Assessing hybrid male fertility in Drosophila species:  Correlation between sperm motility and production of offspring.

Campbell, Roosevelt V., ^1,2 and Mohamed A.F. Noor ^2,*.  ¹ Department of Biology, University of Texas-Pan American, Edinburg, TX 78539 USA;  ² Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 USA;  ^* Corresponding author, FAX 225-578-2597;  E-mail: mnoor@lsu.edu

Abstract

            We have compared the results of two methods used for identifying male sterility in Drosophila species: sperm motility and production of offspring.  In backcross hybrids of D. pseudoobscura and D. persimilis, we found that half of the males scored as fertile by a sperm motility assay fail to produce offspring, and we have confirmed that this observation does not result from a failure in sperm transfer.  Our finding suggests that more factors may contribute to Drosophila hybrid male sterility than has been suggested by some genetic studies.

Introduction

            Hybrid male fertility analyses are often undertaken in studies to understand the genetics of speciation.  Given that in this type of study, sample sizes of 500 to 2500 are often required, a fast and accurate method of assessing male fertility is needed.  Included among these approaches are measurements of testicular length, testicular atrophy, sperm motility, sperm morphology (length and cytological characterization), sperm quantity, sperm transfer during copulation and progeny production (see Table 1).  The various approaches used towards male fertility assessment have different strengths and weaknesses.

            Assessment of male fertility through progeny production analysis is perhaps the most biologically relevant method.  Unfortunately, this method is time-consuming and, for this reason, it is only used occasionally.  In its place, one or a combination of the other methods are commonly used.   Testis size was first used as an indicator of male fertility by Dobzhansky (1933) while studying the genetic basis for Haldane’s Rule, arguing that a high correlation between testis size and fertility exists.  As Orr (1987) pointed out, however, the relationship between testis size and fertility may be questionable since Lancefield (1929) had previously shown that males with normal sized testis could also be sterile.  Thus, whereas small testis size may accurately suggest sterility, large testis size does not necessarily reflect fertility.

            Scoring the presence of motile sperm in an all or none approach as proposed by Coyne (1984) has thus been favored (see Table 1), but this method has not been fully evaluated in how it reflects ability to produce offspring.  As Joly et al. (1997) noted, presence of motile sperm does not always guarantee fertility.

            The purpose of this project is to evaluate the association between male fertility and presence of motile sperm by comparing the relationship between sperm motility and progeny production in backcross hybrids of Drosophila persimilis and D. pseudoobscura.  In a previous study (Noor et al., 2001), it was shown that about half of these backcross hybrid males were able to produce motile sperm and thus they serve as a good model for this project.

Materials and Methods

            The backcross hybrids used in this experiment were derived from D. pseudoobscura collected in Flagstaff and D. persimilis collected in Mount St. Helena, both in 1993.  These strains have been maintained in the laboratory several years and used in several previous studies.

            Fertile F₁ females, produced by crossing D. pseudoobscura females to D. persimilis males, were backcrossed to D. persimilis males.  These crosses were carried out at 20ºC, 85% relative humidity on standard sugar/yeast/agar medium.  The resulting virgin backcross males, designated BCper males, and virgin pure species D. persimilis females were harvested and aged for 8 days in groups of no more than 20 individuals per vial.

            On day 8, BCper males and pure species D. persimilis females were placed in pairs and observed for mating behavior.   If mating took place, the males were separated from the females and assigned a corresponding number.  If mating did not take place, the flies were discarded. 

            Approximately 3 hours after mating, male testes were dissected in insect Ringer’s solution, squashed, and checked for motile sperm using a light compound microscope.  Males showing at least one motile sperm were scored as fertile, while males with immotile sperm were scored as sterile, as suggested by Coyne (1984).

            To ensure that sperm was consistently being transferred during mating, the female oviducts of approximately one third of the females that mated with BCper males bearing motile sperm were also dissected in insect Ringer’s solution, squashed, and checked for transfer of motile sperm using a light compound microscope.  The remainder of the mated females were placed in an incubator and inspected for larvae from the 3^rd to the 7^th day after mating.  If the female died before the 5^th day, data on progeny production for the corresponding male was not recorded.  The results obtained from both male fertility assessment methods – motile sperm assay and progeny production – were then compared and analyzed for significant differences.

            As a control for this experiment, the correlation between motile sperm production and progeny production was also tested in pure species D. persimilis and D. pseudoobscura males, the same way that it was tested in the BCper males.

Results

            Using the sperm motility approach, 75 BCper males were scored as fertile and 106 as sterile.  Females mated to 49 of the 75 fertile males and all 106 of sterile males were checked for progeny production.  Of the 49 males scored as fertile, only 22 (45%) sired offspring and 27 (55%) did not.  Of the 106 males scored as sterile, 1 (1%) sired offspring and 105 (99%) did not. 

            Twenty-six BCper males bearing motile sperm could not be checked for progeny production because the females to which they mated were used in the sperm-transfer test.  Motile sperm was observed in 24 (92%) of these 26 females.  A small quantity of non-motile sperm was detected in the remaining two females.

            D. persimilis and D. pseudoobscura males were also paired with D. persimilis females as a control.  All pure-species males tested bore motile sperm.  Out of 30 D. persimilis males scored as fertile, 29 (97%) of them produced offspring while, out of 9 D. pseudoobscura males scored as fertile, 9 (100%) of them produced progeny.  The sample size for the latter was small because of the reluctance of D. persimilis females to mate with D. pseudoobscura males.

Discussion

         The results from this study show that sperm motility, measured as an all or none property in males, correlates loosely with male ability to produce offspring in backcross hybrids of D. persimilis and D. pseudoobscura hybrids.  While nearly all males scored as sterile by the sperm motility assay failed to produce offspring, only about half of those hybrid males scored as fertile were able to produce offspring.

         This inconsistency likely results from defects associated with sperm motility that are not taken into account by the all or none approach typically used in genetic studies of hybrid sterility.  As Kiefer (1968) pointed out in an early study of spermatogenesis in Drosophila, the process of fertilization involves more than the transfer of motile sperm to the female.  Structurally normal sperm may have physiological defects that may account for the ability of the individual sperm to fertilize the egg (Kiefer, 1968). In addition, certain aspects of sperm morphology may also play an important role.  Sperm length, for instance, may influence its ability to fertilize.

         Our results were probably not influenced by failure of males to transfer motile sperm to the females.  More than 50% of the males scored as fertile by sperm motility failed to produce progeny, but 92% of fertile males transferred motile sperm.

         This observation does not at all suggest that any genetic studies of hybrid sterility are "wrong".  Instead, some of these studies may have not documented some factors that can confer hybrid sterility through means besides rendering sperm immotile.  Hence, the genetic architecture of hybrid male sterility may be more complicated or involve more genes than published studies have suggested.

         Acknowledgments:  This work was supported by the LSU Howard Hughes Medical Institute summer research program.

         References:  Coyne, J.A., 1984,  Proc. Natl. Acad. Sci. USA 81: 4444-4447;  Coyne, J.A., and B. Charlesworth 1988,  Heredity 62: 97-106;  Dobzhansky, Th., 1933, Genetics 19: 950-954;  Joly, D., C. Bazin, L.W. Zeng, and R.S. Singh 1997,  Heredity 78: 354-362;  Kiefer, B.I.,  1969, Genetics 61: 157-166;  Kulathinal, R., and R.S. Singh 1998, Evolution 52: 1067-1079;  Lancefield, D.E., 1929, Zeit. Induk. Abstam. Vererbung. 52: 287-317;  Macdonald, S.J., and D.B. Goldstein 1999, Genetics 153: 1683-1699;  Naveira, H., and A. Fondevila 1991, Heredity 66: 233-239;  Noor, M.A.F., K.L. Grams, L.A. Bertucci, Y. Almendarez, J. Reiland, and K.R. Smith 2001, Evolution 55: 512-521;  Orr, H.A., 1987, Genetics 116: 555-563;  Orr, H.A., and S. Irving 2001, Genetics 158: 1089-1100;  Palopoli, M.F., and C.-I. Wu 1994, Genetics 138: 329-341;  Wu, C.-I., 1983, Genetics 105: 71-86;  Zeng, L.-W., and R.S. Singh 1993, Genetics 134: 251-260.