The impact of overcollection of tarantulas from in situ populations was first documented in 1995 with the protection of the genus Brachypelma (family, Theraphosidae) under the Convention on International Trade in Endangered Species Appendix II.1 Twelve additional species have since been listed as decreasing in number in the wild by the International Union for Conservation of Nature.2 Tarantulas are one of the most commonly exhibited invertebrates in zoological collections; however, they are rarely included in breeding programs within these institutions.3,4 Likewise, numerous tarantula species marketed in the pet trade do not reproduce efficiently in captivity, and to meet market demand, these species continue to be harvested and exported from their native habitats.5–7 Evidence for the waning of wild invertebrate populations and limited efforts to conserve them illustrate the need to explore techniques for advancing conservation of threatened populations.2,8
Captive breeding management is routinely used to facilitate the population growth and long-term survival of endangered animals, a practice that is supported by reproductive technologies such as gamete collection, gamete preservation, and artificial insemination. These breeding programs are based on a comprehensive understanding of fundamental species-specific reproductive biology.9 However, such information regarding tarantulas is currently incomplete.
Chilean rose tarantulas (Grammostola rosea) are native to Chile's Atacama Desert, where this species is harvested in great numbers for the international pet trade. Commercial tarantula suppliers have identified a progressive decrease in age (as determined by body size) of wild-caught individuals, suggesting that over-harvesting and depletion of reproductively mature adults are occurring.10 Despite the abundance of Chilean rose tarantulas in the pet trade, attempts at captive breeding have met with limited success, and continued exportation of wild-caught individuals creates concern for the sustainability of in situ populations.10
Male tarantulas use a fascinating reproductive strategy that permits long-term storage of sperm external to the spider's genital tract and at ambient temperatures.11 At maturity, sperm production occurs in testicular tissue located within the male's opisthosoma (abdomen). In preparation for the breeding season, semen is deposited onto a specialized sperm web and is immediately drawn up into sperm storage organs, the palpal bulbs. The palpal bulbs are modified copulatory organs, located on the distal segment of the male's foremost limbs (the pedipalps). Semen remains within the palpal bulbs until a receptive female is located, whereupon it is deposited into the female's genital tract. The cells remain in the female's genital tract until fertilization and oviposition occur.10,11 The male's unique gamete storage system creates an opportunity for retrieval of viable sperm while avoiding disruption of the delicate opisthosoma.
Previous studies of arachnid reproduction have focused on the use of genital tract anatomic features and gamete morphology to define phylogenetic relationships among spider species.12–18 To the authors' knowledge, all previous investigations required euthanasia of spiders to permit gamete recovery from internal reproductive structures, and no attempt was made to determine sperm viability.12–18 The purpose of the study reported here was to establish a nonterminal semen collection method for use in captive Chilean rose tarantulas and to evaluate tools for investigating morphology and viability of spermatozoa.
Materials and Methods
Animals—Seven mature male Chilean rose tarantulas, presumed to be wild-caught, were obtained from several suppliers. The tarantulas were estimated to be > 1.5 years of age given their reproductive maturity (determined by the presence of tibial hooks and palpal bulbs). Tarantulas were individually housed in 2-L enclosures lined with a vermiculite substrate and supplied with a hide area. Distilled water was provided in a shallow dish in each enclosure, and each tarantula was fed 1 to 3 crickets/wk. On the basis of visual examination, the tarantulas appeared healthy, and all of them consumed feed through the duration of the study. The ambient temperature was maintained at 26° to 28°C with a humidity level of 44% to 52%. The tarantulas were exposed to natural light.
Anesthesia—Tarantulas were not fed for 24 hours prior to an anesthetic event. Each tarantula underwent inhalation anesthesia according to the procedure outlined by Pizzi.19 To limit the stress of handling, a 500mL induction chamber containing a cotton ball saturated with 2 mL of isofluranea was placed directly over the tarantula within the enclosure. When the tarantula had a reduction in purposeful movement and minimal responsiveness to stimuli, it was transferred to an anesthesia maintenance device constructed from a 60-mL syringe case. The case opening was partially occluded, creating an entry point for the tarantula's opisthosoma, and the isoflurane-soaked cotton ball was placed within the device to maintain contact between the anesthetic gas and the external openings of the spider's book lungs. The tarantula was positioned in dorsal recumbency to facilitate semen collection. Following the procedure, the tarantula was returned to its enclosure and placed in sternal recumbency for recovery from anesthesia; it was not given access to water until after the recovery period.
Semen collection—Semen was collected from 7 mature tarantulas 2 to 5 times over a 12-month experimental period. For each collection, the left or right pedipalp of a given tarantula was isolated and a forceps was used to gently extend the palpal bulb, which is naturally held in a flexed position on the distal segment of the limb. A glass slide was placed in contact with the narrow opening of the palpal bulb (the embolus), and the forceps was used to apply pressure to the palpal bulb (Figure 1) until the desired amount of semen was deposited onto the glass slide or until it was determined that no extrudable semen remained within the palpal bulb. The procedure was repeated on the contralateral limb as needed. All extrudable semen was collected from both palpal bulbs of 2 tarantulas early in the experimental period, followed by a second complete collection approximately 12 weeks later.
Photograph of a Chilean rose tarantula (Grammostola rosea) during semen collection. The tarantula's left palpal bulb (arrow) is extended by use of forceps, and a glass slide held in contact with the bulb facilitates semen collection.
Citation: American Journal of Veterinary Research 75, 10; 10.2460/ajvr.75.10.929
Semen characterization—A total of 4 semen samples were collected from 3 individuals over a period of 12 weeks for evaluation via light microscopy. Samples were submerged immediately in approximately 100 μL of neutral-buffered 10% formal–saline solution (10 mL of formalin, 90 mL of deionized water, and 0.85 mg of NaCl) or McDowell and Trump formaldehyde-glutaraldehyde (4:1) fixative and covered with a coverslip to prevent drying. Samples were observed immediately after collection via light microscopy to confirm the presence of coenospermia. To examine individual spermatozoa, the spherical sperm packets (coenospermia) were disrupted by applying light pressure to the coverslip directly over the specimens.
Transmission electron microscopy was performed on 2 semen samples each collected from a different tarantula according to the procedure outlined by Dykstra.20 Each sample was transferred from a glass slide to a microcentrifuge tube containing 0.5 mL of McDowell and Trump formaldehyde-glutaraldehyde (4:1) fixative; a razor blade was used to elevate the sticky sample from the slide. Following fixation, fixative was poured off, and the sample was embedded in agar and then immersed in 1% osmium tetroxide and 0.1M phosphate buffer. The sample was rinsed with distilled water and dehydrated in a graded ethanol series. The sample was placed in fresh 100% resin in molds and polymerized at 70°C. Semithin (0.25- to 0.5-μm) sections were cut with a glass knife and stained with 1% toluidine blue-O stain in 1% sodium borate solution. Ultrathin (70- to 90-nm) sections were cut with a diamond knife and stained with methanolic uranyl acetate followed by lead citrate for examination with a transmission electron microscope.b
Scanning electron microscopy was performed on 2 semen samples each collected from different tarantulas according to the procedure outlined by Dykstra.20 The sample was collected onto a glass slidec and submerged in approximately 100 μL of McDowell and Trump formaldehyde-glutaraldehyde (4:1) fixative. Following fixation, the sample was rinsed with distilled water and dehydrated in a graded ethanol series. The sample was immersed in hexamethyldisilazane and covered for 3 hours, then immersed in fresh hexamethyldisilazane uncovered to allow for evaporation over a 24-hour period. The sample was mounted on specimen stubs with colloidal silver, sputter-coated with gold-palladium, and examined with a scanning electron microscope.d
A fluorescent membrane-permeant nucleic acid dye and propidium iodide sperm viability kite was used to assess sperm cell membrane integrity (ie, to distinguish live from dead spermatozoa) of 2 semen samples each collected from different spiders. The coenospermia were disrupted by applying pressure to a coverslip placed over the semen sample and then covered with 200 μL of saline (0.9% NaCl) solution pipetted directly onto the glass slide. A 20μM solution of the nucleic acid dye was prepared in saline solution. One microliter of this solution was applied to the sample, resulting in a final nucleic acid dye concentration of 100nM. The sample and dye were incubated at room temperature (approx 21°C) in the dark for 5 minutes. One microliter of the counter stain, propidium iodide, was applied to the sample, resulting in a final concentration of 12μM. The sample was incubated at room temperature in the dark for 5 minutes and underwent fluorescent microscopy.f
Statistical analysis—No statistical comparisons of data were made in this descriptive study. Mean and SD of measured variables were calculated.g
Results
Semen collection—The anesthetic protocol used for the study resulted in a smooth induction period and induced an appropriate plane of anesthesia in all tarantulas. No adverse reactions were observed. For each tarantula, normal behavior resumed following recovery from anesthesia with no apparent residual effects.
Semen was collected successfully from all 7 tarantulas. During a 12-month period, the mean number of collection procedures for an individual tarantula was 2.75 (range, 2 to 5 collections); the mean intercollection interval was 10 weeks (range, 24 hours to 29 weeks). For all tarantulas, the palpal bulb was extended easily from the distal tarsal segment of the foremost limbs, and pressure applied to the widest portion of the bulb with forceps resulted in extrusion of sperm through the embolus.
The tarantulas did not appear to react to the collection procedure, which ranged in duration from approximately 2 to 10 minutes. A small fracture in the external surface of the right palpal bulb of a single tarantula was observed during a collection procedure; this was presumed to be the result of excess force applied to the forceps during the procedure. This tarantula had no change in behavior or feeding patterns following anesthetic recovery, and semen samples were collected successfully from both palpal bulbs during subsequent procedures.
Grossly, the semen samples were white in color, had a wax-like consistency, and adhered readily to the glass slides. These characteristics precluded determination of exact semen volumes, although the largest sample was estimated to be < 1 μL. The amount of semen collected during a single procedure was determined by the volume required for analysis and did not result in complete emptying of the palpal bulb in most instances.
Sequential collection of all extrudable semen was successfully completed for 2 male tarantulas in the study, indicating that the tarantulas were able to refill their palpal bulbs within a relatively short period and confirming observations (made by the investigators [KEA and CSB]) of periodic sperm web formation and sperm deposition by several tarantulas during the study period. Volumes of semen were comparable between subsequent collections, with maximum volumes estimated to be < 1 μL for all samples.
Semen characterization—The semen samples were composed of numerous coenospermia, characterized by clusters of nonmotile sperm encased in a spherical sheath (Figure 2). Prior to manipulation, coenospermia were adhered in a single mass or several clumped masses. Intermixed with the coenospermia was a minimal amount of amorphous material, which appeared to bind the spheres together. Without further processing, the capsular sheath material did not allow evaluation of individual sperm cells. Light pressure applied to a coverslip overlying the semen sample caused disruption of the capsular sheath and release of spermatozoa. Cells were dispersed by dilution in either a fixative (for light and electron microscopy) or saline solution (for the sperm viability assay). Examination by light microscopy revealed numerous loosely coiled clusters of spermatozoa; each cluster represented a group of cells released from a single coenospermia sheath. Under the conditions of the study, spermatozoa did not appear to have the discrete head, midpiece, and tail regions characteristic of mammalian sperm cells. Individual spermatozoa had a spiral-shaped cell body that tapered gradually into the tail region with no obvious flagellar structure.
Representative photomicrographs of semen samples collected from Chilean rose tarantulas. A—Light microscopy revealed that the semen contains multiple spherical coenospermia, characterized by clusters of nonmotile sperm encased in a spherical sheath. Bar = 20 μm. B—Application of light pressure to the coverslip directly over the semen specimen disrupted the coenospermia, thereby releasing the spermatozoa. Multiple spermatozoa are present in each coenospermia capsule, as illustrated in this image. Bar = 10 μm. C and D—Individual sperm cells have a spiral-shaped cell body that tapered into the tail region with no flagellar structure. Bar in both panels = 5 μm.
Citation: American Journal of Veterinary Research 75, 10; 10.2460/ajvr.75.10.929
Ultrastructure imaging of semen samples revealed the spherical nature of the coenospermia. The mean ± SD diameter of the coenospermia was 10.3 ± 1.6 μm; the mean number of sperm cells per capsule was approximately 18.5 ± 3.8. At the widest point, the diameter of the anterior region of the spermatozoa was 1.5 ± 0.14 μm; mean length of the spermatozoa was 16.7 ± 1.4 μm. The spiral-shaped cell body was evident in SEM images (Figure 3). External morphological distinction between the cell body and tail was not evident by means of light microscopy or SEM. The contents of coenospermia identified by TEM included tightly packed spermatozoa containing numerous glycogen granules and abundant mitochondria surrounding a discrete electron-lucent structure (Figure 4). The coenospermia sheath appeared as a highly regular multilaminar structure (mean width, 1.03 ± 0.14 μm). Coenospermia and sperm cell dimensions were summarized (Table 1).
Representative SEM images of coenospermia and spermatozoa in semen samples collected from Chilean rose tarantulas. A—Image of numerous coenospermia in a semen sample. Bar = 100 μm. B—Release of spermatozoa from a rupture in the sheath of a single coenospermia. Bar = 5 μm. C—Spermatozoa released from a coenospermia capsule have a spiral-shaped cell body. Bar = 1 μm. D—Spermatozoa do not appear to have the discrete head, midpiece, and tail regions characteristic of mammalian sperm cells. Bar = 5 μm.
Citation: American Journal of Veterinary Research 75, 10; 10.2460/ajvr.75.10.929
Representative TEM images of coenospermia in semen samples collected from Chilean rose tarantulas. A—Spermatozoa are visible packaged within the coenospermia. Bar = 2 μm. B—The capsular sheath of each coenospermia has a laminar arrangement. Bar = 0.2 μm.
Citation: American Journal of Veterinary Research 75, 10; 10.2460/ajvr.75.10.929
Characteristics of the coenospermia and spermatozoa in semen samples collected from 7 Chilean rose tarantulas (Grammostola rosea).
| Variable | No. of sperm cells or coenospermia examined | Mean ± SD | Range |
|---|---|---|---|
| Sperm cell anterior width (μm) | 25 | 1.5 ± 0.14 | 1.3–1.7 |
| Sperm cell length (μm) | 5 | 16.7 ± 1.4 | 15.3–19.1 |
| Number of cells within coenospermia | 6 | 18.5 ± 3.8 | 14–25 |
| Coenospermia diameter (μm) | 105 | 10.3 ± 1.6 | 6.8–14.9 |
| Coenospermia capsular width (μm) | 13 | 1.03 ± 0.15 | 0.76–1.22 |
Application of the fluorescent membrane-permeant nucleic acid stain to undisrupted coenospermia did not result in stain penetration of the encapsulated sperm mass, although nonspecific staining of the capsular sheath was evident. Thus, the viability assessment of sperm cells within intact coenospermia was precluded. Therefore, disruption of the coenospermia capsule was required to allow staining of individual cells. Application of the membrane-permeant nucleic acid stain and propidium iodide to cells following coenospermia disruption resulted in differential staining of spermatozoa (ie, live vs dead; Figure 5). Live cells, represented by green fluorescence, were rarely identified under the staining conditions of the study.
Results of fluorescent staining to distinguish live from dead spermatozoa in semen samples collected from Chilean rose tarantulas. In general, live cells were rarely identified by means of the staining technique used; the number of dead sperm cells was large relative to the number of live sperm cells. A—Fluorescent green spermatozoa represent viable cells. In the coenospermia capsule, nonspecific uptake of the membrane-permeant nucleic acid stain is evident. B—Fluorescent orange spermatozoa represent nonviable cells. C—Nonviable cells were more common than viable cells. Fluorescent membrane-permeant nucleic acid stain and propidium iodide stain; bar = 20 μm.
Citation: American Journal of Veterinary Research 75, 10; 10.2460/ajvr.75.10.929
Discussion
Owing to the increasing awareness of the decline of native tarantula populations and the challenges of captive breeding, the investigation of tarantula gamete collection was undertaken to potentially benefit species conservation and advance the knowledge of arachnid reproductive physiology. Semen was collected successfully from all 7 tarantulas included in the present study. There were no detectable adverse effects associated with anesthesia or the collection technique applied. To the authors' knowledge, the present report represents the first description of a nonterminal procedure for semen collection and characterization in tarantulas, specifically G rosea.
The palpal bulb morphology of spiders has been studied extensively for the purpose of taxonomic classification, and palpal bulbs lack both musculature and innervation.21,22 The proposed mechanism of semen deposition into the female's gonopore at the time of mating is through the creation of hemolymphatic hydrostatic pressure within the bulb to facilitate semen expulsion.22 The collection technique employed in the present study produced increased pressure within the palpal bulb by applying force to the external surface. It is believed that this procedure mimicked the normal physiologic process of semen expulsion and therefore did not disrupt the function of the palpal organs; however, natural breeding of the study tarantulas was not attempted after semen collection.
The initial stages of tarantula spermatogenesis have a progression similar to that of mammals, in that peripherally located spermatogonial germ cells undergo a series of divisions that culminate in the maturation of spermatids and luminal release of spermatozoa.23 In contrast to mammals, spider spermatogenesis is completed in the deferent ducts following the production of an extracellular sheath secreted around clusters of cells, which results in the formation of coenospermia.23 The characteristics of Chilean rose tarantula semen are typical of other mygalomorph spiders, notably the presence of coenospermia comprised of clusters of numerous nonmotile sperm cells surrounded by a spherical capsule.24 The coenospermia structure is theorized to protect the inactive sperm cells from external insults during the prolonged period between gamete production and the time of spermatozoal activation and fertilization, which may span several months.25
Scanning electron micrographs of Chilean rose tarantula semen revealed coenospermia adhered together in the ejaculate mass by amorphous seminal secretions. Sperm cells released from disrupted coenospermia had a spiral-shaped cell body and a tail, with no discrete morphological delineation between these 2 regions (which is evident in flagellated spermatozoa). The function of the spiral-shaped cell body is unknown. This distinctive morphology may have a role in long-term membrane stability, aid in packaging spermatozoa tightly within the coenospermia, or contribute to effective fertilization. The lack of a distinctly defined tail and absence of microtubular flagellar structures identified on TEM imaging were surprising. In other tarantula species, postmortem evaluation of semen, by use of fixation and processing techniques similar to those used in the present study, revealed identifiable flagellar structures.24,26 Therefore, the unique appearance of the spermatozoal tail region observed in the present study was unlikely to be related to technique artifact.
With regard to TEM findings, G rosea semen resembled semen of other mygalomorph spiders. However, discrete areas of nuclear material were not apparent in the space interpreted as the anterior portion of the cell. This region appeared to contain a large electron-lucent region, a notable difference from findings for other tarantula species. The electron-lucent material is suspected to represent lipid storage, possibly as a source of energy that can be used either prior to or after insemination. Extensive glycogen particles and numerous mitochondria in the G rosea spermatozoa were expected findings and are features that have been previously identified in spermatozoa of spiders.24,26 Flagellar microtubule structures were not identified in TEM images of the Chilean rose tarantula spermatozoa, which supported the SEM and light microscopy findings. It is not known whether G rosea sperm cells are motile at the time of fertilization. Future studies exposing freshly collected semen to secretions produced by a female during oviposition may help determine whether motility is induced at the time of fertilization in this species.
In mammalian semen evaluations, quantification of the percentage of motile spermatozoa is routinely used as an indicator of cell viability. Viability analysis is critical to the assessment of individual ejaculates and also for optimizing various semen storage and cryopreservation techniques. Because tarantula spermatozoa are nonmotile at the time of collection, an alternate method of viability detection is necessary. Fluorescent stains to distinguish live from dead cells were applied to freshly collected semen samples as an indicator of sperm viability in the present study. This staining protocol has been validated in several invertebrate species27–29 and resulted in differential staining of live and dead sperm collected from the study tarantulas. The detection of both green fluorescent cells and orange fluorescent cells representing viable and nonviable cells, respectively, confirmed that a proportion of spermatozoa were alive after extrusion from the coenospermia in semen retrieved from the palpal bulbs. The quantification of the percentage of live cells was not possible under the staining conditions of the present study because of the large number of dead sperm cells relative to the number of live sperm cells. The fact that live cells were rarely identified under the staining conditions of the study was attributed to cell membrane damage associated with the mechanical disruption of coenospermia, rapid changes in environmental moisture and osmolarity after submersion in saline solution, and expiration of cells during the stain incubation period. Despite the low number of live cells identified with this staining technique under the described conditions, the successful identification of viable cells suggests that staining to distinguish live from dead sperm cells can be applied in future investigations. This method will aid in designing appropriate media for Chilean rose tarantula gamete analysis and storage by facilitating accurate quantification of live cells and may be used for semen evaluation prior to artificial insemination in the future.
Spermatogenesis in spiders begins as the male approaches sexual maturity and continues through the remainder of the male's life.23 The ability of Chilean rose tarantulas to refill the palpal bulbs after semen collection was identified in 2 tarantulas at the onset of the present study and was suspected in the remaining spiders on the basis of the appearance of sperm webs within individual enclosures during the experimental period. Additionally, sperm web production and loading of the palpal bulbs after semen collection was observed for a single mature male Haitian brown tarantula (Phormictopus cancerides) that was used as a pilot study animal and not included in the present investigation. The ability of Chilean rose tarantulas to refill the palpal bulbs has positive implications for repeated semen collections in this species and indicates that this procedure would likely have minimal effects on subsequent mating and reproductive success in both captive and wild tarantulas.
Chilean rose tarantulas are seasonal breeders in the wild. Mating occurs primarily in September and October, and during this time, the male relies heavily on pheromonal signals to locate a receptive female.30 It is plausible that proximity to a female and seasonal variations such as light, temperature, and humidity influence sperm production in tarantulas. The effect of environmental cues was not evaluated in the present study. By use of the collection technique described in this report, further investigations could be undertaken to explore the effect of seasonal and pheromonal cues on production and morphology of spermatozoa. Such information may help optimize both natural and assisted breeding techniques in captive tarantulas.
In the present study of Chilean rose tarantulas, collection of quiescent, encapsulated spermatozoa that are adapted for storage in an organ devoid of obvious metabolic support products suggested that the retrieved cells were in a relatively stable and environmentally resistant state. This characteristic may permit G rosea semen collection and transport and artificial insemination without the use of semen extender or temperature manipulation. Further investigation into the mechanisms enabling prolonged sperm cell survival at ambient temperatures and outside of a tarantula's reproductive tract may aid in the development of long-term semen storage techniques for mammalian gamete preservation in the absence of liquid nitrogen, an area of research currently receiving considerable interest.31–33
The procedure for tarantula gamete acquisition performed in the present study avoided the necessity of euthanasia and may be applied for taxonomic assessments in field settings, the study of tarantula gamete physiology, and the development of assisted reproductive technologies in spiders. All semen samples were highly concentrated, but the small sample volume of collected semen (≤ 1 μL) limited the opportunity for evaluation and further manipulation. Future studies involving Chilean rose tarantulas will require preparation of appropriate extension media to sustain sperm cell viability for analysis, further characterization of spermatozoal quiescence and activation, and techniques for artificial insemination. Expansion of the scientific understanding of tarantula reproductive physiology and development of effective breeding tools will promote conservation and awareness for this family of arachnids.
ABBREVIATIONS
| SEM | Scanning electron microscopy |
| TEM | Transmission electron microscopy |
Forane, Baxter, New Providence, NJ.
FEI/Philips EM 208S/Morgagni, FEI, Hillsboro, Ore.
SuperFrost Plus, Fisher Scientific, Waltham, Mass.
JEOL JSM-6360LV, JEOL, Peabody, Mass.
LIVE/DEAD Sperm Viability Kit (L-7011), Molecular Probes, Eugene, Ore.
Zeiss AxioImager M-1, Carl Zeiss Microscopy, Thornwood, NY.
Microsoft Excel 97-2003, Microsoft, Redmond, Wash.
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