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Fluorescein-dextran sequestration in the reproductive tract of the migratory grasshopper Melanoplus sanguinipes (Orthoptera, Acridiidae)
Micron 46 (2013) 80–84
Contents lists available at SciVerse ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Short communication
Fluorescein-dextran sequestration in the reproductive tract of the migratory grasshopper Melanoplus sanguinipes (Orthoptera, Acridiidae) Nathan Jones a,b , Tina Taub-Montemayor b,∗ , Mary Ann Rankin b a b
Brackenridge Field Laboratory, The University of Texas at Austin, 2907 Lake Austin Boulevard, Austin, TX 78703, United States Section of Integrative Biology, The University of Texas at Austin, 1 University Station #C0930, Austin, TX 78712, United States
a r t i c l e
i n f o
Article history: Received 8 August 2012 Received in revised form 3 December 2012 Accepted 3 December 2012 Keywords: Grasshopper Fluorescein-dextran Vas deferens Testis Sperm tube Aedegus
a b s t r a c t The fluid dynamics of the reproductive system of the migratory grasshopper, Melanoplus sanguinipes F. (Orthoptera: Acrididae) was examined by the introduction of fluorescein-dextran (FD) into the hemocoel and observing its tissue specific sequestration. Male grasshoppers were observed to sequester FD first in the apical end of each sperm tube. FD then moved into the vasa deferentia and ejaculatory duct. This suggests that materials, that transit the hemolymph could be a component of the spermatophore in M. sanguinipes. Female grasshoppers were observed to sequester hemolymph FD into vitellogenic oocytes and to sometimes reuptake the FD during the resorption of oocytes in the formation of yellow bodies or corpora lutea. Female M. sanguinipes who performed long duration flight sequestered FD in their oocytes to a greater degree than controls as determined by fluorescence intensity data collected as the mean gray value of the flourescein emission channel. Transfer of hemolymph FD from males to females was observed at the pores along the margin of the operculum of eggs in the female common oviduct following mating. FD has the potential to be an effective tracker of male reproductive secretions and as a tool for the observation of insect reproductive tract development. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Fluid transfer plays an important role in the development of reproductive tissues in insects (females reviewed in Wheeler, 1996; males reviewed in Happ, 1992). The fluid transport and sequestration of hemolymph resources in the developing reproductive system is an integral component of understanding reproduction (Englemann, 1970; Heming, 2003). A recently developed technique for examining fluid sequestration in insect reproductive development is the use of fluorphore conjugated dextrans as a tracer (Peel and Akam, 2007). Fluorescein-dextran (FD) is a complex, branched glucan with a fluorophore covalently bound to it (Singleton, 2002). Sequestration of FD, which has been introduced into the hemolymph of an experimental subject, can be used to follow uptake of hemolymph resources into the reproductive system of an insect (Peel and Akam, 2007). Melanoplus sanguinipes have panoistic ovaries; the synthesis of yolk proteins, lipids, and carbohydrates mostly takes place in the fat body before transport through the hemolymph to the developing oocyte (reviewed in Heming, 2003). These resources that
∗ Corresponding author. Tel.: +1 512 471 6905; fax: +1 512 471 3878. E-mail addresses: [email protected] (N. Jones), [email protected] (T. Taub-Montemayor), [email protected] (M.A. Rankin). 0968-4328/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.micron.2012.12.003
transit through the hemolymph are thought to be sequestered into only the terminal (proximal, basal) vitellogenic oocyte undergoing vitellogenesis (Englemann, 1970). In male grasshoppers the synthesis and sequestration of reproductive materials has been thought to be primarily local (Friedal and Gillott, 1977). The upper reproductive tract of male grasshoppers consists of a fused pair of testes. Each testis is composed of numerous sperm tubes in which spermatogenesis occurs (Heming, 2003). The sperm tubes have been reported to be impermeable in the basal half when incubated with tracer materials (Szolloisi and Marcaillou, 1977). The sperm tubes join at a pair of vasa deferentia leading from the testes to the lower reproductive tract that contains the secretory tissues referred to as the accessory and white glands (Klowden, 2007; Heming, 2003). The accessory glands of M. sanguinipes have been examined via electron microscopy by Couche and Gillott (1988). Their observations indicated a high degree of protein synthesis and secretory capacity by virtue of the highly developed Golgi complexes and endoplasmic reticulum in the accessory glands and white glands (Couche and Gillott, 1988). Other observations from Friedal and Gillott (1976) have indicated that hemolymph material may be incorporated into the developing accessory or white glands in a manner analogous to that of female fat body vitellogenin synthesis, hemolymph transport and oocyte sequestration. The ultrastructure of the accessory glands and white glands are indicative of high secretory activity and it
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remains unclear what if any the role of hemolymph fluid transport in male M. sanguinipes gonad development may be. Here we present an examination of fluorescein-dextran sequestration in male and female reproductive tract development as well as the acceleration of ovarian sequestration in M. sanguinipes following performance of long duration tethered flight. Previous studies have demonstrated that female individuals who experience a long duration flight have a significantly reduced time to first oviposition than controls (McAnelly and Rankin, 1986b; Rankin and Burchsted, 1992; Min et al., 2004). 2. Methods 2.1. Animal collection and rearing Adult M. sanguinipes were collected from the San Carlos Apache Reservation near Globe, Arizona and transported to the University of Texas at Austin. Colonies were maintained as in McAnelly and Rankin (1986a). Individuals were collected upon eclosion and separated into individual cages. The individuals were maintained at 30 ◦ C with a 16:8 h photo cycle. 2.2. Flight assay Female grasshoppers were assayed on the fourth day after eclosion using a tethered-flight apparatus (McAnelly and Rankin, 1986a). A small stick was attached to the pronotum of each grasshopper with wax, and the insect was then suspended in front of a fan. The fan, an electric heater, and incandescent lamps simulated conditions of wind speed, illumination, and temperature that are associated with migratory flights in the field (Parker et al., 1955). The grasshoppers that flew to voluntary cessation after a flight of at least 4 h (LF-E, N = 5) were compared to individuals whose flight was terminated by the observer at 1 h (LF-1, N = 4). Male grasshoppers were not tested for flight propensity in this study.
Fig. 1. Increased sequestration of hemocoel FD into terminal oocytes following performance of long duration, tethered flight of 4 h or more. (A) Lateral ovary of migrant whose flight was terminated after 1 h. (B) Lateral ovary of migrant that performed a long duration flight. LF-E green = AlexaFluor 488-dextran. Scale bars = 1 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
2.3. Injection of fluorescein-dextran Five l of 10 mg/ml 10,000 MW fixable AlexaFluor 488-dextran (Molecular Probes) in sterile ultra pure water was injected per individual (males, N = 7; non-flight tested females, N = 7 and flight tested females: LF-E, N = 4; LF-1, N = 5). Injections were performed via a 10 l Hamilton syringe (Hamilton Company, Reno, Nevada) that was washed in 100% ethanol and sterile, distilled water prior to and between each injection. Injections were performed between the 3rd and 4th abdominal sternite. Needle penetration was kept to a minimum to reduce the chance of injection of FD into nonhemocoel spaces. No mortality was observed in grasshoppers injected with FD during the study. 2.4. Dissections and fixation of tissue Female grasshoppers were injected five days following flight and dissected six days following flight performance (N = 9) while males and non-flight tested females were dissected 24 h following injection of fluorescein-dextran (the age of males and non-flight tested females varied, N = 7 for each gender). Tissues were excised and washed 3 times under ice-cold locust saline before fixation. Fixation was performed using the MEMFA formulae previously described for the fixation of Xenopus laevus embryos (Lee et al., 2008). Tissues were placed in the fixative (0.1 M MOPS, 2 mM EGTA, 1 mM MgSO4 pH 7.4, and 3.7% formaldehyde) for 1 h at room temperature and then washed three times in ice-cold phosphate buffered saline pH 7.5. Fixed tissues were kept at 5 ◦ C in PBS containing 0.01% (w/v) NaN3 until imaging was performed. For male to female transfer experiments 8 males were injected and allowed
to copulate for 48 h with a female. The female’s reproductive tract was then removed and analyzed as above for the presence of FD (N = 6). 2.5. Microscopy and imaging Micrographs were collected using a Leica MZ16 fluorescence stereomicroscope using the fluorescein dichromatic filter cube (Leica Wetzler, Germany) at the University of Texas Institute for Cellular and Molecular Biology core microscopy facility. Fluorescence exposures were kept between 0.1 and 0.2 s for male tissues and at 0.1 s for female tissues. Autofluorescence of noninjected tissues was observed when exposures were increased to 1.5 s, especially in those tissues on which the cuticular intima were sclerotized, such as the lateral oviduct, oocytes in the common oviduct, spermatheca and trachea. Four individuals of each sex were screened for autofluorescence. No autofluorescence was observed at the exposure parameters used in the study. Image processing was performed via the native Leica image software suite (Leica Wetzler, Germany). 3. Results 3.1. Sequestration of FD in female reproductive tissues When FD was introduced into the hemocoel of female M. sangunipes it was detected in the terminal oocytes within 24 h (Fig. 1). Furthermore terminal oocytes sometimes undergo resorption,
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Fig. 2. FD that is injected into the hemocoel of female M. sanguinipes is sometimes sequestered into the oocyte resorption bodies of M. sanguinipes. (A) Color micrograph of the lateral oviduct showing oocyte resorption bodies. (B) Composite micrograph of the sequestration of hemocoel FD into the oocyte resorption bodies. Green in B = fluoresceindextran. Scale bars = 0.5 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Table 1 Frequency of observation of FD in female reproductive tissues. Terminal oocyte
Oocyte resorption body
Intra-ovariole transfer
16/16
7/16
1/16
presumably of the materials that were sequestered during oogenesis (Highnam et al., 1963; Lusis, 1963; Phipps, 1966). FD concentration was highest in resorption bodies (corpora lutea) when they were present. That is, when FD was injected into females that were resorbing oocytes, the terminal oocytes that were undergoing this resorption, sequestered the FD into the so-called oocyte resorption (yellow) body or corpora lutea to a greater degree than surrounding tissue (Fig. 2). We did not observe oviposition in these females so the possibility of accumulation at the base of each ovariole is most likely due to resorption of oocytes rather than ovipositional scarring. Movement of FD from the terminal oocyte to the penultimate developing oocyte was also observed, and there appeared to be further transfer or sequestration between each subsequent oocyte as well (Fig. 3). This was only observed in one of the ovaries examined however and will be a subject of future work. The frequency of these observations is shown in Table 1. Performance of long-duration flight significantly increased the degree of sequestration of FD from hemolymph to oocytes relative to controls by the mean gray value of their respective ovaries in the flourescein channel (t(7) = 5.656, P < 0.001) (Fig. 1B and Table 2). 3.2. Sequestration of FD in male reproductive tissues The testes of M. sanguinipes are composed of a number of sperm tubes that join at the vas deferens. FD injected into the hemocoel of male M. sanguinipes was sequestered into the reproductive tract (Fig. 4 and Table 3). Beginning at the apical end of the sperm tube where the cyst progenitor cells reside (Fig. 4A). FD was observed in the testis via the vas deferens (Fig. 4B). FD was also observed to sequester in the ejaculatory duct of M. sanguinipes (Fig. 4C), but was never observed in the accessory glands or the white glands of the Table 2 Mean gray value of fluorescence micrographs from flight-tested individuals.
lower male reproductive tract (Fig. 4C). This observation does not preclude the transfer of hemolymph fluid to the accessory gland or white gland as the FD found in the aedegus may have been transferred from glands to the aedegus prior to our dissections. FD from injected male grasshoppers who were allowed to copulate with a female for 48 h was ambiguously observed in the pores (either aeropyles or micropyles) located at the margins of eggs found in the common oviduct of the female (Fig. 5). FD was not found in all eggs within the common oviduct and the high degree of autofluorescence that develops as the oocyte chorion sclerotizes made reliable detection of transferred FD challenging. 4. Discussion Understanding the sequestration of fluids from hemocoel to tissues is integral to our understanding insect reproductive development. This study shows that female M. sanguinipes performing long duration flight differ in how hemolymph material is sequestered into the vitellogenic terminal oocyte during oogenesis. Further it was demonstrated that material transfer occurs from basal to more distal oocytes within each ovariole. To our knowledge this is the first hint of intra-ovariolar transfer of material in a panoistic ovary. This observation suggests that during oogenesis, transfer of hemolymph fluids to the developing oocyte could be tracked but also that transfer may perhaps occur sequentially along the developing ovariole in the panoistic ovary of M. sanguinipes. The potential for transfer of fluids and possibly yolk components from one oocyte to the next is interesting in light of the developmental pattern of the more derived, meroistic ovariole in which the developing oocyte is either connected to, or accompanied by, sibling cells from the germarium. Fluid resource sharing between developing oocytes could perhaps even be a primitive form of the resource provisioning provided by the sibling trophocytes of the more derived meroistic ovary. The transfer of fluid during male M. sanguinipes reproductive development takes a significantly different form than that of the female. We observed that FD injected into the hemocoel enters the apical end of the sperm tube within the testicular sheath and is Table 3 Frequency of observation of FD in male reproductive tissues.
Group
N
Mean
Std Dev
SEM
LF-E LF-1
5 4
101.8 44.4
12 18.5
5.4 9.3
Sperm tube
Vas deferens
White gland
Accessory gland
Aedegus
5/7
2/7
0/7
0/7
6/7
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Fig. 3. Composite photomicrograph of the sequestration of FD in the terminal oocyte of M. sanguinipes (A) as well as its transfer to the penultimate oocyte (arrow in A). FD was also detected in the inter-oocyte follicular stalk (arrows in B). Green = fluorescein-dextran. Scale bars = 0.5 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
transferred to the vas deferens. This is somewhat different from the apical basal sperm tube barrier observed by Szolloisi and Marcaillou (1977) where in vitro incubation of sperm tubes with electron opaque tracer molecules did not result in penetration of the basal compartment. Our observation of FD entry and passage into the vas deferens may be indicative of a potential for utilizing male orthopterans as a vehicle for the delivery of developmental disruptors such as double stranded RNA to the developing embryos of a female with which the injected male mates (Peel and Akam, 2007). Hemocoel injection of fluorescein-dextran can be a powerful tool to track hemolymph material transfer and sequestration dynamics. We show here that the introduction of this molecule to the hemocoel of M. sanguinipes can be used in the examination of fluid transfer and sequestration during reproductive development in males and females. Finally some of our observations may indicate that hemolymph sequestration and transfer of fluids within the
Fig. 4. FD that is injected into the hemocoel of male M. sanguinipes is sequentially taken up into the apical end of the sperm tubes of each lobe of the testes (A). FD was sometimes found in the vas deferens where the sperm tubes from each testicular lobe join en route to the lower reproductive tract (B). FD was also observed in the ejaculatory duct or aedegus but never in the accessory glands or white glands (C). Scale bars A = 100 m; B and C = 1 mm.
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Acknowledgements The authors wish to thank Ryan Grey and Howard Wang for advice on early experiments to develop the methods. We thank Terry Heckmann for technical assistance and proofreading of the manuscript. Angela Bardo at the UT ICMB microscopy core facility also deserves our thanks for consulting and training NTJ on fluorescence stereomicroscopy. We thank two anonymous reviewers for helpful comments on an earlier version of this manuscript. This work was supported in part by NSF grant #: 0235892 to MAR. References
Fig. 5. Fluorescein-dextran from male M. sanguinipes injected with FD was faintly observed in the eggs of females with which they mated. FD was observed in pores (either aeropyles or micropyles) at the margins of the egg (B). Control eggs from the common oviduct of uninjected grasshoppers (A). Autofluorescence from the sclerotizing egg chorion (green cell borders in A and B) presents an obstacle to the use of this technique as a reliable paternity tag. Scale bars = 50 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
reproductive tract may be more complex than previously thought, such as in the case of inter-oocyte transfer. Conflicts of interest No conflicts of interest, financial or otherwise, are declared by the author(s).
Couche, G.A., Gillott, C., 1988. Development of secretory activity in the seminal vesicle of the male migratory grasshopper, Melanoplus sanguinipes (Fabr.) (Orthoptera: Acrididae). International Journal of Insect Morphology & Embryology 17 (1), 51–61. Englemann, R., 1970. The Physiology of Insect Reproduction. Pergamon Press, New York. Friedal, T., Gillott, C., 1976. Extraglandular synthesis of accessory reproductive gland components in male Melanoplus sanguinipes. Journal of Insect Physiology 22 (10), 1309–1311, 1313–1314. Friedal, T., Gillott, C., 1977. Contribution of male produced proteins to vitellogenesis in Melanoplus sanguinipes. Journal of Insect Physiology 23, 145–151. Happ, G.M., 1992. Maturation of the male reproductive system and its endocrine regulation. Annual Review of Entomology 37, 303–320. Heming, B.S., 2003. Insect Development and Evolution. Cornell University Press, Ithaca, NY. Highnam, K.C., Lusis, O., Hill, L., 1963. Factors affecting oocyte resorption in the desert locust Schistocerca gregaria (Forskal). Journal of Insect Physiology 9 (6), 827–837. Klowden, M.J., 2007. Reproductive systems. In: Physiological Systems in Insects, second ed. Elsevier Academic Press, Boston (Chapter 4), pp. 181–238. Lee, C., Kieserman, E., Gray, R.S., Park, T.J., Wallingford, J., 2008. Wholemount fluorescence immunocytochemistry on Xenopus embryos. Cold Spring Harbor Protocols, http://dx.doi.org/10.1101/pdb/prot4957. Lusis, O., 1963. The histology and histochemistry of development and resorption in the terminal oocytes of the desert locust, Schistocerca gregaria. Quarterly Journal of Microscopical Science, London 104, 57–68. McAnelly, M.L., Rankin, M.A., 1986a. Migration in the grasshopper Melanoplus sanguinipes (Fab.). I. The capacity for flight in non-swarming populations. Biological Bulletin 170, 368–377. McAnelly, M.L., Rankin, M.A., 1986b. Migration in the grasshopper Melanoplus sanguinipes (Fab.). II. Interactions between flight and reproduction. Biological Bulletin 170, 378–392. Min, K.J., Jones, N.T., Borst, D.W., Rankin, M.A., 2004. Increased juvenile hormone levels following long flight in the grasshopper, Melanoplus sanguinipes. Journal of Insect Physiology 50, 531–537. Parker, J.R., Newton, R.C., Shotwell, R.L., 1955. Observations on mass flights and other activities of the migratory grasshopper. USDA Technical Bulletin No. 1109. Peel, A.D., Akam, M., 2007. Dynamics of yolk deposition in the desert locust Schistocerca gregaria. Journal of Insect Physiology 53, 436–443. Phipps, J., 1966. Ovulation and oocyte resorption in Acridoidae (Orthoptera). Proceedings of the Royal Entomological Society of London – Series A 41 (4–6), 78–86. Rankin, M.A., Burchsted, J.C.A., 1992. The cost of migration in insects. Annual Review of Entomology 37, 533–559. Singleton, V., 2002. Review article: advances in techniques of dextran analysis – a modern day perspective. International Sugar Journal 104, 132–136. Szolloisi, A., Marcaillou, C., 1977. Electron microscope study of the blood–testis barrier in an insect: Locusta migratoria. Journal of Ultrastructure Research 59, 158–172. Wheeler, D., 1996. The role of nourishment in oogenesis. Annual Review of Entomology 41, 407–431.
Contents lists available at SciVerse ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Short communication
Fluorescein-dextran sequestration in the reproductive tract of the migratory grasshopper Melanoplus sanguinipes (Orthoptera, Acridiidae) Nathan Jones a,b , Tina Taub-Montemayor b,∗ , Mary Ann Rankin b a b
Brackenridge Field Laboratory, The University of Texas at Austin, 2907 Lake Austin Boulevard, Austin, TX 78703, United States Section of Integrative Biology, The University of Texas at Austin, 1 University Station #C0930, Austin, TX 78712, United States
a r t i c l e
i n f o
Article history: Received 8 August 2012 Received in revised form 3 December 2012 Accepted 3 December 2012 Keywords: Grasshopper Fluorescein-dextran Vas deferens Testis Sperm tube Aedegus
a b s t r a c t The fluid dynamics of the reproductive system of the migratory grasshopper, Melanoplus sanguinipes F. (Orthoptera: Acrididae) was examined by the introduction of fluorescein-dextran (FD) into the hemocoel and observing its tissue specific sequestration. Male grasshoppers were observed to sequester FD first in the apical end of each sperm tube. FD then moved into the vasa deferentia and ejaculatory duct. This suggests that materials, that transit the hemolymph could be a component of the spermatophore in M. sanguinipes. Female grasshoppers were observed to sequester hemolymph FD into vitellogenic oocytes and to sometimes reuptake the FD during the resorption of oocytes in the formation of yellow bodies or corpora lutea. Female M. sanguinipes who performed long duration flight sequestered FD in their oocytes to a greater degree than controls as determined by fluorescence intensity data collected as the mean gray value of the flourescein emission channel. Transfer of hemolymph FD from males to females was observed at the pores along the margin of the operculum of eggs in the female common oviduct following mating. FD has the potential to be an effective tracker of male reproductive secretions and as a tool for the observation of insect reproductive tract development. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Fluid transfer plays an important role in the development of reproductive tissues in insects (females reviewed in Wheeler, 1996; males reviewed in Happ, 1992). The fluid transport and sequestration of hemolymph resources in the developing reproductive system is an integral component of understanding reproduction (Englemann, 1970; Heming, 2003). A recently developed technique for examining fluid sequestration in insect reproductive development is the use of fluorphore conjugated dextrans as a tracer (Peel and Akam, 2007). Fluorescein-dextran (FD) is a complex, branched glucan with a fluorophore covalently bound to it (Singleton, 2002). Sequestration of FD, which has been introduced into the hemolymph of an experimental subject, can be used to follow uptake of hemolymph resources into the reproductive system of an insect (Peel and Akam, 2007). Melanoplus sanguinipes have panoistic ovaries; the synthesis of yolk proteins, lipids, and carbohydrates mostly takes place in the fat body before transport through the hemolymph to the developing oocyte (reviewed in Heming, 2003). These resources that
∗ Corresponding author. Tel.: +1 512 471 6905; fax: +1 512 471 3878. E-mail addresses: [email protected] (N. Jones), [email protected] (T. Taub-Montemayor), [email protected] (M.A. Rankin). 0968-4328/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.micron.2012.12.003
transit through the hemolymph are thought to be sequestered into only the terminal (proximal, basal) vitellogenic oocyte undergoing vitellogenesis (Englemann, 1970). In male grasshoppers the synthesis and sequestration of reproductive materials has been thought to be primarily local (Friedal and Gillott, 1977). The upper reproductive tract of male grasshoppers consists of a fused pair of testes. Each testis is composed of numerous sperm tubes in which spermatogenesis occurs (Heming, 2003). The sperm tubes have been reported to be impermeable in the basal half when incubated with tracer materials (Szolloisi and Marcaillou, 1977). The sperm tubes join at a pair of vasa deferentia leading from the testes to the lower reproductive tract that contains the secretory tissues referred to as the accessory and white glands (Klowden, 2007; Heming, 2003). The accessory glands of M. sanguinipes have been examined via electron microscopy by Couche and Gillott (1988). Their observations indicated a high degree of protein synthesis and secretory capacity by virtue of the highly developed Golgi complexes and endoplasmic reticulum in the accessory glands and white glands (Couche and Gillott, 1988). Other observations from Friedal and Gillott (1976) have indicated that hemolymph material may be incorporated into the developing accessory or white glands in a manner analogous to that of female fat body vitellogenin synthesis, hemolymph transport and oocyte sequestration. The ultrastructure of the accessory glands and white glands are indicative of high secretory activity and it
N. Jones et al. / Micron 46 (2013) 80–84
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remains unclear what if any the role of hemolymph fluid transport in male M. sanguinipes gonad development may be. Here we present an examination of fluorescein-dextran sequestration in male and female reproductive tract development as well as the acceleration of ovarian sequestration in M. sanguinipes following performance of long duration tethered flight. Previous studies have demonstrated that female individuals who experience a long duration flight have a significantly reduced time to first oviposition than controls (McAnelly and Rankin, 1986b; Rankin and Burchsted, 1992; Min et al., 2004). 2. Methods 2.1. Animal collection and rearing Adult M. sanguinipes were collected from the San Carlos Apache Reservation near Globe, Arizona and transported to the University of Texas at Austin. Colonies were maintained as in McAnelly and Rankin (1986a). Individuals were collected upon eclosion and separated into individual cages. The individuals were maintained at 30 ◦ C with a 16:8 h photo cycle. 2.2. Flight assay Female grasshoppers were assayed on the fourth day after eclosion using a tethered-flight apparatus (McAnelly and Rankin, 1986a). A small stick was attached to the pronotum of each grasshopper with wax, and the insect was then suspended in front of a fan. The fan, an electric heater, and incandescent lamps simulated conditions of wind speed, illumination, and temperature that are associated with migratory flights in the field (Parker et al., 1955). The grasshoppers that flew to voluntary cessation after a flight of at least 4 h (LF-E, N = 5) were compared to individuals whose flight was terminated by the observer at 1 h (LF-1, N = 4). Male grasshoppers were not tested for flight propensity in this study.
Fig. 1. Increased sequestration of hemocoel FD into terminal oocytes following performance of long duration, tethered flight of 4 h or more. (A) Lateral ovary of migrant whose flight was terminated after 1 h. (B) Lateral ovary of migrant that performed a long duration flight. LF-E green = AlexaFluor 488-dextran. Scale bars = 1 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
2.3. Injection of fluorescein-dextran Five l of 10 mg/ml 10,000 MW fixable AlexaFluor 488-dextran (Molecular Probes) in sterile ultra pure water was injected per individual (males, N = 7; non-flight tested females, N = 7 and flight tested females: LF-E, N = 4; LF-1, N = 5). Injections were performed via a 10 l Hamilton syringe (Hamilton Company, Reno, Nevada) that was washed in 100% ethanol and sterile, distilled water prior to and between each injection. Injections were performed between the 3rd and 4th abdominal sternite. Needle penetration was kept to a minimum to reduce the chance of injection of FD into nonhemocoel spaces. No mortality was observed in grasshoppers injected with FD during the study. 2.4. Dissections and fixation of tissue Female grasshoppers were injected five days following flight and dissected six days following flight performance (N = 9) while males and non-flight tested females were dissected 24 h following injection of fluorescein-dextran (the age of males and non-flight tested females varied, N = 7 for each gender). Tissues were excised and washed 3 times under ice-cold locust saline before fixation. Fixation was performed using the MEMFA formulae previously described for the fixation of Xenopus laevus embryos (Lee et al., 2008). Tissues were placed in the fixative (0.1 M MOPS, 2 mM EGTA, 1 mM MgSO4 pH 7.4, and 3.7% formaldehyde) for 1 h at room temperature and then washed three times in ice-cold phosphate buffered saline pH 7.5. Fixed tissues were kept at 5 ◦ C in PBS containing 0.01% (w/v) NaN3 until imaging was performed. For male to female transfer experiments 8 males were injected and allowed
to copulate for 48 h with a female. The female’s reproductive tract was then removed and analyzed as above for the presence of FD (N = 6). 2.5. Microscopy and imaging Micrographs were collected using a Leica MZ16 fluorescence stereomicroscope using the fluorescein dichromatic filter cube (Leica Wetzler, Germany) at the University of Texas Institute for Cellular and Molecular Biology core microscopy facility. Fluorescence exposures were kept between 0.1 and 0.2 s for male tissues and at 0.1 s for female tissues. Autofluorescence of noninjected tissues was observed when exposures were increased to 1.5 s, especially in those tissues on which the cuticular intima were sclerotized, such as the lateral oviduct, oocytes in the common oviduct, spermatheca and trachea. Four individuals of each sex were screened for autofluorescence. No autofluorescence was observed at the exposure parameters used in the study. Image processing was performed via the native Leica image software suite (Leica Wetzler, Germany). 3. Results 3.1. Sequestration of FD in female reproductive tissues When FD was introduced into the hemocoel of female M. sangunipes it was detected in the terminal oocytes within 24 h (Fig. 1). Furthermore terminal oocytes sometimes undergo resorption,
82
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Fig. 2. FD that is injected into the hemocoel of female M. sanguinipes is sometimes sequestered into the oocyte resorption bodies of M. sanguinipes. (A) Color micrograph of the lateral oviduct showing oocyte resorption bodies. (B) Composite micrograph of the sequestration of hemocoel FD into the oocyte resorption bodies. Green in B = fluoresceindextran. Scale bars = 0.5 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Table 1 Frequency of observation of FD in female reproductive tissues. Terminal oocyte
Oocyte resorption body
Intra-ovariole transfer
16/16
7/16
1/16
presumably of the materials that were sequestered during oogenesis (Highnam et al., 1963; Lusis, 1963; Phipps, 1966). FD concentration was highest in resorption bodies (corpora lutea) when they were present. That is, when FD was injected into females that were resorbing oocytes, the terminal oocytes that were undergoing this resorption, sequestered the FD into the so-called oocyte resorption (yellow) body or corpora lutea to a greater degree than surrounding tissue (Fig. 2). We did not observe oviposition in these females so the possibility of accumulation at the base of each ovariole is most likely due to resorption of oocytes rather than ovipositional scarring. Movement of FD from the terminal oocyte to the penultimate developing oocyte was also observed, and there appeared to be further transfer or sequestration between each subsequent oocyte as well (Fig. 3). This was only observed in one of the ovaries examined however and will be a subject of future work. The frequency of these observations is shown in Table 1. Performance of long-duration flight significantly increased the degree of sequestration of FD from hemolymph to oocytes relative to controls by the mean gray value of their respective ovaries in the flourescein channel (t(7) = 5.656, P < 0.001) (Fig. 1B and Table 2). 3.2. Sequestration of FD in male reproductive tissues The testes of M. sanguinipes are composed of a number of sperm tubes that join at the vas deferens. FD injected into the hemocoel of male M. sanguinipes was sequestered into the reproductive tract (Fig. 4 and Table 3). Beginning at the apical end of the sperm tube where the cyst progenitor cells reside (Fig. 4A). FD was observed in the testis via the vas deferens (Fig. 4B). FD was also observed to sequester in the ejaculatory duct of M. sanguinipes (Fig. 4C), but was never observed in the accessory glands or the white glands of the Table 2 Mean gray value of fluorescence micrographs from flight-tested individuals.
lower male reproductive tract (Fig. 4C). This observation does not preclude the transfer of hemolymph fluid to the accessory gland or white gland as the FD found in the aedegus may have been transferred from glands to the aedegus prior to our dissections. FD from injected male grasshoppers who were allowed to copulate with a female for 48 h was ambiguously observed in the pores (either aeropyles or micropyles) located at the margins of eggs found in the common oviduct of the female (Fig. 5). FD was not found in all eggs within the common oviduct and the high degree of autofluorescence that develops as the oocyte chorion sclerotizes made reliable detection of transferred FD challenging. 4. Discussion Understanding the sequestration of fluids from hemocoel to tissues is integral to our understanding insect reproductive development. This study shows that female M. sanguinipes performing long duration flight differ in how hemolymph material is sequestered into the vitellogenic terminal oocyte during oogenesis. Further it was demonstrated that material transfer occurs from basal to more distal oocytes within each ovariole. To our knowledge this is the first hint of intra-ovariolar transfer of material in a panoistic ovary. This observation suggests that during oogenesis, transfer of hemolymph fluids to the developing oocyte could be tracked but also that transfer may perhaps occur sequentially along the developing ovariole in the panoistic ovary of M. sanguinipes. The potential for transfer of fluids and possibly yolk components from one oocyte to the next is interesting in light of the developmental pattern of the more derived, meroistic ovariole in which the developing oocyte is either connected to, or accompanied by, sibling cells from the germarium. Fluid resource sharing between developing oocytes could perhaps even be a primitive form of the resource provisioning provided by the sibling trophocytes of the more derived meroistic ovary. The transfer of fluid during male M. sanguinipes reproductive development takes a significantly different form than that of the female. We observed that FD injected into the hemocoel enters the apical end of the sperm tube within the testicular sheath and is Table 3 Frequency of observation of FD in male reproductive tissues.
Group
N
Mean
Std Dev
SEM
LF-E LF-1
5 4
101.8 44.4
12 18.5
5.4 9.3
Sperm tube
Vas deferens
White gland
Accessory gland
Aedegus
5/7
2/7
0/7
0/7
6/7
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Fig. 3. Composite photomicrograph of the sequestration of FD in the terminal oocyte of M. sanguinipes (A) as well as its transfer to the penultimate oocyte (arrow in A). FD was also detected in the inter-oocyte follicular stalk (arrows in B). Green = fluorescein-dextran. Scale bars = 0.5 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
transferred to the vas deferens. This is somewhat different from the apical basal sperm tube barrier observed by Szolloisi and Marcaillou (1977) where in vitro incubation of sperm tubes with electron opaque tracer molecules did not result in penetration of the basal compartment. Our observation of FD entry and passage into the vas deferens may be indicative of a potential for utilizing male orthopterans as a vehicle for the delivery of developmental disruptors such as double stranded RNA to the developing embryos of a female with which the injected male mates (Peel and Akam, 2007). Hemocoel injection of fluorescein-dextran can be a powerful tool to track hemolymph material transfer and sequestration dynamics. We show here that the introduction of this molecule to the hemocoel of M. sanguinipes can be used in the examination of fluid transfer and sequestration during reproductive development in males and females. Finally some of our observations may indicate that hemolymph sequestration and transfer of fluids within the
Fig. 4. FD that is injected into the hemocoel of male M. sanguinipes is sequentially taken up into the apical end of the sperm tubes of each lobe of the testes (A). FD was sometimes found in the vas deferens where the sperm tubes from each testicular lobe join en route to the lower reproductive tract (B). FD was also observed in the ejaculatory duct or aedegus but never in the accessory glands or white glands (C). Scale bars A = 100 m; B and C = 1 mm.
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Acknowledgements The authors wish to thank Ryan Grey and Howard Wang for advice on early experiments to develop the methods. We thank Terry Heckmann for technical assistance and proofreading of the manuscript. Angela Bardo at the UT ICMB microscopy core facility also deserves our thanks for consulting and training NTJ on fluorescence stereomicroscopy. We thank two anonymous reviewers for helpful comments on an earlier version of this manuscript. This work was supported in part by NSF grant #: 0235892 to MAR. References
Fig. 5. Fluorescein-dextran from male M. sanguinipes injected with FD was faintly observed in the eggs of females with which they mated. FD was observed in pores (either aeropyles or micropyles) at the margins of the egg (B). Control eggs from the common oviduct of uninjected grasshoppers (A). Autofluorescence from the sclerotizing egg chorion (green cell borders in A and B) presents an obstacle to the use of this technique as a reliable paternity tag. Scale bars = 50 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
reproductive tract may be more complex than previously thought, such as in the case of inter-oocyte transfer. Conflicts of interest No conflicts of interest, financial or otherwise, are declared by the author(s).
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