. elegans: initiation, release, continued transfer, and cessation.
unlike the other steps of mating behavior, which occur almost instantaneously upon stimulation, the sub-steps of sperm transfer occur over a 90-second period.
this observation suggests that a slower acting signal-transduction pathway may be involved in aspects of this behavior.
to gain insight into genetic control of the individual sub-steps of sperm-transfer behavior, we screened for mutants in this process.
in a screen for sperm-transfer-defective mutants, we isolated two mutants, sy <dig>  defective in sperm-transfer initiation, and sy <dig>  defective in the continued transfer of sperm.
we cloned and characterized sy <dig> and determined it to be an allele of unc- <dig>  unc- <dig> is a critical component of the vesicle exocytosis machinery.
we infer that the sperm-transfer defects of unc- <dig> result from a failure in exocytosis.
unc- <dig> has mild locomotor defects, while most other alleles of unc- <dig> have severe movement defects.
we used unc- <dig> as a tool to further define the circuitry necessary for initiation of sperm-transfer behavior by determining in which tissue/cells unc- <dig> acts during this process.
we have shown that neuronal expression of unc- <dig> in the ventral nerve cord is required for initiation.
through a combination of transgene and mutant analyses, the input from the ventral-nerve cord is possibly from the male-specific ca motor neurons.
also possible, and not ruled out by our experiments, is that the b-type motor neurons are responsible for the initiation of sperm transfer.
how is the male's ventral nerve cord communicating with the gonad to initiate sperm transfer?
initially it was reported that there was no innervation of the c. elegans male gonad.
however, reconstruction of the male nervous system has shown neuronal connections to the gonad, for example, through the spc spicule motor neurons  <cit> .
the connections of the male-specific ca motor neurons are not yet reconstructed, and it will be interesting to see if they, or the b-type ventral-cord motor neurons, innervate the gonad.
in drosophila melanogaster, the release of sperm and seminal fluid has been shown to be controlled by a small group of male-specific cholinergic abdominal neurons that innervate male internal reproductive organs  <cit> .
it will be interesting to see if c. elegans parallels the configuration in drosophila.
although restoration of neuron-specific unc- <dig> function was able to rescue the initiation defect in sy <dig> males, we determined that animals transferred very few sperm, appearing to initiate, but not continue transfer.
through a series of experiments, we were able to show that non-neuronal expression of unc- <dig> in the male gonad is required for correct continued transfer.
non-neuronal expression of unc- <dig> had not been previously reported in c. elegans; however, in other systems, the unc- <dig> homologue, munc- <dig>  is known to function in non-neuronal cells such as the pancreatic β-cells and also chromaffin cells  <cit> .
in these cells, munc- <dig> is involved in the exocytosis of dense-core vesicles that mediate the release of hormones and possibly other humoral factors.
molecules potentially involved in non-neuronal exocytosis, in particular unc- <dig> , have been described as expressed in non-neuronal tissue in c. elegans  <cit> .
the mechanism underlying the ability of gonadally expressed unc- <dig> to rescue the continuation of transfer remains to be explored.
garcia et al found that the male somatic gonad, the germ line, and the connection between the vas deferens  and the cloaca helps to sustain prolonged spicule protraction  <cit> .
because sustained spicule protraction is necessary for proper continuation and cessation of sperm-transfer behavior, the coordination between sperm transfer and spicule protraction suggests that multiple sensory inputs and feedback are necessary for the proper execution of this behavior, especially from the gonad.
exploring these mechanisms in the future will enhance our understanding of the regulation of these behavioral outputs.
CONCLUSIONS
our work provides further understanding of how sperm transfer in c. elegans is achieved.
we have separated the behavior of sperm transfer into four sub-steps: initiation, release, continued transfer, and cessation.
in a genetic screen, we isolated an allele of unc- <dig> defective for the initiation sub-step of sperm transfer , and we determined that the site of action for unc- <dig> in the initiation of transfer was in the ventral-cord motor neurons, most likely the male-specific ca neurons.
in addition, we have also assigned a role for unc- <dig> in the gonad for the proper completion of another sub-step of sperm transfer, the continuation of transfer.
we have shown not only that unc- <dig> is required in two distinct tissue types for two sub-steps of sperm-transfer behavior , but also that the spv neurons that inhibit premature sperm transfer act either upstream or in parallel to the requirement for unc- <dig> in initiation.
further analysis of sperm transfer will hopefully provide novel information about the genetic control of behavior.
because many parasitic nematodes require sperm transfer for reproduction  <cit> , the sub-steps of sperm transfer are potential targets for new antehelminthics.

 to execute a complex behavior, an animal must perceive and integrate information from various sources, process this information and perform the appropriate task. in many animals, one such complex behavior is mating, as different aspects of this behavior must be coordinated to ensure sexual reproduction. male mating behavior in c. elegans affords the opportunity to study a complex multi-step behavior in an organism with a relatively simple nervous system. when mating, c. elegans males exhibit a highly reproducible series of behavioral steps, culminating in the fertilization of hermaphrodite eggs. briefly, when the male comes in contact with a hermaphrodite, the male responds by placing the ventral side of his tail against the hermaphrodite, and moves backward along the body, scanning for the vulva. if the male's tail reaches the end of the hermaphrodite before encountering the vulva, the male turns and continues scanning the other side. when the vulva is located, the male ceases backward motion, prods with his copulatory spicules, inserts them into the vulva, and transfers sperm into the uterus. although the steps of reproductive behavior have been described in many organisms, relatively little is known about the genes that specifically control these behaviors. we and others have begun molecular genetic analyses of the c. elegans male response, turning, vulval location, spicule insertion, and most recently, sperm-transfer behaviors. by dissecting each step, their eventual integration as a complex series of overlapping sub-behaviors can be understood, and provide insight into nervous system function. in addition, the study of each individual step, each with its own unique aspects, may provide knowledge about the regulation of behavior not gotten from studying the other steps of mating. for example, the sperm-transfer sub-behavior in c. elegans is slower than any of the other mating steps, and does not occur instantaneously upon spicule insertion; therefore, its analysis will further our understanding of behavior. to generate a behavior, an animal must employ its nervous system to perceive and integrate stimuli, and often coordinate this information with non-neuronal targets necessary for its proper execution. what is known about the neuronal contribution to sperm-transfer behavior comes from systematic ablation of the sexually dimorphic neurons in the male to assign them function during mating behavior. each of the two male spicules contains three neurons: two sensory neurons, designated spv and spd, and the motor neuron spc. ablation of the spv spicule neurons leads to premature sperm transfer at the vulva; therefore, the spv neurons regulate the timing of sperm transfer by acting as a negative regulator. less is known about non-neuronal contribution to sperm-transfer behavior. gower et al have recently shown that signaling mediated by inositol,5-triphosphate is required for efficient sperm transfer and propose that the site of action for this signaling is the male somatic gonad. here we present an analysis of the sperm-transfer step of male mating behavior. direct observation suggests that sperm transfer comprises four sub-steps: initiation, release, continued transfer, and cessation. in a genetic screen for mutations affecting successful copulation, we isolated mutants in the sperm transfer process. of the mutants we isolated, unc- the focus of this work, has defects in two distinct sub-steps of sperm transfer: initiation and continued transfer. unc- is a member of the sm family of highly conserved proteins involved in membrane-trafficking pathways, particularly in regulated exocytosis. the mammalian unc- homologue has been reported to be expressed in neurons, as well as in non-neuronal cell types. in c. elegans, unc- expression and function has been previously reported only in neuronal cells. here we show that, while the unc- site of action for initiation involves neuronal expression, the continuation of transfer involves non-neuronal expression of unc- in the male gonad.  steps of transfer and timing as a first step toward understanding the molecular genetic processes underpinning sperm-transfer behavior and its regulation in c. elegans, and to establish a context to characterize sperm transfer mutants, we further defined this behavior. our observation of wild-type sperm transfer and subsequent genetic screening has defined at least four steps in the transfer process: initiation, release, continued transfer, and cessation. once a male has inserted his spicules into the hermaphrodite's vulva, there is movement of sperm out of the seminal vesicle, which we call'initiation'. the initiation step, from tonic insertion of spicules to sperm exit from the seminal vesicle, takes ± seconds. no gross changes in morphology were observed, except for the exit of sperm from the seminal vesicle, through the'valve region', and into the tube formed by the vas deferens. the sperm leave the seminal vesicle, travel through the vas deferens, and are released from the animal through the cloaca, a sub-step termed'release'. the anal sphincter is contracted at this time, presumably to open the cloaca. release of sperm takes ± seconds from the time of insertion, and failure at this step causes sperm to collect in the vas deferens. failure to release is rarely observed in wild-type animals; however, if a male's spicules fall out of the vulva while transferring sperm, sperm will sometimes collect in the vas deferens. sperm continue to exit the seminal vesicle in a genetically separable step that we call'continued transfer'. when all sperm that exit the seminal vesicle have been released, the spicules retract into the male. this is the'cessation' sub-step and the termination of sperm transfer. the entire process from spicule insertion to cessation takes ± seconds. liu and sternberg reported that it took approximately seconds, compared to our reported seconds, to transfer sperm. we attribute the discrepancy between the results to the microscope used for behavioral analysis. because the majority of sperm to be transferred are ejaculated in the beginning of the transfer process, it is difficult to observe without the use of a high-magnification dissecting microscope that as the behavior progresses, the male still continues to intermittently transfer a few sperm. screen for mating behavior mutants to begin to answer fundamental questions about how the sub-steps of sperm transfer are regulated, we performed a genetic screen designed to isolate males defective for this behavior. briefly, plg- males will extrude a gelatinous substance that puts a'plug' over the hermaphrodite vulva after sperm transfer. the plg- allele is a dominant allele derived from a wild c. elegans isolate that was introgressed into the n wild-type strain. by screening for the lack of copulatory plugs in a clonal population of ems mutagenized worms, we were able to isolate mutants defective in all steps of mating. secondary screening of putative mutants by observation of mating behavior allowed us to determine those mutants with defects in sperm transfer. to isolate only those with non-developmental mating defects, we screened through the mutants we isolated for defects in morphology, focusing on those with wild-type anatomy. one recessive mutant isolated, sy is defective in the initiation step of sperm transfer. while sy mutant males will insert their spicules into the hermaphrodite vulva and remain tonically inserted for ± seconds from wild type), they rarely initiate sperm transfer. a second recessive mutant isolated, sy allows initiation and release, but is defective in the continuation of sperm transfer. sy males transfer very few sperm, although sperm are clearly visible in the seminal vesicle, and sy males keep their spicules tonically inserted during the transfer behavior for ± seconds. although the duration that sy males remain with their spicules inserted in the hermaphrodite vulva is less than wild type, they should transfer significantly more sperm, as the bulk of transfer should have already occurred by this time. the sy mutant allows us to genetically separate the continued-transfer step from initiation and release steps. for the remainder of this study, we will focus on the analysis of sy a for all genotypes carrying an extrachromosomal array, at least three independent lines were assayed except for unc-18; ex, for which only two lines each were tested. these results are the combined data from individual lines. see additional file for results of individual lines. b wild type is strain ps plg-1; him- c rescue is defined as being different from unc- using anova with a tukey-kramer multiple comparisons test. sy acts downstream or in parallel to spv as mentioned above, the spv neurons regulate sperm transfer by inhibiting release until the spicules have penetrated the vulva: spv-ablated wild-type males prematurely transfer sperm before spicule insertion. because sy has the opposite phenotype we ablated the spv neurons in the sy mutant to see if this could suppress the initiation defect. in particular, we ablated the b.β cell, the precursor of both spv neurons. although the b.β ablation also eliminates four of structural cells, the sheath cells are not necessary for spicule morphology, and the function of the socket cells is redundant. in addition, ablation of the b.β cell causes the same defect in sperm transfer as the ablation of the spv alone. we found that ablation of the b.β cell in the sy mutant did not suppress the initiation defect. this observation suggests that the defect in our mutant lies downstream or in parallel to the spv regulation in the sperm-transfer pathway. a number of animals assayed. b scored as animals that transfer sperm without tonic contraction of spicules in the vulva. c percentage altered is the number of animals with altered transfer divided by the total number of animals tested. altered transfer for wild type includes premature transfer and no transfer. altered transfer for sy includes premature transfer and wild-type transfer. d wild type is strain ps plg-1; him- molecular characterization of sy671 to understand the sperm-transfer initiation defect in sy males, we cloned this locus. we employed single-nucleotide polymorphism mapping to map sy to a 120-kb interval between the polymorphisms f26a10: and c47c12:. injection of cosmids and pcr fragments narrowed the sy locus to a -kb region containing f27d. we sequenced the predicted coding and non-coding regions of unc- from the sy mutant, and found that the sy mutant contains a g→a missense mutation in an unc- exon, which changes the amino-acid arg to his. in c. elegans, unc- which encodes a member of the sec1/munc- family of proteins, is required in neurons for synaptic-vesicle exocytosis. for exocytosis to occur, secretory vesicles must dock, be primed, and then fuse with the plasma membrane. unc- has been proposed to function in each of these steps of exocytosis, based on work in various systems, and may also act as a trafficking factor for other proteins necessary for exocytosis. although unc- has been postulated to act in various steps of the exocytosis pathway, in c. elegans it has been shown that there is a reduction of docked vesicles at the active zone in unc- mutants, indicating that unc- functions at least as a facilitator of vesicle docking in c. elegans. mutations in unc- cause locomotor defects and acetylcholine accumulation, and therefore unc- has been implicated in the release of acetylcholine. unc- interacts with syntaxin, a protein believed to mediate fusion of synaptic vesicles to the plasma membrane and hence neurotransmitter release. while most alleles of unc- have severe locomotor defects, sy males have only mild locomotor defects. although the amino-acid change in sy occurs in a residue conserved among unc- orthologs, this residue lies outside the syntaxin-binding region. we were intrigued by the unique position of the molecular lesion in the sy allele of unc- all reported sequenced alleles of unc- have a molecular defect in either exon or exon; however, sy has its defect in exon. of the other unc- mutants, only two, md and md move sufficiently well to allow sperm transfer to be assayed. although their movement is indistinguishable from sy both mutants are able to initiate transfer. this difference among alleles leads us to speculate that the mutation in sy has some specificity for sperm transfer through a mechanism different from the function of unc- in vesicle release. to address if a perturbation of vesicle release is the likely defect in sy males, we assayed males that were transheterozygous for sy and for a severe allele of unc- b the unc- allele has a single amino-acid substitution that causes the accumulation of acetylcholine, the inability to bind syntaxin, and a severe locomotor defect, all characteristics of defective vesicle release. if sy does have a defect in a yet uncharacterized unc- function, we would expect that when placed in trans to an allele that perturbs vesicle release through a single amino-acid substitution, we would see compensation for the sperm-transfer defect. however, the b allele fails to complement sy for the initiation of transfer, and therefore it seems likely that the mutation in sy also alters vesicle release. unc- is expressed in the male gonad and nervous system previous characterization of unc- expression using polyclonal antisera showed that unc- was present in ventral-cord motor neurons and some unidentified head neurons in the adult hermaphrodite. to replicate the expression in the hermaphrodite and determine expression in the male, we fused the unc- upstream sequences used in the mutant-rescue experiment to a yfp reporter. in transgenic animals, this reporter recapitulated the previously observed expression in the hermaphrodite. in the male, unc-18::yfp showed the same expression in the ventral cord and head, but surprisingly also had strong expression in the gonad. the expression was strongest in the'valve region', a non-neuronal tissue that lies at the junction of the seminal vesicle and the vas deferens. expression was also present in most, if not all neurons in the male tail. site of action: initiation to define the site of action of unc- in sperm-transfer behavior, we created a yfp-tagged version of unc- under the regulation of the unc- upstream regulatory region, which was able to rescue the sy sperm transfer initiation defect. this construct was also able to confer the null allele, unc- with the ability to initiate sperm transfer. next we expressed the unc-18::yfp functional protein under different tissue-specific, transcriptional-control regions to define its site of action in sperm-transfer behavior, beginning with the two most probable candidates: the gonad and the spicule neurons. because unc- expression in the male gonad coincides with the region where sperm exit from the seminal vesicle to the vas deferens, this seemed a likely candidate for the site of action of unc- in the initiation of sperm-transfer behavior. an equally attractive candidate is the spv spicule neurons in the male tail, as they are known regulators of sperm transfer. to express unc- under a gonad-specific promoter, we used a portion of the described itr- gene. it was previously reported that two introns and one exon of the itr- gene gave expression in the intestine, pharyngeal isthmus, and gonad of hermaphrodites. through further dissection of this region, called the pc promoter, we were able to drive expression in the seminal vesicle, the valve region, and the vas deferens of the male. this expression coincides with that described for the entire pc promoter in the male. we call this element int2itr- we then expressed unc- under the control of the itr- gonadal enhancer, but were not able to rescue the mutant phenotype in sy. to express unc- under a spicule neuron promoter, we used kb of 5' upstream sequence of the gpa- gene that drives expression in all three types of spicule sensory neurons, including the spv neurons. the gpa-1::unc-18::yfp transgene was also unable to rescue the sperm-transfer initiation defect in sy. after initial testing of possible gonadal and spicule neuron contributions, we chose to express unc- more broadly to see if unc- was at least acting in neurons to initiate the transfer of sperm. we used a pan-neuronal promoter to drive expression of unc- in neurons, and also used a body-wall muscle promoter to rule out non-specific rescue effects. the neuronal expression of unc- was able to rescue the initiation defect. these results suggest that although unc- is expressed in the male gonad, its expression in this tissue is not required for the initiation of sperm transfer, but it is required in neurons. to confirm this result, we used the 5' regulatory region of the aex- gene, another pan-neuronal promoter, to express unc-. as expected, this transgene was able to rescue the sperm-transfer initiation defect in sy. the negative result using gpa-1::unc-18::yfp to drive expression in the spicule neurons does not exclude an unc- contribution from these neurons; therefore, we decided to further define the site of action of unc- in this behavior using various neuronal promoters. unc- was initially described to be expressed in all ventral-cord motor neurons. the ventral cord has been divided into eight classes of motor neurons: four classes that innervate ventral muscles and four classes that innervate dorsal muscles. the d-type ventral cord neurons are gamma-aminobutyric acid ergic and the others are cholinergic. the vc cholinergic neurons are hermaphrodite-specific, while in males, the homologous cells divide to give the male-specific cp and ca motor neurons. to test whether unc- was acting exclusively in either cholinergic or gabaergic ventral cord motor neurons to initiate sperm transfer, we drove expression specifically in each type. to express unc- in all gabaergic neurons in the sy mutant, we used the promoter region from the unc- gene to drive expression. expression in only the gabaergic neurons was unable to rescue our mutant. a for all genotypes tested, at least three independent lines were assayed. these results are the combined data from individual lines. see additional file for results of individual lines. b rescue is defined as being different from unc- in table using anova with a tukey-kramer multiple comparisons test. c nd, not done. these lines were not assayed because the introduction of the transgene caused severe mating behavioral defects in steps prior to sperm transfer assay. to express unc- in all cholinergic neurons in the sy mutant, we initially tried using promoters from both unc- and cho- to drive expression. however, the use of either of these promoters, caused penetrant locomotion and mating behavioral defects in the steps prior to sperm transfer, and therefore the worms could not be assayed for their ability to transfer sperm. these defects, not seen in non-transgenic worms, could be caused by overexpression of unc- or possible titration of promoter regulatory proteins caused by our transgenic arrays. next we tried the acr- promoter, which drives expression in the va, vb, da, and db cholinergic ventral-cord motor neurons. the acr- construct, acr-2::unc-18::yfp, when expressed in sy mutants, was able to rescue initiation. because acr- is expressed in cholinergic neurons throughout the ventral cord and not in the male spicule sensory neurons, these ventral-cord motor neurons are strong candidates for contributing to the initiation of sperm transfer. to further test ventral-cord input for the initiation of sperm-transfer behavior and to further pinpoint the site of action to a specific subset of cells, we used additional promoters to attempt to rescue the sy defect. we initially tested two classes of cholinergic ventral-cord motor neurons, the a-type neurons, va and da and the b-type neurons, vb and db. we used the unc- upstream sequences to drive expression in the a-type neurons and the acr- promoter to drive expression in b-type neurons. by expressing unc- under the acr- regulatory region, we were able to rescue the sy initiation defect, but were unable to do so with the unc- promoter. this result suggests that unc- in the b-type neurons could be necessary for initiation, again with the exception of the neurons outside the ventral cord that express acr- we determined that acr- is expressed in the male-specific ray sensilla using the acr- regulatory region to drive expression of dsred. the expression in the rays appears to be in the a-type ray neurons, as acr- expression in the tail does not overlap with pkd-2::gfp, which is expressed in the b-type ray neurons. acr- also shows expression in a cell in the right preanal ganglion, which we have tentatively identified as pga or pvz. however, input from the acr- expressing cells in the male tail in initiating transfer is very unlikely, as there is no overlap with acr- expression in the tail, yet both promoters driving unc- expression can rescue the initiation defect in unc- mutants. the overlap in expression of acr- and acr- is in the ventral cord, making a strong case for it being necessary for the initiation of sperm transfer. although these data also suggest it is specifically the b-type motor neurons in the ventral cord that are relevant for the initiation of sperm transfer, further experiments indicate that the male-specific ca ventral-cord motor neurons are stronger candidates. in the adult male ventral cord we noticed that acr-5::yfp was expressed not only in the b-type neurons, but also in additional neurons. because the male has additional ventral cord neurons, the cps and cas, we thought that these may account for the additional cells showing expression, and it may be these cells that are necessary for initiation of sperm transfer. because the cp neurons are necessary for efficient turning behavior, we wanted to test if the acr-5-expressing cells were cp neurons. we used a strain carrying a tph-1::gfp transgene that drives expression in the cp neurons, and crossed it into animals expressing acr-5::dsred the additional cells in the male ventral cord that express acr-5::dsred did not overlap with the cp-expressing cells, but in many cases these cells were anterior to the cp neurons, suggesting that they could be ca neurons. because there are no reported markers for the ca neurons, we tested the possible contribution of these neurons to sperm-transfer initiation by assaying lin- mutant males. the lin- gene, which encodes a homeobox gene, is required for mid-body region-specific development in c. elegans. in wild-type males, p.aap divide during late l to generate male-specific motor neurons. the posterior cells, p.aapp, are the cp neurons and the anterior cells, p.aapa, are the ca neurons. in lin- mutant males, p.aap do not divide, but instead have a compact neuron-like morphology or die. also affected in lin- mutant males are p.aapp, which take on a more posterior fate, as judged by the loss of serotonin staining in cp and cp. this observation suggests that p.a might be transformed into p a-like cells, thus also affecting ca and ca if the ca neurons are necessary for initiation of sperm transfer, we would expect lin- males to be unable to initiate this behavior, as these cells have been genetically ablated or misfated. we tested two alleles of lin- and both were unable to initiate sperm transfer, further supporting a role for the ca neurons and confirming ventral cord involvement, limiting it to the mid-body region. to further corroborate that the unassigned ventral cord motor neurons expressing acr- in the wild-type males could be ca neurons, we analyzed the expression of acr-5::gfp in lin- mutant males. as expected, we saw fewer cells expressing acr-5::gfp in the ventral cord of lin- mutant males. site of action: continued transfer although the sy mutants can be rescued for the initiation of sperm transfer using a transgene to drive neuronal expression of unc- in a subset of ventral cord cholinergic neurons, these animals did not transfer as many sperm as wild type. we observed that by using the unc- promoter to drive unc- expression, the animals would initiate, and usually the sperm that were released from the seminal vesicle were released from the animal, but the animals transferred much less sperm and thus appeared to be defective in the continuation of transfer. this observation raised the possibility that unc- functions in two separate sub-steps of the sperm-transfer process, and that neuronal expression of unc- was responsible for the initiation of transfer, while the gonad expression may be necessary for continued transfer. to formally test this possibility, we hypothesized that if we could add back both neuronal and gonadal expression of unc- using different 5' control regions, we should restore wild-type sperm transfer to the unc- mutants. to do this, we again used our neuronal construct, and remade our gonadal transgene with a cfp marker to facilitate the scoring of worms carrying both transgenes. to quantify the amount of sperm transferred, we devised a'continuation assay'. this assay uses the percentage of cross progeny as a proxy for the amount of sperm transferred. as a control for the effectiveness of our continuation assay, we tested unc- mutants carrying a rescuing transgene to determine if they could sire a wild-type percentage of cross progeny. not only did we observe that these transgenic animals appeared to transfer a similar amount of sperm to that of wild type, but as expected they sired an equivalent percentage of cross progeny. this construct also was able to completely restore wild-type sperm transfer in the unc- null mutant. in addition, as expected based on observation of sperm transfer, animals carrying the unc-119-driven transgene sired much fewer progeny than wild type in our assay, because they initiated transfer and released sperm, but did not continue to transfer. in addition, lines carrying only gonadally expressed unc- did not sire cross progeny, consistent with an inability to initiate transfer. we then tested if we could restore wild-type sperm transfer by expressing unc- in both neuronal and gonadal tissues. indeed, when unc- and int2itr- promoter elements are used to drive unc- expression simultaneously in the sy mutant, sperm transfer is restored completely. these transgenes are lost at some frequency during meiotic cell divisions, and if either transgene is lost, wild-type sperm transfer no longer occurs in that animal: if the unc-119::unc-18::yfp transgene is lost, animals do not initiate, and if the int2itr-1::unc-18::cfp transgene is lost animals will initiate, but no longer continue to transfer and hence sire fewer crossprogeny. the results from the continuation assays support our hypothesis that unc- functions in two different tissues to mediate two sub-steps of sperm transfer. we also assayed unc- animals carrying either the acr-2::unc-18::yfp, the acr-5::unc-18::yfp, or the aex-3::unc-18::yfp transgene using our continuation assay. both acr- and acr-5-driven transgenes sired fewer progeny as expected, similar to our results using the unc- regulatory region, however aex-3::unc-18::yfp was able to fully restore wild-type transfer. we then checked to see if aex-3:: unc-18::yfp was also expressed in the male gonad, which would explain the complete rescue. aex-3:: unc-18::yfp is indeed expressed in the gonad, while acr-2::yfp and acr-5::yfp are not. although sy mutant animals carrying the acr-5::unc-18::yfp transgene mostly transferred less sperm, occasionally an animal transferred wild-type amounts. considering that the acr- regulatory region we used does not drive detectable expression in the gonad, expression in the gonad may not be absolutely required, but important for proper continued transfer. one caveat for the use of the continuation assay as a proxy for sperm transfer is that the sperm being transferred must be fertile. mature spermatozoa that are capable of fertilization develop from spermatids following ejaculation by the male. as it is possible to activate spermatids in vitro to test whether they are competent to mature, we isolated and activated sperm from unc- males. spermatids from unc- males were capable of developing into mature spermatozoa and there appeared to be similar quantities of spermatids released upon dissection compared with wild-type males. this observation suggests that there is no sperm formation or activation defect in this mutant. although the sperm from unc- males are capable of being activated in vitro, we cannot rule out the possibility that the vas deferens in these mutant males does not release the secreted material necessary to activate the sperm in vivo. because unc- is involved in regulated exocytosis, such a secretory function in the vas deferens could be postulated. although this defect would account for a decrease in cross progeny, we do observe a coincident decrease in sperm being transferred. this observation means that even if there is a defect in seminal release, there must be some feedback to the gonad to tell it to stop the continuation of transfer. in either case, unc- is necessary for the proper execution of this step. discussion understanding how genes function within neural circuits and networks to coordinate a behavioral output is a central focus in neurogenetics. we are using c. elegans mating behavior as a model to address how genes control the ability of an animal to execute a stereotyped behavior. during c. elegans mating behavior, the male must perceive sensory cues, integrate and process information, and generate the appropriate motor responses in order to successfully mate. here we have defined the motor outputs, one of the molecules, and parts of the circuitry involved in c. elegans male sperm transfer, the last step in mating behavior. our results indicate that there are at least four sub-steps in the sperm-transfer behavior in c. elegans: initiation, release, continued transfer, and cessation. unlike the other steps of mating behavior, which occur almost instantaneously upon stimulation, the sub-steps of sperm transfer occur over a 90-second period. this observation suggests that a slower acting signal-transduction pathway may be involved in aspects of this behavior. to gain insight into genetic control of the individual sub-steps of sperm-transfer behavior, we screened for mutants in this process. in a screen for sperm-transfer-defective mutants, we isolated two mutants, sy defective in sperm-transfer initiation, and sy defective in the continued transfer of sperm. we cloned and characterized sy and determined it to be an allele of unc- unc- is a critical component of the vesicle exocytosis machinery. we infer that the sperm-transfer defects of unc- result from a failure in exocytosis. unc- has mild locomotor defects, while most other alleles of unc- have severe movement defects. we used unc- as a tool to further define the circuitry necessary for initiation of sperm-transfer behavior by determining in which tissue/cells unc- acts during this process. we have shown that neuronal expression of unc- in the ventral nerve cord is required for initiation. through a combination of transgene and mutant analyses, the input from the ventral-nerve cord is possibly from the male-specific ca motor neurons. also possible, and not ruled out by our experiments, is that the b-type motor neurons are responsible for the initiation of sperm transfer. how is the male's ventral nerve cord communicating with the gonad to initiate sperm transfer? initially it was reported that there was no innervation of the c. elegans male gonad. however, reconstruction of the male nervous system has shown neuronal connections to the gonad, for example, through the spc spicule motor neurons. the connections of the male-specific ca motor neurons are not yet reconstructed, and it will be interesting to see if they, or the b-type ventral-cord motor neurons, innervate the gonad. in drosophila melanogaster, the release of sperm and seminal fluid has been shown to be controlled by a small group of male-specific cholinergic abdominal neurons that innervate male internal reproductive organs. it will be interesting to see if c. elegans parallels the configuration in drosophila. although restoration of neuron-specific unc- function was able to rescue the initiation defect in sy males, we determined that animals transferred very few sperm, appearing to initiate, but not continue transfer. through a series of experiments, we were able to show that non-neuronal expression of unc- in the male gonad is required for correct continued transfer. non-neuronal expression of unc- had not been previously reported in c. elegans; however, in other systems, the unc- homologue, munc- is known to function in non-neuronal cells such as the pancreatic β-cells and also chromaffin cells. in these cells, munc- is involved in the exocytosis of dense-core vesicles that mediate the release of hormones and possibly other humoral factors. molecules potentially involved in non-neuronal exocytosis, in particular unc-, have been described as expressed in non-neuronal tissue in c. elegans. the mechanism underlying the ability of gonadally expressed unc- to rescue the continuation of transfer remains to be explored. garcia et al found that the male somatic gonad, the germ line, and the connection between the vas deferens and the cloaca helps to sustain prolonged spicule protraction. because sustained spicule protraction is necessary for proper continuation and cessation of sperm-transfer behavior, the coordination between sperm transfer and spicule protraction suggests that multiple sensory inputs and feedback are necessary for the proper execution of this behavior, especially from the gonad. exploring these mechanisms in the future will enhance our understanding of the regulation of these behavioral outputs.  our work provides further understanding of how sperm transfer in c. elegans is achieved. we have separated the behavior of sperm transfer into four sub-steps: initiation, release, continued transfer, and cessation. in a genetic screen, we isolated an allele of unc- defective for the initiation sub-step of sperm transfer, and we determined that the site of action for unc- in the initiation of transfer was in the ventral-cord motor neurons, most likely the male-specific ca neurons. in addition, we have also assigned a role for unc- in the gonad for the proper completion of another sub-step of sperm transfer, the continuation of transfer. we have shown not only that unc- is required in two distinct tissue types for two sub-steps of sperm-transfer behavior, but also that the spv neurons that inhibit premature sperm transfer act either upstream or in parallel to the requirement for unc- in initiation. further analysis of sperm transfer will hopefully provide novel information about the genetic control of behavior. because many parasitic nematodes require sperm transfer for reproduction, the sub-steps of sperm transfer are potential targets for new antehelminthics. 
based on observation of wild-type males and on genetic analysis, we have divided the sperm-transfer step of mating behavior into four sub-steps: initiation, release, continued transfer, and cessation. by a series of transgenic site-of-action experiments, we found that motor neurons in the ventral nerve cord require unc- for the initiation of sperm transfer, and that unc- acts downstream or in parallel to the spv sensory neurons in this process. here we extend our understanding of the sperm-transfer step of male mating behavior. in addition to this neuronal requirement, we found that non-neuronal expression of unc- in the male gonad, is necessary for the continuation of sperm transfer. our allele, sy is defective in two distinct sub-steps of sperm transfer: initiation and continued transfer. we have shown both a neuronal and non-neuronal requirement for unc- in distinct sub-steps of sperm-transfer behavior. our division of sperm-transfer behavior into sub-steps has provided a framework for the further detailed analysis of sperm transfer and its integration with other aspects of mating behavior. by determining the site of action of unc- in sperm-transfer behavior, and its relation to the spv sensory neurons, we have further defined the cells and tissues involved in the generation of this behavior. this behavior has been divided into the following steps: response, backing, turning, vulva location, spicule insertion, and sperm transfer. we isolated an allele of unc- a previously reported member of the sec1/munc- family of proteins that is necessary for regulated exocytosis in c. elegans motor neurons.
 the caenorhabditis elegans male exhibits a stereotypic behavioral pattern when attempting to mate. this behavior has been divided into the following steps: response, backing, turning, vulva location, spicule insertion, and sperm transfer. we and others have begun in-depth analyses of all these steps in order to understand how complex behaviors are generated. here we extend our understanding of the sperm-transfer step of male mating behavior.  based on observation of wild-type males and on genetic analysis, we have divided the sperm-transfer step of mating behavior into four sub-steps: initiation, release, continued transfer, and cessation. to begin to understand how these sub-steps of sperm transfer are regulated, we screened for ethylmethanesulfonate -induced mutations that cause males to transfer sperm aberrantly. we isolated an allele of unc- a previously reported member of the sec1/munc- family of proteins that is necessary for regulated exocytosis in c. elegans motor neurons. our allele, sy is defective in two distinct sub-steps of sperm transfer: initiation and continued transfer. by a series of transgenic site-of-action experiments, we found that motor neurons in the ventral nerve cord require unc- for the initiation of sperm transfer, and that unc- acts downstream or in parallel to the spv sensory neurons in this process. in addition to this neuronal requirement, we found that non-neuronal expression of unc- in the male gonad, is necessary for the continuation of sperm transfer.  our division of sperm-transfer behavior into sub-steps has provided a framework for the further detailed analysis of sperm transfer and its integration with other aspects of mating behavior. by determining the site of action of unc- in sperm-transfer behavior, and its relation to the spv sensory neurons, we have further defined the cells and tissues involved in the generation of this behavior. we have shown both a neuronal and non-neuronal requirement for unc- in distinct sub-steps of sperm-transfer behavior. the definition of circuit components is a crucial first step toward understanding how genes specify the neural circuit and hence the behavior. 