METHODS FOR USING SPERMIOGRAMS TO SCORE VIABILITY OF SPERMATOZOA IN FRUIT FLY STERILE MALES

Abstract

A method for determining the relative viability of sterilized male fruit flies, focusing on examination of the male fruit fly teste or testes to enable consideration of the spermatozoa extruding from the vas deferens of the teste. The evaluation may determine relative viability of the spermatozoa; spermatogenesis; activity or motility of the spermatozoa; or morphological or physiological sperm cell abnormalities. The spermatozoa activity may also be captured in still or moving images, to allow further consideration based on the images.

Claims

1. A method of determining viability of sterile male fruit fly spermatozoa, comprising: (a) obtaining a sterile male fruit fly from a particular sterilized population, (b) isolating from the sterile male fruit fly at least one fruit fly teste together with its respective vas deferens, and (c) examining the condition of spermatozoa extruding from the vas deferens.

2. The method of claim 1, wherein the examining is conducted under magnification.

3. The method of claim 2, wherein the magnification is provided by use of a microscope.

4. The method of claim 1, wherein the teste and vas deferens are placed on a microscope slide for examination using a microscope.

5. The method of claim 4, wherein the examination considers the spermatozoa's activity and motility.

6. The method of claim 4, wherein the examination is conducted to analyze spermatogenesis of the spermatozoa.

7. The method of claim 5, wherein the examination includes capturing still or moving images of the spermatozoan activity.

8. The method of claim 6, wherein the examination includes capturing still or moving images of spermatozoan activity.

9. A method of determining viability of sterile male fruit fly spermatozoa, comprising (a) isolating at least one male fruit fly teste together with its respective vas deferens, and (b) examining spermatozoa extruding from the vas deferens to detect morphological or physiological sperm cell abnormalities.

10. The method of claim 9, wherein the examination also determines relative activity and motility of the spermatozoa extruding from the vas deferens.

11. The method of claim 9, wherein the examination evaluates spermatogenesis.

12. The method of claim 11, wherein the examination evaluates a level of morphological or physiological sperm cell abnormalities.

13. The method of claim 10, wherein the examination includes capturing still or moving images of the spermatozoan activity to permit further analysis of the activity.

14. The method of claim 11, wherein the examination includes capturing still or moving images of spermatozoan activity.

15. The method of claim 12, wherein the examination includes capturing still or moving images of spermatozoan activity.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 shows a male fly in a petri dish with sterile saline solution or Ringer's Solution, ready for visualization under a dissecting microscope.

[0021] FIG. 2 shows a fly grasped by the thorax using angled forceps, with Vannas scissors being used to make an incision through the wall of the abdomen along the dorsal midline, from the apex to the base.

[0022] FIG. 3 shows the fly, with reproductive organs exposed, on a microscope slide, ready for dissection of the testes.

[0023] FIG. 4 shows the magnified vas deferens, and the free spermatozoa extruding from the vas deferens.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present method provides a technique for determining the effectiveness of sterile male fruit flies that are intended to be used in a SIT program, to mate with wild female fruit flies without resulting in fertilized eggs and viable offspring. This new technique uses spermiogram analysis of sterile male fruit flies to determine their viability, and the attractiveness of the sterile male fruit fly spermatozoa to wild female fruit flies. This use of spermiogram analysis provides a means to reliably determine quality assurance and quality control regarding the sterile male fruit flies to be released for mating with wild female fruit flies, in an attempt to control the wild fruit fly population.

The method involves micro-dissection of the male gonads (testes) of sterile male fruit flies, and in vivo visualization of sperm movement at the end of the vas deferens from the sterile male fruit flies. The fruit fly spermiogram is carried out undiluted, also known as a wet technique. The cover slipped area is used to determine the volume required to allow accurate measurements of the concentration of spermatozoa, under microscopic examination.

[0025] This evaluation enables consideration of the concentration, structure, and activity of the spermatozoa, to help determine the laboratory colonies' performance in real-time.

[0026] The process analyzes the viability of sterile male fruit fly spermatozoa, as well as appropriate spermatogenesis, aiding the early detection of morphological/physiological sperm cell abnormalities within the testes. The results of this analysis can help suggest and guide the use of timely corrective measures to help ensure the release of viable, competitive sterile insects into the field, improving the prospects of the SIT program successfully resulting in the reduction of the wild population.

[0027] This process helps fill a gap in the current methodology. Typical SIT programs do not account in this manner for the ability of the physiology of wild female fruit flies to discern between viable sperm and sperm that is not viable, and potentially to eject sperm from sterile male fruit flies in favor of wild male spermatozoa. This analysis of the viability of sterile male fruit fly spermatozoa will help strengthen the field practitioner's ability to reliably prepare and release effective sterile male fruit flies into the subject population.

[0028] One embodiment of the present subject matter is summarized in FIGS. 1-4. A fruit fly to be examined is placed in a petri dish with sterile saline solution or Ringer's Solution. This enables visualization under the dissecting microscope during the procedure. FIG. 1. The fly's abdomen is cut open along the dorsal midline from the apex to the base. FIG. 2. This exposes the reproductive organs of the fly. Next, the testes are separated from the fly's body, by using forceps to grasp the testes at a point near the vas deferens, and gently pulling the testes away from the fly's abdomen. Each teste is placed on a microscope slide, with a drop of Ringer's solution or sterile water, and gently covered with a slide cover-slip. See FIG. 3. The specimen can then be examined, such as by using a compound microscope. In order to evaluate the viability and movement of spermatozoa, the specimen may be examined under successively increasing magnification up to 1000, using an oil immersion lens. FIG. 4 shows free spermatozoa extruding from the vas deferens. If possible, recording an in vivo video provides active record of the spermatozoa activity and motility.

[0029] One embodiment of the present subject matter is a method for determining the viability of sterile male fruit fly spermatozoa. The method involves isolating the testes of the male fruit fly, and examination under magnification to determine activity and motility.

[0030] Another embodiment of the present subject matter is a method for determining the viability of sterile male fruit fly spermatozoa, and considering appropriate spermatogenesis, to help detect morphological and physiological sperm cell abnormalities. The method involves use of a dissecting microscope to enable dissection of the testes from the fly and use of a compound microscope to examine the spermatozoa exuding from the vas deferens, and optionally the use of a digital camera to capture still or active images of the spermatozoan activity.

[0031] Another embodiment of the present subject matter is a method for determining the viability of sterile male fruit fly spermatozoa. The method involves recordation of in vivo video of the spermatozoa activity, to document the sperm motility and activity.

[0032] The sperm analyses, or spermiograms, enable in vivo or in vitro evaluation of the concentration, structure, and activity of the spermatozoa directly from the male's testes.

[0033] A spermiogram is the most reliable test to directly measure, in real time, male fertility. The spermiograms provide real-time results that allow the detection of abnormalities, the determination of the presence or absence of pathogens within the testes, including for example parasites, bacteria, or protozoans, and provide reliable, timely remediation recommendations that will serve as scientific evidence to assist upper management with a better-informed decision-making process. This analysis provides a valuable added quality control test, evaluating the sterile male flies in order to help determine the laboratory colonies performance in real-time.

[0034] The process may be used to analyze qualitative and/or quantitative factors regarding the viability of spermatozoa, as well as appropriate spermatogenesis, aiding the early detection of morphological/physiological sperm cell abnormalities within the testes. Qualitative factors may include, for example, sperm morphology, physiology, and motility. Quantitative factors may include, for example, total spermatozoa count, sperm volume, and spermatozoa/seminal fluid volume ratio.

[0035] The results of this analysis can help guide the potential recommendation of timely corrective measures intended to ensure the release of viable, competitive sterile insects into the field. This can be used to help maximize the chances of the SIT program resulting in the desired effect on the wild population.

[0036] Normal results of a spermiogram test, or normospermia, indicate normal spermatogenesis, including normal quantity, morphology, and physiology of the spermatozoa. Male flies with these normal QA/QC values are the most effective in the field. Such male flies will generally transfer adequate volumes of sperm and seminal fluids into the female recipients. This will avoid female re-mating and result in effectiveness of the Sterile Insect Techniques by achieving suppression of the wild populations in the field.

[0037] Assessments conducted during a spermiogram can include sperm motility; sperm morphology; and concentration of volume, seminal fluid:spermatozoa ratio.

Assessment of Sperm Motility

[0038] Category A: Normal or Typical motility is indicated by active movement and linear/circular displacement of the fruit fly spermatozoa.
Category B: Abnormal or Atypical motility is indicated by the non-progressive movement from the flagellum to the linear head of the fruit fly spermatozoa.
Category C: Immotility is indicated by the absence of spermatozoa movement.

Assessment of Morphology

[0039] Category 1. Normal or Typical linear head and normal flagellum demonstrated by the fruit fly spermatozoa.
Category 2. Abnormal or Atypical head (curved, loop) or flagellum demonstrated by the fruit fly spermatozoa.

Concentration of Volume, Seminal Fluid/Spermatozoa Ratio

[0040] This parameter measures the fruit fly's testicular capacity to produce sperm. The total sperm count in the ejaculate is multiplied by the seminal fluid volume, if we need to be exact. For practicality and daily practice, the volume of seminal fluid should not exceed the total number of spermatozoa in the cover slipped specimen. This visual assessment is based on training and daily practice.

[0041] If the sterile male's spermatozoa are of perceptively lower quality, or abnormal, the fertile females are likely to re-mate with another maleoften a wild mate rather than another sterile male. Re-mating with a wild mate displaces the inferior sperm and replaces it with good quality fertile sperm from wild mates. This, of course, tends to defeat the purpose of releasing sterile males in a particular area, and to result in the establishment and growth of wild populations and larvae-infested areas.

[0042] Abnormal results of a spermiogram test can indicate one or more of at least the following possibilities: [0043] Oligospermia: abnormally low sperm cell count. For male flies prepared to be infertile, this could result from issues such as abnormal or inadequate irradiation processes as one of the most common causes, but this finding just opens the door to many other possible causal factors. [0044] Atypical sperm morphology: For male flies prepared to be infertile, this could result from issues such as improper irradiation, genotypic mutation, or genetic recombinants in the mass colony. [0045] Atypical decreased sperm motility: The most probable cause in male flies prepared to be infertile would be improper irradiation, but it could also indicate genetic recombinants or substandard quality due to entomopathogens. [0046] Decreased seminal fluid: This is most likely due to entomopathogens.

The Examples

Materials and Methods

[0047] We used the following materials and methods in practicing our new method.

[0048] Equipment

Basic Microscopy System

[0049] a) Dissecting stereo-microscope, magnification required from 0.8 to 50.
b) Compound microscope, magnification from 40 to 1000 (using immersion oil).
c) Digital camera for capturing the images.

Micro-Dissection Tools

[0050] a) Micro-dissection scissors, Vannas style, cutting edge 2 mm.
b) Micro-dissection forceps, straight tip, 0.0250.005 mm.
c) Micro-dissection forceps, 45-degree angle, serrated tip.

Microscope Slides

[0051] a) Adhesion super frost slides, 25750.1 mm.
b) Cover glass 1818 mm.

Immersion Oil

[0052] a) Low viscosity immersion oil for 1000 microscopy.

Methodology

Example 1Determination of Spermatozoa Viability in Sterile Male Flies

[0053] 1. Place the fly in a petri dish with sterile saline solution or Ringer's Solution. Visualize under the dissecting microscope. FIG. 1.
2. Grasp the fly by the thorax using the angled forceps. Use the Vannas scissors to cut open the abdomen, making an incision along the dorsal midline through the wall of the abdomen from the apex to the base. FIG. 2. This exposes the reproductive organs of the fly.
3. Dissect the testes by using the straight-tip forceps to grasp the testes at a point near the vas deferens, and gently pulling the testes away from the abdomen. Place each teste on the microscope slide, apply a drop of Ringer's solution or sterile water, and gently cover with a slide cover-slip. See FIG. 3.
4. Examine the specimen on the slide using the compound microscope objective at 20 magnification. To evaluate the viability and movement of spermatozoa, view the specimen under successively increasing magnification up to 1000 (oil immersion lens) and obtain in vivo video if possible. FIG. 4 shows free spermatozoa extruding from the vas deferens. The specimen may be viewed under successively increasing magnification, up to 1000 (using an oil immersion lens). Any specific magnification within the range of 40 to 1000 is contemplated as within the scope of the present subject matter, including but not limited to 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, and 900. Further, any contemplated range of magnifications can use any of the above as endpoints of the range. Thus, the specimen can be viewed at one magnification level, or at multiple magnification levels. FIG. 10 shows a magnified, ruptured ventral receptacle with spermatozoa having spilled from the ruptured alveoli. A live view (rather than a still photograph) may also enable evaluation of the degree of activity of any spermatozoa that are present.

[0054] It is to be understood that the new method described here is not limited to the specific embodiments described above, but instead encompasses any and all embodiments within the scope of the generic language of the following claims enabled by the embodiments described herein, or otherwise shown in the drawings or described above in terms sufficient to enable one of ordinary skill in the art to make and use the claimed subject matter.