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Effects of ultraviolet radiation on gametic function during fertilization in zebra mussels (Dreissena polymorpha).

ABSTRACT The gamete-specific effects of UV radiation on fertilization in the invasive freshwater zebra mussel (Dreissena polymorpha) are addressed. Both eggs and sperm of zebra mussels were highly sensitive to UVB. Sperm irradiated with 118 mJ/ [cm.sup.2] UVB showed decreased fertilization rates when inseminating nonirradiated eggs. Sperm binding was unaffected; however, acrosomal integrity and sperm incorporation into the egg cytoplasm were reduced by UVB irradiation. There was a decrease in first zygotic cleavage. The level of UVB needed to inhibit fertilization was lower than that required for inhibition of sperm motility. UVB-irradiated eggs also showed reduced function when inseminated by nonirradiated sperm. UVB-induced damage in eggs most commonly resulted in a stoppage or delay of meiosis. The levels effective in killing zebra mussel larvae should be sufficient to inhibit fertilization.

KEY WORDS: ultraviolet, fertilization, Dreissena, gametes, zebra mussel

INTRODUCTION

Since their introduction into North America in the mid 1980s, the dreissenid species Dreissena polymorpha Pallas, 1771 (zebra mussels) (Hebert & Muncaster 1989) and the later arriving Dreissena bugensis (quagga mussels) (May & Marsden 1992) have had profound ecological and economic effects on colonized waterways. Their spread into the western United States has further increased interest and concern regarding these invasive species (Stokstad 2007, Benson 2009). The distribution of these species as they invade North American waterways has varied somewhat from the spread observed in Europe (Nichols 1996). A better understanding of the biology of these species and the environmental factors that limit their spread should aid in possible control mechanisms and identification of waters at risk for future infestation.

Zebra mussels broadcast spawn, externally fertilize, and have direct larval development in the water column. These characteristics are in contrast to most North American freshwater bivalve species, in which fertilization and larval brooding occurs within the female mantle cavity (Glaubrecht et al. 2006). In many aspects, reproduction in zebra mussels more closely resembles reproductive strategies used by marine bivalves (Longwell & Stiles 1968, Longo & Anderson 1969a; Longo & Anderson 1969b, Ackerman et al. 1994).

Within the last 15 y, several studies have focused on various aspects of reproduction in zebra mussels including maturation (Ram et al. 1996), spawning (Ram et al. 1993, Claxton & Mackie 1998), fertilization (Misamore et al. 1996, 2006), and early developmental events (Luetjens & Dorresteijn 1998a, 1998b). The effects of several environmental parameters on zebra mussel reproduction have also been addressed, including salinity, temperature, and food availability (Borcherding 1995, Fong et al. 1995, Sprung 1995). One aspect that has received little attention is the potential effect of UV radiation (UVR) on zebra mussel reproduction.

Ultraviolet radiation is conventionally subdivided into three categories: UVC (200-280 nm), UVB (280-320), and UVA (320-400). Only UVA and UVB are able to penetrate the atmosphere and reach the Earth's surface and UVB (280-320 nm) is considered to be the most damaging to biological systems under natural conditions (Bonaventura et al. 2006). It is well established that UVR induces DNA and protein damage and, without protection, gametic stages are especially vulnerable. Previous studies have addressed the effect of UVR on adults and later larval stages in zebra mussels (Chalker-Scott et al. 1993; Chalker-Scott et al. 1994, Wright et al. 1997, Chalker-Scott & Scott 1998). Chalker-Scott et al. (1993) found that adult zebra mussels were resistant to acute exposure to UVB; however, chronic exposure ultimately resulted in death. Wide-range UVR (UVA, B, C) at 350 mJ/[cm.sup.2] was 100% effective in killing larval zebra mussels (Chalker-Scott et al. 1994). They also found that UVB, not UVC, was the most important component of UVR in controlling zebra mussels. Moreover, veligers and postveligers were more susceptible to UVB than young adults. Similarly, Wright et al. (1997) reported nearly 100% mortality of 16-day-old quagga mussel larvae exposed to UVB.

Ultraviolet radiation has two potential implications with regards to zebra mussel reproduction. First, UVR has been proposed as a potential method for control, especially for fouling prevention in water intake pipes (Chalker-Scott et al. 1994, Wright et al. 1997). Second, naturally occurring UVB may negatively effect zebra mussel reproduction. Planktonic organisms are especially vulnerable to environmental UV radiation (Epel et al. 1999). UVB has detrimental effects on planktonic organisms and larvae across a diverse taxa (Hader et al. 1998, Rozema et al. 2002, Hader & Sinha 2005) including zooplankton (Leech & Williamson 2001, Dattilo et al. 2005, Cooke & Williamson 2006, Kessler et al. 2008), larval echinoderms (Lesser et al. 2003), molluscan embryos (Longo & Scarpa 1991, Li et al. 2000a; Li et al. 2000b, Przeslawski et al. 2004, Przeslawski et al. 2005; Wraith et al. 2006), algae (Holzinger & Lutz 2006) and coral larvae (Wellington & Fitt 2003). This is particularly true for broadcast spawning species in which eggs and early developmental stages remain suspended in the UV-penetrated portion of the water column (Adams et al. 2001, Au et al. 2002, Karentz et al. 2004, Lu & Wu 2005, Lesser et al. 2006). Whereas UVB induced DNA damage leading to disrupted embryonic or larval development has been frequently reported (Adams & Shick 2001, Adams et al. 2001, Lesser et al. 2003), fewer studies have looked at the effects of UV irradiation on eggs and sperm prior to fertilization (Bonaventura et al. 2006; Nahon et al. 2008).

To avoid the damaging effects of UVR, aquatic broadcast spawning species use a variety of defensive mechanisms including extraembryonic shielding cells, spawning behavior, DNA repair mechanisms, and UV absorbing molecules (Epel et al. 1999). Whereas UV absorbing compounds have been identified in molluscan eggs (Przeslawski et al. 2005), none of these mechanisms have been addressed in dreissenid species. Moreover, zebra mussels have several reproductive characteristics that may render them susceptible to UVR. Zebra mussel eggs are relatively small, have highly transparent cytoplasm, exhibit little pigmentation, and should remain suspended in the water column because of their near neutral buoyancy. Zebra mussel development is fairly rapid, limiting time for potential DNA repair. Also, spawned oocytes are not surrounded by protective accessory cells and UV protection via brooding is absent. It is unknown whether zebra mussels might spawn nocturnally to reduce UV exposure.

To date, there are no studies involving the effects of UVR on zebra mussel gametes. We addressed the effects of UVB radiation separately on eggs and sperm to determine its effects on gamete function and zygote development to first cleavage and identified the dose required to disrupt gamete function.

METHODS

Spawning and Gamete Collection

Zebra mussels (Dreissena polymorpha) were collected from Ann Arbor, MI and kept in aquaria filled with artificial pond water (PW) (Dietz et al. 1994) chilled to 10[degrees]C. Animals used for spawning ranged form 0.5-2 cm in length. Animals were red weekly with Shellfish Diet 1800 (Reed Mariculture, Campbell CA). Handling and spawning of animals followed (Misamore et al. 2006). Individual animals were isolated 24 h prior to use and allowed to warm to room temperature (~21[degrees]C). Spawning was induced by placing individual animals into 25-mL test tubes and adding 5-10 mL of 2 x [10.sup.-4] M serotonin, sufficient to just cover each animal. The mussels remained submerged for 12 min followed by two washes with PW. Males typically spawned within 5-10 min whereas females spawned 60 min after serotonin treatment. Males were removed from their test tubes once sufficient quantities of sperm were released to avoid damage to the sperm associated with continued filtering of the media. Once a female began spawning, she was transferred to a 70 x 50-mm crystallizing dish containing approximately 50 mL of PW to minimize clumping and damage to the eggs being released. To ensure freshly spawned sperm for fertilization trials, a second group of animals was spawned once females from the first group started spawning. Sperm from this second spawn were used to inseminate the eggs of the first. Thus, sperm were typically used within 30 min of spawning and eggs within 1 h of spawning.

Exposure of Gametes to UVB

Gametes were exposed to UVB emitted from a Westinghouse FS20 lamp (290-320 nm). A Solarmeter 6.2 Digital UV Radiometer (Solartech Inc., Harrison Twp, MI) with a spectral response between 280-320 nm was used to measure irradiance. The bulb was allowed to stabilize for at least 15 min prior to use. Samples were placed at a distance of 20 cm with an average irradiance of 263 [micro]W/[cm.sup.2] at this distance.

To expose individual gamete types, 10-mL suspensions of sperm or egg in PW were placed in a 25-mL watch glass to a total fluid depth of 4 mm. In sperm exposures, sperm concentrations were standardized to 2 x [10.sup.7] cells/mL based on hemocytometer counts to reduce any potential turbidity effects. Turbidity effects were not a concern in the egg exposure experiments, because the eggs settled in a single layer at the bottom of the dish at an equivalent depth. Sperm motility was observed in the cases of sperm exposure experiments.

Evaluation of UVB levels on Sperm Motility

In preliminary trials, sperm were subjected to a range of UVB and qualitative measures of sperm motility were made (Table 1). Observations on general sperm motility, swimming pattern, and sinusoidal flagellar beating were made. Sperm were scored as exhibiting normal sinusoidal swimming, nonsinusoidal/twitching movement (abnormal), or immotility. The first appreciable decline in sperm motility was observed ata dosage of 118 mJ/[cm.sup.2]. By 222 mJ/[cm.sup.2], a larger percentage of sperm exhibited disrupted (abnormal or immotile) swimming behavior. At 333 mJ/[cm.sup.2], most sperm were immotile with a few still exhibiting abnormal swimming.

With the primary focus of the present study on the disruptive effects of UVB on fertilization rather than general gamete immobilization, we selected UVB levels at or below dosage of first observable decline in sperm motility. For fertilization experiments, eggs or sperm were irradiated for 0, 2.5, 5, or 7.5 min with an average irradiance of 263 [micro]W/[cm.sup.2] resulting in total dosage for the four treatments of 0, 39, 79, 118 mJ/[cm.sup.2].

Fertilizations Between Irradiated and Nonirradiated Gametes

Fertilizations between irradiated and nonirradiated gametes were performed by mixing 400 [micro]L of sperm with 4 mL of eggs in a 50-mL beaker. In sperm-irradiated trials, 2 x [10.sup.7] cells/mL sperm from a single male was used to inseminate nonirradiated eggs from a single female. Three independent fertilizations involving different males and females were preformed. In egg-irradiated trials, eggs from a single female were inseminated with sperm pooled from several males. Three independent fertilizations were preformed involving different females and sperm pooled from different males. Fertilization samples were fixed at 0, 15, 30, 45, 70, and 90 min time points postinsemination (PI). Samples were fixed in 3.2% paraformaldehyde in mussel buffer (Misamore et al. 2006). To label DNA, fixed samples were stained with 1[micro]g/mL Hoechst 33342 for 15 min then washed twice with mussel buffer (Misamore et al. 2006).

Timing of Fertilization Events in Zebra Mussels

Fertilization events in zebra mussels have been previously reported (Misamore et al. 1996, Ram et al. 1996, Misamore & Lynn 2000, Misamore et al. 2006). Sperm bind uniformly across the egg surface (Fig. 1A,A') within 1-4 min after insemination. After binding, sperm are incorporated into the egg cytoplasm and the sperm nucleus begins to decondense (Fig. 1B,B') approximately 5 min PI. Eggs are arrested at metaphase 1 prior to insemination and meiosis resumes with the formation of the first polar body approximately 5-7 min PI (Fig. 1 C, C'). Pronuclear formation begins approximately 20 min PI and the pronuclei are evident in the eggs (Fig. 1C,C') until shortly before first cleavage (Fig. 1 D, D') at approximately 60 min PI.

For sperm-irradiated trials, the number of bound sperm and number of sperm incorporated into the egg cytoplasm were determined at 15 min PI. Observations distinguished between unbound, bound, and incorporated sperm. Incorporated sperm were identified based on the decondensation of the sperm chromatin observed shortly after entry into the egg cytoplasm (Misamore et al. 2006). Pronuclear formation and polar body formation were determined using the 45-min time points, whereas cleavage was determined at 90 min time points. In egg-irradiated trials, polar body formation, and the meiotic stage of female genetic material were determined at 30 min PI. Pronuclear formation was determined at 45 min PI and cleavage was determined at 90 min PI. In determining frequency of the various fertilization events, a minimum of 40 eggs from each of three independent fertilizations involving different males and females were scored in all experimental crosses.

[FIGURE 1 OMITTED]

Evaluation of Acrosomal Vesicles

To determine the potential effects of UVB on acrosomal integrity, three groups of UVB irradiated sperm were evaluated based on the presence of intact sperm acrosomes. To aid in distinguishing intact from disrupted acrosomes, sperm were labeled with 20 [micro]g/mL FITC-conjugated wheat germ agglutinin (WGA) (EY Laboratories, San Mateo, CA). WGA labels the outer plasma membrane including intact acrosomes (Misamore et al. 2006). When acrosomes react or are disrupted the inner portion of the acrosome is exposed allowing labeling of the inner acrosomal membrane and the acrosomal filament by WGA. Thus, acrosomal state was determined using phase and epifluorescent microscopy. Fifty sperm of both control and 118-mJ/[cm.sup.2] treatments were scored from three different trials.

Microscopy and Statistical Analysis

A Zeiss Axiovert equipped with phase contrast and epifluorescent optics was used for visual observations. A Zeiss Axiocam MRm camera and Axiovision software were used for image capture. Final image processing was done using Adobe Photoshop. Instat (GraphPad Software, Inc., La Jolla, CA) was used for statistical analysis. Unless indicated otherwise, one-way analysis of variance (ANOVA) was preformed with Tukey multiple comparisons when significant differences were observed. Microsoft Excel was used for generating graphs.

RESULTS

Fertilization Success of UV-Irradiated Sperm and Nonirradiated Eggs

To evaluate the effects of UVB on sperm function, three independent fertilizations between irradiated sperm and nonirradiated eggs were performed and fixed at various time points described earlier. Forty eggs from each treatment and time point were scored for the various fertilization events. When UVB irradiated sperm were used to inseminate nonirradiated eggs, there was no significant difference in the total number of bound sperm between the control (0 min) and UVB treatments (39, 79, and 118 mJ/[cm.sup.2]) based on a one-way analysis of variance (ANOVA; F = 0.1722; df = 3,8; P = 0.9122) (Fig. 2). However, there was a significant difference between treatments when looking at sperm incorporation into the egg cytoplasm (ANOVA; F = 8.464; df = 3,8; P = 0.0073) (Fig. 3). There was no significant difference in sperm incorporation between the 39- or 79-mJ/[cm.sup.2] UVB treatments and the control (Fig. 3) based on Tukey multiple comparisons (P > 0.05). However, there was a significant decrease in sperm incorporation in the 118-mJ/[cm.sup.2] treatments relative to the control (P < 0.01) even though there was no difference in number of sperm bound. Likewise, there was a significant decrease in intact acrosomes in the 118-mJ/[cm.sup.2] irradiated sperm relative to the control based on a t-test (t = 4.7, df = 4, P = 0.0093) (Fig. 4).

The ability of UVB irradiated sperm to induce egg activation, as indicated by the resumption of meiosis and the formation of the first polar body, was examined (Fig. 5A). There was a significant difference between the four treatments (ANOVA; F = 4.795; df = 3,8; P = 0.0339). There was a significant decrease in egg activation by 118-mJ/[cm.sup.2] irradiated sperm as compared with the control (Tukey; P < 0.05). Of the 120 total eggs counted in control treatments, none of the eggs without incorporated sperm formed polar bodies because of spontaneous egg activation.

By 45 min PI both male and female pronuclei have formed in normally developing eggs. There was a significant decrease in numbers of eggs exhibiting two pronuclei (one male, one female) between the treatments (ANOVA; F = 7.603; df = 3,8; P = 0.0100) (Fig. 5B). Moreover, the significant decrease observed in the 118-mJ/[cm.sup.2] irradiated sperm treatment for the presence of two pronuclei (Tukey, P < 0.05) parallels the similar decrease observed in sperm incorporation (Fig. 3).

The first zygotic cleavage is typically complete by 60 min PI in normally developing eggs. By 90 min PI, greater than 80% of control eggs completed first cleavage (Fig. 5C) and a number of these had completed second cleavage. There was a significant difference in first cleavage between the treatments (ANOVA; F = 17.275; df = 3,8; P = 0.0007) (Fig. 5C). Significantly fewer eggs inseminated with either the 79- or 118-mJ/[cm.sup.2] treated sperm had two pronuclei as compared with the control (Tukey; P < 0.05). There was no significant difference between the control and 39-mJ/[cm.sup.2] irradiated sperm treatments (P > 0.05).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Fertilization Success of UV-Irradiated Eggs and Nonirradiated Sperm

To evaluate the effects of UVB on egg function, three independent fertilizations between irradiated eggs and nonirradiated sperm were performed and fixed at various time points described earlier. Forty eggs from each treatment and time point were scored for the various fertilization events. Bound and incorporated sperm were observed in eggs exposed to all three UVB levels. Normally by 30 minutes PI, meiotic divisions in the egg are complete and the first and second polar bodies have formed (Misamore et al. 1996). In the present study, there was a significant difference in egg activation and polar body formation between the treatments (ANOVA; F : 24.778; df = 3, 8; P = 0.0143). Greater than 85% of the nonirradiated (control) and lower (39 and 79 mJ/[cm.sup.2]) irradiated eggs were activated and formed polar bodies by 30 min PI (Fig. 6A). However, there was a significant decrease in polar body formation in eggs irradiated with the highest UVB dosage (Tukey; P < 0.05).

To understand irridiated eggs were able to complete meiosis, the percentage of eggs completing meiosis at 45 min PI was determined for three independent trials of forty eggs per trial (Fig. 6B). There was a significant difference between the treatments for completion of meiosis (ANOVA; F = 69.824; df = 3,8; P < 0.0001). All of the control eggs completed meiosis (Fig. 6B). There was a significant decrease in eggs completing meiosis in the 79- and 118-mJ/[cm.sup.2] dosages relative to the control (Tukey; P < 0.01).

The percentage of eggs possessing two pronuclei (one male, one female) as part of the normal fertilization process was also examined (Fig. 6C). Percentage of eggs exhibiting two pronuclei at 45 min PI was determined for three independent trials of 40 eggs per trial. There was a significant difference in numbers of eggs with two pronuclei between the treatments (ANOVA; F = 106.987; df = 3,8; P < 0.0001). In the control, greater than 95% of the eggs had two pronuclei (one male, one female) present. The percentage of eggs with two pronuclei decreased significantly in each of the UVB treatments relative to the control (P < 0.05) (Fig. 6B).

[FIGURE 4 OMITTED]

To determine if UVB irradiation affected the egg's ability to undergo first cleavage, the number of divided eggs was determined at 90 min PI (Fig. 6D). With first cleavage typically occurring around 60 min PI, there was a significant difference in cleaved eggs between the treatments (ANOVA: F = 154.321; df = 3, 8; P < 0.0001). Greater than 60% of the control eggs divided by 90 min PI and many had completed second cleavage. Conversely, there was a significance decrease in numbers of zygotes that were able to divide in the UVB treatments (Tukey; P < 0.05) with less than 10% of the 79-mJ/[cm.sup.2] irradiated and none of the 118-mJ/[cm.sup.2] irradiated eggs dividing.

To evaluate the effects of UVB on the point at which meiotic development stopped, eggs were assigned 1 of 5 stages of failure to complete meiosis based on chromosome pattern: (1) metaphase I, (2) anaphase/telophase I, (3) prophase/metaphase II, (4) anaphase/telophase II, and (5) completed meiosis (Fig. 7). A four levels-of-UVB dosage by five points-of-failure contingency table analysis indicated a statistically significant difference in failure points among the exposure levels (Likelihood Ratio [chi square] = 339.9; df = 12; P < 0.0001). To determine at what dose levels these failure points changed, 3 successive two-dose levels X five failure point contingency table analyses were performed testing (1) 0 versus 39-mJ/[cm.sup.2] (2) 39 versus 79-mJ/[cm.sup.2] and (3) 79 versus 118-mJ/[cm.sup.2]. Significance levels for these nonindependent tests was set at P = 0.0167 (0.05 hypothesis wide alpha/3 independent tests). These tests indicate statistically significant differences in failure points between 0 versus 39-mJ/[cm.sup.2] (LR [chi square] = 21.8; df = 3, P < 0.0001), 39 versus 79-mJ/[cm.sup.2] (LR [chi square] = 30.2; df = 4, P < 0.0001), and 79 versus 118-mJ/[cm.sup.2] (LR [chi square] = 21.8; df = 3, P < 0.0001). Thus, UVB affected the failure point of meiosis with each increased dosage having an additional effect.

[FIGURE 5 OMITTED]

DISCUSSION

Zebra mussel gametes were highly sensitive to UV radiation and exposure resulted in reduced gametic function during fertilization. Progression of eggs and sperm through fertilization events were used as a measure of gametic function and viability. Eggs and sperm were similarly susceptible to levels of UVB, especially at the 118-mJ/[cm.sup.2] dosage. Inseminations involving unexposed eggs and irradiated sperm showed significant decreases in acrosomal integrity, sperm incorporation, polar body formation, pronuclear formation, and first cleavage. However, there was no associated decrease in sperm binding. These data suggest that sperm binding alone is insufficient for induction of egg activation. Inseminations involving irradiated eggs and unexposed sperm showed decreases in polar body formation, pronuclear formation, and cleavage. Disruption of meiosis was significantly higher in UVB irradiated eggs with each increased dosage resulting in earlier failures of progression.

[FIGURE 6 OMITTED]

Effects of UV Irradiation on Sperm Function Exposure of zebra mussel sperm to UVB negatively affected sperm motility. Although motility was not the primary focus of this study, it is worth noting that the first discernable decrease in motility occurred at 118 mJ/[cm.sup.2] and at 333 mJ/[cm.sup.2] most motility was disrupted. Because we were interested in better understanding how UVB might disrupt fertilization rather than sperm motility alone, we chose UVB levels at and below levels initially affecting sperm motility. This study suggests that the level of UV irradiance needed to disrupt gametic function is lower than that required to inhibit sperm motility, meaning that irradiated sperm may be motile but functionally sterile. These levels are below the proposed UV-treatment levels for controlling zebra mussel larvae (Chalker-Scott et al. 1994), suggesting that the level of irradiance necessary to kill zebra mussel larvae or juveniles should be sufficient in disrupting gametic function.

[FIGURE 7 OMITTED]

In all fertilization trials, most sperm exhibited normal motility. Additionally, there was no significant effect on sperm binding to eggs in any of the treatments (Fig. 2). However, there was a significant decrease in the ability of 118-mJ/[cm.sup.2] irradiated sperm to enter into the egg cytoplasm (Fig. 3). Thus, UVB-induced decrease in fertilization was caused by effects on postbinding sperm function. Our findings correspond with previous UVR studies using several marine bivalve species. Using comparable UV intensities, decreased fertilization (first cleavage) rates were observed with UV-irradiated sperm in the scallop Patinopectin yessoensis (Li et al. 2000b) and the oyster Crassostrea gigas (Li et al. 2000a). Here we show that not only is first cleavage reduced (Fig. 5C), but also there is a decrease in sperm entry into the egg cytoplasm (Fig. 3).

In P. yessoensis and C. gigas, sperm acrosomes were morphologically damaged at higher UV intensities (Li et al. 2000a, 2000b). Similar findings were observed in the present study. Although a significant portion of sperm in the 118-mJ/ [cm.sup.2] treatment had disrupted acrosomal vesicles, there was no significant decrease in sperm binding. However, there was a significant decrease in sperm entry into the cytoplasm after binding by sperm that had lost their acrosomal vesicles prior to binding. The enzymes contained in the sperm acrosome are believed to play an important role in penetration through the egg coat in numerous species (Reviewed in Hirohashi et al. 2008). The decrease in sperm incorporation into the egg cytoplasm in UVB irradiated sperm in zebra mussels may be caused in part to the loss of the sperm acrosome prior to penetrating the vitelline envelope. A greater understanding of the specific role of the acrosomal contents in the passage through the vitelline envelope leading to gamete fusion is needed to further support the hypothesis that the UV-induced decrease in zebra mussel sperm incorporation is caused by the loss of acrosomal vesicles.

Like many other species, it is unclear what specific fertilization event induces egg activation and the resumption of meiosis in zebra mussels. Several generalized models regarding the induction of egg activation have been proposed (Reviewed in Runft et al. 2002). In the contact hypothesis, the binding of receptors on the surfaces of sperm and eggs is sufficient to induce egg activation. In other models, fusion of sperm and egg plasma membranes is required. In the present study, UVB did not affect the ability of sperm to bind to the eggs but did decrease their ability to induce egg activation. One explanation is that UVB irradiation damaged a hypothetical sperm receptor for inducing egg activation without damaging the sperm's ability to adhere to the egg surface. Alternately, sperm incorporation may be essential for egg activation and the UVB-induced decrease in sperm incorporation accounts for the decrease in egg activation.

There was a decrease in eggs exhibiting two pronuclei in fertilizations involving the high UVB-irradiated sperm (Fig. 5B). This is expected, given the similar decrease in sperm incorporation. Alternatively, the decrease in two pronuclei may be attributed to incorporated sperm stopping pronuclear formation at the brief latent period after sperm nuclear decondensation where detection is more difficult (Misamore et al. 2006). The UVB-treated sperm that were incorporated may have failed to form sperm pronuclei. However, no obvious condensed sperm were observed in these later time points and Longo & Scarpa (1991) showed that UVR did not effect transformation of sperm into the male pronuclei in Mulina lateralis. Similar results of UV-treated sperm forming normal pronuclei have been reported in other bivalve species (Li et al. 2000a, Pan et al. 2004a).

There was a significant decrease in first cleavage in the inseminations involving the 79 and 118-mJ/[cm.sup.2] irradiated sperm. These UVB induced decreases in fertilization are similar to decreased fertilization rates reported for other species (Guo et al. 1993, Li et al. 2000a, Li et al. 2000b). Moreover, UV dosages ranging from 80-100 mJ/[cm.sup.2] resulting in significant decreases in first cleavage ate similar for zebra mussels and these other bivalve species (Guo et al. 1993, Fairbrother 1994, Li et al. 2000a; Li et al. 2000b, Pan et al. 2004a, Pan et al. 2004b).

Effects of UVB Irradiation on Egg Function

In the present study, we show that UVB exposure of zebra mussel eggs negatively affects their function when fertilized by nonirradiated sperm. UVB irradiation of eggs did not seem to alter the egg's ability to bind with sperm or allow sperm incorporation into the egg cytoplasm. Similarly, UV-irradiated urchin eggs were able to bind and fuse with sperm (Nahon et al. 2008). The most dramatic effects of UVB on egg function were associated with chromosomal separation during meiosis and subsequent first cleavage. Chromosomal separation leading to first polar body formation was effected only at the highest UVB level (Fig. 6A). However, subsequent formation of the second polar body and completion of meiosis (Fig. 6B) decreased significantly with each increasing UVB dosage. Ultimately, the normal zebra mussel development of two pronuclei and first zygotic cleavage was significantly effected by UVB. With increasing levels of UVB, the point at which meiosis failed occurred earlier in development (Fig. 7A). Nondisjunction of chromosomes was frequently observed in the UVB-arrested eggs. Morphologically similar chromosomal nondisjunction leading to abnormal cleavage has been reported in other molluscs (Longo & Scarpa 1991). Additionally, Nahon et al. (2008) found that sea urchin eggs irradiated with UVB prior to fertilization exhibited discernable chromatin damage. DNA damage in fertilized eggs irradiated shortly after fertilization resulting in abnormal development have been reported for several other species of urchin (Lesser & Barry 2003, Lesser et al. 2003).

Implications for Zebra Mussels

Although dosage comparisons between UV studies are often difficult because of nonuniformity in measuring UV intensity, there is clearly a trend in increased sensitivity to UV damage in earlier developmental stages (Bonaventura et al. 2006). In zebra mussels, levels needed to reduce fertilization efficiency (118 mJ-[cm.sup.2]) seem less than levels needed to kill larval stages (350 mJ/[cm.sup.2], Chalker-Scott et al. 1994). Thus, the recommended UVB intensities lethal to larval zebra mussels should be sufficient to disrupt gamete function (Chalker-Scott et al. 1994, Wright et al. 1997). Moreover, disruption of fertilization can be achieved at UVB levels less than required for complete immobilization of sperm.

The potential role of UVR on the distribution and survival of zebra mussels is more difficult to ascertain. The penetration of UVB in freshwater is highly variable and dependent on dissolved organic carbon and particulate material (Morris et al. 1995, Hader et al. 1998). However, UVB penetration in freshwater lakes can reach levels that have detrimental effects on aquatic biota (Arts et al. 2000, Kessler et al. 2008). As shown here with gametes and in similar studies focusing on later larval stages, early developmental stages in zebra mussels are more susceptible to UV damage (Chalker-Scott et al. 1994, Wright et al. 1997, Chalker-Scott & Scott 1998). Zebra mussel eggs, sperm, and early embryos are potential candidates of environmental UVB damage. The near-neutral buoyancy of the eggs should allow them to remain suspended in the water column. Whereas it is unlikely that gametes released by the deeper populations of zebra mussels especially in eutrophic lakes might be exposed to harmful UV, the potential for shallow-dwelling populations to be exposed to UVR is possible.

At present, the ability to predict limitations on zebra mussel reproduction because of environmental UVR is limited by two factors. First, more information regarding in situ reproduction of zebra mussels is needed. Our knowledge of the location, timing, and depth of actively spawning populations, the location of fertilization within the water column, and the distribution of larval stages throughout the water column are unknown of limited. Additionally, larval development is highly variable between locations with development times for some stages ranging from a few days to nearly a year (Nichols 1996). The second limiting factor for accurately projecting potential distribution limitations caused by UVR is accurate measurement of in situ intensities at the depths were the various stages of zebra mussel development occurs. Nevertheless, zebra mussel gametes, like the later developmental stages are highly susceptible to UVR.

ACKNOWLEDGMENTS

The authors acknowledge Dr. John Pinder, Texas Christian University, for his assistance with this project. Texas Christian University Research and Creative Activities Fund and the Undergraduate Research Grant Programs (SERC and URCAI) provided funding for this project.

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RYAN W. SEAVER, GARY W. FERGUSON, WILLIAM H. GEHRMANN AND MICHAEL J. MISAMORE *

Department of Biology, Texas Christian University, Forth Worth, Texas 76129

* Corresponding author. E-mail: m.misamore@tcu.edu
TABLE 1.
Qualitative observations of UVB effects on sperm motility.

                             Observations on Sperm
Irradiance                          Motility

  0 mJ/[cm.sup.2]    Full motility (normal swimming sperm)
 39 mJ/[cm.sup.2]    Full motility (normal swimming sperm)
 79 mJ/[cm.sup.2]    Full motility (normal swimming sperm)
118 mJ/[cm.sup.2]    Most sperm swimming normally, some
                       abnormal swimming
222 mJ/[cm.sup.2]    Some normal swimming, some abnormal
                       swimming, some immotile
333 mJ/[cm.sup.2]    Few abnormal swimming,
                       Most immotile

Sperm were exposed to varying levels of UVB and visual observations of

general motility and swimming patterns were made. Normal swimming
was defined as typical sinusoidal beating of the flagella. Abnormal
swimming was defined as movement that deviated from typical sinusoidal
beating of the flagella. Immotility was defined as no observable
movement of sperm.
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Author:Seaver, Ryan W.; Ferguson, Gary W.; Gehrmann, William H.; Misamore, Michael J.
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:1USA
Date:Aug 1, 2009
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