Mikihiko Kumashiro

Auto-spermatophore extrusion in male crickets

SUMMARY The reproductive cycle of the male cricket consists of the mating stage and the sexually refractory stage. The latter is further divided into the first refractory stage (RS1) from spermatophore extrusion in copulation to spermatophore preparation after copulation, and the second refractory stage (RS2) from spermatophore preparation to recommencement of a calling song. RS2 is time-fixed and unaffected by the female or by stress, hence RS2 is assumed to be controlled by the reproductive timer. Previously, we suggested that the timer is located in the terminal abdominal ganglion (TAG), because functional inactivation of the TAG by local cooling lengthened RS2 in proportion to cooling time. To obtain further evidence of timer localization and to examine the operation of the timer in dissected animals, we investigated the characteristics of auto-spermatophore extrusion, a phenomenon in which males eject the mature spermatophore themselves without any prior courtship. The occurrence of auto-spermatophore extrusion was 100% in dissected males with the TAG separated, compared to 1.7% in intact males. The time interval (SPaSE) between spermatophore preparation and auto-spermatophore extrusion was comparable to RS2 measured by the calling song. Spike recording from a genital motor neurone in the separated TAG indicated that burst discharge associated with auto-spermatophore extrusion occurred with a SPaSE comparable to RS2. Other efferent neurones, some of which were identified as dorsal unpaired median (DUM) neurones, showed a time-dependent spike frequency increase during SPaSE. These results strengthen our previous conclusion that the reproductive timer is located within the TAG, and demonstrate that the timer functions normally even when the TAG is separated from the central nervous system.

Copulation in the cricket is performed by chain reaction.

Abstract The male and female genitalia are finely designed to match each other for copulation in the cricket Gryllus bimaculatus. Copulatory acts of the male, stereotyped and time-fixed, are elicited by stimulation of mechanoreceptors on particular regions of the abdomen, cerci and genitalia. Sequential execution of each motor act proceeds as a chain reaction in which one act stimulates some receptors which in turn elicits another act and so on, while the female remains immobile on the males back. Each key stimulus for a motor act appears as a result of the males own act, except for copulatory papilla protrusion by the female. The final sequence of spermatophore extrusion and transfer are irreversible fixed motor actions which are triggered when the female copulatory papilla stimulates the epiphallic hairs. They proceed without continual central drive from the brain, and apparently without sensory feedback. In addition, they are well coordinated with movement and posture in the entire body. Some neural mechanisms of controlling mating behavior and switching the reproductive cycle are discussed.

Genital Autocleaning in the Male Cricket Gryllus bimaculatus (1): Structure and Function of the Genital Membrane

We found that the genitalia of the male cricket Gryllus bimaculatus are equipped with an autocleaning system. The cricket keeps its genitalia clean by removing foreign matter and endogenous waste. Morphological study showed that the membrane complex consists of a median pouch and a genital chamber floor covered by small scales, each of which has a base of approximately 10 µm in width and a fringe with 5–10 spines 3–20 µm in length. The scales are arranged symmetrically about the midline, curving gradually in the lateral direction and continuing to the lateral pouch serving as a trash container. Observation of cleaning revealed that a small piece of artificial dirt placed on the membrane complex was conveyed over a distance of 1.3 mm to the lateral pouch in 12 minutes. Inspection of the dorsal pouch just after spermatophore extrusion in the mating stage revealed that there were patchy remnants of spermatophore material on the inner surface of the pouch, but that these were evacuated in a few minutes. Surgical elimination of the median pouch caused the formation of abnormal spermatophores with the sperm tube and attachment plate being deformed. These results suggest that genital autocleaning is indispensable for the production of a normal spermatophore in the male cricket.

Genital Autocleaning in the Male Cricket Gryllus bimaculatus (2): Rhythmic Movements of the Genitalia and Their Motor Control.

Three types of genital movement, their neural controls, and functional roles were investigated to gain a better understanding of the mechanism underlying autocleaning in the male cricket. The membrane complex consisting of the median pouch and genital chamber floor shows peculiar undulation that is composed of two types of movements: a right-left large shift and small crease-like movements. The large shift was caused by contraction of a pair of muscles (MPA) located anterior to the median pouch, while the crease-like movements were caused by numerous muscle fibers extending over the membrane complex. The MPA and muscle fibers were each innervated by efferent neurons in the terminal abdominal ganglion. Experiments with artificial dirt mimicking a foreign object revealed that the crease-like movements were responsible for dirt transport, while the large shift participated in sweeping the dirt into the lateral pouch as a trash container. On the other hand, the dorsal pouch serving as a template for the spermatophore showed a jerky bending movement. Simultaneous monitoring of the membrane complex and dorsal pouch activities suggested that their movements cooperate to enable the efficient evacuation of waste in the dorsal pouch. Based on the results, we conclude that genital autocleaning supports the production of the spermatophore.

Reproductive Behavior and Physiology in the Cricket Gryllus bimaculatus

Gryllus bimaculatus males have a reproductive cycle consisting of a mating stage and a sexually refractory stage. During the mating stage, the male exhibits distinct behavior that encompasses three main stages: calling, courtship, and copulation. The last stage, copulation, is carried out in a fixed manner by the stimulus-response chain. The final copulatory act, spermatophore extrusion, is caused by stimulation of mechano-sensilla in the epiphallus during genitalia coupling, which terminates the mating stage. The sexually refractory stage starts with spermatophore extrusion, during which the male is rather aggressive and does not exhibit any mating behavior. A male first shows spermatophore preparation when stimulated by the female, then forms the new spermatophore, and finally recommences the calling song, i.e., the start of the mating stage. Physiological investigations reveal that the male mating behavior is mainly controlled by the brain and the terminal abdominal ganglion (TAG), which exerts three types of inhibition on the pattern generators for mating behavior. The brain also facilitates sexual excitation via octopamine. One of the conspicuous features of the reproductive behavior in Gryllus bimaculatus is that the sexually refractory stage between spermatophore preparation and the start of calling song is time fixed at around 1 h. Experiments utilizing the targeted cooling of the central nervous system indicate a presence of a time-measuring mechanism (“timer”) that is located within the TAG. Long-term spike recordings of neurons also support the presence of such a timer within the TAG. Finally, the occurrence of mating-like actions in larval nymphs is discussed. All of these findings have now generated a large body of work that will help establish Gryllus as a new experimental system for studying reproductive behavior and physiology in crickets and other insect species.