A scientific note on amoeboid movement of honey bee semen

Abstract

honeybee /Apismellifera /spermatozoa /amoeboidmovement /spermcooperationHoney bee (Apis mellifera) semen is a tan, viscousliquid containing tightly packed and slowly movingspermatozoa (Stort & Goncalves 1986). Immediatelyafter transfer of spermatozoa to the queen’s sperma-theca, they move faster, but after 1 h, the movementstops (Kerr et al. 1962) and later during storage thespermatozoa remain immotile (Lensky & Schindler1967). The immotility is reversible as long as thespermatozoa are alive (Verma 1978). In nature, honeybee semen is diluted with spermathecal fluid but thespermatozoa survive also in many human madediluents (Moritz 1984; Taylor et al. 2009).Here, I report that diluted honey bee semenbehaves in an unusual way; it produces pseudopodiaand is able to move. The movement differs from theamoeboid movement of a single spermatozoon(Nelson & Ward 1981) and involves the coordinatedbehaviour of a group of spermatozoa.Honey bee drones were collected at hive entrancesand were stored in a cage attended by workers. Toinitiate ejaculation, the abdomen of a drone waspressed with fingers, a standard method (Laidlaw &Page 1997). The semen was collected with a syringedesigned for instrumental insemination of honey beequeens. The semen was evacuated from the syringeonto a microscopic slide. From there, it was collectedpassively into a MEDLAB glass capillary (insidediameter 0.7 mm, outside diameter 1.3 mm andlength 50 mm). The semen inside the capillary wasdiluted 1:1 (v/v) with spermathecal fluid obtained froma young uninseminated honey bee queen. Its sperma-theca wasdissectedfrom the queen immediately beforethe experiment and crushed on a microscope slide. Thespermathecalfluidwascollectedusingtheinseminationsyringe, and a droplet of it was evacuated onto anothercleanmicroscopicslide.Whenthecapillarywithsemenwas brought into contact with the droplet, it was drawninto the capillary and diffused through the semen. Thecapillary was positioned horizontally, and it wasphotographed every hour with a Nikon D70 digitalcameratodeterminethespeedofsemenmovement.Theexperiment was repeated 10 times using the semen ofdifferent drones.The behaviour of diluted semen on a microscopicslide was observed with a Nikon Eclipse E600microscope. Semen movement was recorded with aJVC colour video camera (resolution 720×576) at-tached to the microscope. In some experiments, thedroplet of diluted semen was surrounded by six smalldroplets of spermathecal fluid. There was a distance ofabout 0.2 mm between the droplet of semen and thedroplets of spermathecal fluid. If a pseudopodiumformed near a droplet of diluent, it was video recorded.In all experiments, the capillaries or microscopeslides with semen were kept in a transparent plasticcontainer with a tissue wetted with distilled water. Thewet tissue provided high humidity and prevented thesemen from drying. In some experiments, the semenwasdilutedinKievbufferinsteadofspermathecalfluid.The Kiev buffer consisted of 0.3 g D+ glucose, 0.41 g