Effects of light and temperature on the circadian system controlling sperm release in moth Spodoptera littoralis

Reproductive physiology of male moths is regulated by a peripheral circadian system, which controls the timing of sperm release from the testis into the upper vas deferens (UVD) and timing of sperm transfer from the UVD to the seminal vesicles. We investigated various effects of light and temperature on sperm release and transfer rhythms in the moth Spodoptera littoralis. We report that both rhythms persist for up to 1 week in constant darkness without significant dampening and are also temperature compensated in the range from 20°C to 30°C. However, the duration of sperm retention in the UVD is temperature-dependent; consequently, temperature exerts a masking effect on the rhythm of sperm transfer. Experimental manipulations of light and temperature regime demonstrated that light dominates over temperature in entraining the timing of sperm release and transfer. Nevertheless, temperature plays a critical role in the absence of light Zeitgeber. Sperm release and transfer are arrhythmic in constant light (LL); however, both rhythms are restored by temperature cycles.     

Temporal and spatial expression of the period gene in the reproductive system of the codling moth

The authors examined patterns of spatial and temporal expression of Drosophila per gene homologue in the codling moth, Cydia pomonella. Since sperm release in moths is regulated in a circadian manner by an autonomous clock that is independent from the brain, the authors investigated per expression in male reproductive system along with its expression in moth heads. per mRNA is rhythmically expressed with the same phase and amplitude in both tissues under light-dark (LD) conditions. The levels of per mRNA are low during the day, start to increase before lights-off, reach the peak in dark, and decrease after lights-on. In constant darkness (DD), cycling of per mRNA continued in heads with severely blunted amplitude. No cycling of per mRNA was detected in testis in DD. In situ hybridization and immunocytochemistry revealed distinct spatial patterns of per expression in the moth reproductive system. There is no expression of per in cells forming the wall of testes or in sperm bundles. However, per mRNA and protein are rhythmically expressed in the epithelial cells forming the wall of the upper vas deferens (UVD) and in the cells of the terminal epithelium, which are involved in the circadian gating of sperm release. Increase in per mRNA in the UVD coincides with sperm accumulation in this part of the insect reproductive system.     

Daily rhythm in myogenic contractions of vas deferens associated with sperm release cycle in a moth

In the gypsy moth, Lymantria dispar, release of sperm bundles from the testis into the upper vas deferens (UVD) and subsequent transfer of sperm bundles into the seminal vesicles (SV) occurs in a daily rhythm. The UVD undergoes different types of contractions despite the fact that its musculature appears to receive no innervation. Patterns of the UVD movements were recorded throughout the daily sperm release and transfer cycle. In males kept in light-dark cycles, transfer of sperm from the UVD to the SV was accompanied by a characteristic pattern of UVD contractions of high frequency and amplitude. In males kept in constant light, which fail to transfer sperm, this contraction pattern was absent. It is concluded that the vas deferens muscles undergo daily changes in contraction pattern in phase with the light-dark cycle. The increased muscular contractions appear to be a causal factor in the gated sperm transfer from the UVD to the SV.Abbreviations LD light-dark - LL constant light - SV seminal vesicle - UVD upper vas deferens     

Ontogeny of the circadian system controlling release of sperm from the insect testis.

In the gypsy moth, the release of sperm bundles from the testis into the vas deferens is rhythmic and is controlled by a circadian pacemaker located in the reproductive system. However, in males kept since pupation in constant darkness (DD) and temperature, the release of sperm was arrhythmic. The release of sperm became rhythmic when males were transferred from a light-dark cycle (LD 16:8) to DD 6-7 days after pupation. To further investigate the development of the circadian system during the pupal stage, we exposed DD pupae to a single 8-hr pulse of light or 8-hr pulse of a 4 degrees C temperature increase on different days after pupation. The pattern of sperm release was determined 5-6 days after the pulse. Males that were exposed to light or temperature pulses 5 days after pupation subsequently showed nonrhythmic sperm release. However, about half of the pupae that received the pulse on day 6 and most of the pupae that received it on day 7 subsequently showed synchronized sperm release. These results suggested that the clock underlying rhythmic release of sperm becomes operational at approximately 6 days after pupation--that is, 2 days prior to initiation of rhythmic sperm release from the testis.     

Circadian rhythm of sperm release in the gypsy moth,Lymantria dispar: ultrastructural study of transepithelial penetration of sperm bundles

Release of mature bundles of spermatozoa from the testis into the vas deferens is a critical but poorly understood step in male insect reproduction. In moths, the release of sperm bundles is controlled by a circadian clock which imposes a temporal gate on the daily exit of bundles through the terminal epithelium-a layer of specialized epithelial cells separating testis follicles from the vas deferens. The sequence of cellular events associated with the daily cycle of sperm release was investigated by scanning and transmission electron microscopy. In the hours preceding sperm release, there is a solid barrier between the testis and the vas deferens formed by the interdigitation of cytoplasmic processes of adjacent terminal epithelial cells. At the beginning of the sperm release cycle, sperm bundles protrude through this barrier while the terminal epithelial cells change their shape and position relative to the bundles. Subsequently, the cyst cells enveloping the sperm bundles break down and spermatozoa move out of the testis through the exit channels formed between the epithelial cells. Afterwards, cyst cell remnants and other cellular debris are released into the vas deferens lumen, and the epithelial barrier is reconstructed due to phagocytic activity of its cells. These data provide a foundation on which to build an understanding of the cellular mechanisms of clock-controlled sperm release in insects.     

Circadian rhythm of glycoprotein secretion in the vas deferens of the moth, Spodoptera littoralis

Background  

Reproductive systems of male moths contain circadian clocks, which time the release of sperm bundles from the testis to the upper vas deferens (UVD) and their subsequent transfer from the UVD to the seminal vesicles. Sperm bundles are released from the testis in the evening and are retained in the vas deferens lumen overnight before being transferred to the seminal vesicles. The biological significance of periodic sperm retention in the UVD lumen is not understood. In this study we asked whether there are circadian rhythms in the UVD that are correlated with sperm retention.     

Changes in protein patterns in sperm and vas deferens during the daily rhythm of sperm release in the gypsy moth

In the gypsy moth, Lymantria dispar, the release of sperm bundles from the testis into the upper vas deferens (UVD) is precisely timed within each 24 h period by a circadian mechanism located in the reproductive system. In males kept under light:dark cycles of 16:8, release of sperm bundles is limited to the 3 h period that starts before lights off. Sperm released from the testis remains in the UVD for about 12 h and then moves into the seminal vesicles, so that the UVD stays empty until the next cycle of sperm release begins. The rhythm of release appears to play a role in the terminal stages of sperm maturation and is essential for the fertility of males. Sperm bundles undergo substantial morphological changes during the release from the testis and while they are retained in the UVD. In this study, using gel electrophoresis, we compared protein patterns in sperm and in the UVD during the daily cycle of sperm release and maturation. Several protein bands evident in the sperm bundles contained in the testis were missing from the sperm bundles that had passed from the testis into the UVD. Furthermore, a number of new proteins appeared in the sperm bundles as they remained in the UVD. Some of these proteins appeared to be secreted from the UVD epithelium into the UVD lumen before being incorporated into sperm bundles. Correlations between changes in protein patterns and ultrastructural changes in sperm during the cycle of sperm release and maturation are discussed. © 1994 Wiley-Liss, Inc.     

Compatibility of codling moths Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) from South Africa with codling moths shipped from Canada

The sterile insect technique (SIT) has been successfully applied against codling moth Cydia pomonella (Linnaeus) (Lepidoptera; Tortricidae) in British Columbia since 1992 where the mass‐rearing facility produces between 15 and 16 million moths per week. Due to the seasonality of this pest, the facility is only fully utilized for part of the year. The time and expense of implementing SIT against codling moth in South Africa may be substantially reduced if moths from Canada were sexually compatible with those from South Africa. In addition, because the pome fruit‐growing season in both countries is opposite, the programme in Canada might benefit by maintaining moth production year‐round and selling moths to South Africa. Semi‐field studies in small cages and release‐recapture studies were conducted in an unsprayed apple orchard in South Africa to assess mating compatibility of laboratory‐reared codling moth from Canada and wild codling moths from South Africa. The results suggest that Canadian codling moth males were equally attracted to calling Canadian and South African females despite the fact that Canadian moths had been transported (from Canada to South Africa) for 48 h as both pupae and adults. The data also suggest that at lower field temperatures Canadian moths were more active than South African moths. Results from the release‐recapture field trials indicated that Canadian and South African males were equally attracted to Canadian and South African females. These results suggest that codling moths from Canada and South Africa are fully compatible and indicate that Canadian moths can be used for SIT studies in South Africa. As these studies were conducted with moths from two very different climatic and time zones, it is proposed that populations of codling moth in other pome fruit production areas may also be compatible with the Canadian moths.     

Diurnal rhythm in expression and release of yolk protein in the testis of Spodoptera littoralis (Lepidoptera: Noctuidae)

Circadian clocks (oscillators) regulate multiple life functions in insects. The circadian system located in the male reproductive tract of Lepidoptera is one of the best characterized peripheral oscillators in insects. Our previous research on the cotton leafworm, Spodoptera littoralis, demonstrated that this oscillator controls the rhythm of sperm release from the testis and coordinates sperm maturation in the upper vas deferens (UVD). We demonstrated previously that a protein that functions as yolk protein in females is also produced in cyst cells surrounding sperm bundles in the testis, and is released into the UVD. Here, we investigated the temporal expression of the yolk protein 2 (yp2) gene at the mRNA and protein level in the testis of S. littoralis, and inquired whether their expression is regulated by PER-based molecular oscillator. We describe a circadian rhythm of YP2 accumulation in the UVD seminal fluid, where this protein interacts with sperm in a circadian fashion. However, we also demonstrate that yp2 mRNA and YP2 protein levels within cyst cells show only a diurnal rhythm in light/dark (LD) cycles. These rhythms do not persist in constant darkness (DD), suggesting that they are non-circadian. Interestingly, the per gene mRNA and protein levels in cyst cells are rhythmic in LD but not in DD. Nevertheless, per appears to be involved in the diurnal timing of YP2 protein accumulation in cyst cells.     

Adrenergic response patterns in vas deferens isolated from rats under diurnal rhythms. Influence of stress

The aim of this study was to verify, by means of functional methods, whether the circadian rhythm changes adrenergic response patterns in the epididymal half of the vas deferens isolated from control rats as well as from rats submitted to acute stress. The experiments were performed at 9:00 a.m., 3:00 p.m., 9:00 p.m., and 3:00 a.m. The results showed a light-dark dependent variation of the adrenergic response pattern on organs isolated from control as well as from stressed rats. In the control group, only the phenylephrine sensitivity was changed throughout the circadian rhythm. Under the stress condition, both norepinephrine and phenylephrine response patterns were changed, mainly during darkness. The maximal contractile response to both alpha- and beta-agonist and alpha1-agonist was increased in the dark phase, corresponding to high plasmatic concentrations of endogenous melatonin. The vas deferens isolated from stressed rats during the light phase simultaneously incubated with exogenous melatonin showed the same pattern of response obtained in the dark phase, thus indicating a peripheric action of melatonin on this organ. Therefore, the circadian rhythms are important to the adrenergic response pattern in rat vas deferens from both control and stressed rats. In conclusion, we suggest a melatonin modulation on alpha1-postsynaptic adrenergic response in the rat vas deferens.     

Quality of mass-reared codling moth (Lepidoptera: Tortricidae) after long-distance transportation: 1. Logistics of shipping procedures and quality parameters as measured in the laboratory

The sterile insect technique (SIT) is a proven effective control tactic against lepidopteran pests when applied in an areawide integrated pest management program. The construction of insect mass-rearing facilities requires considerable investment and moth control strategies that include the use of sterile insects could be made more cost-effective through the importation of sterile moths produced in other production centers. For codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), this is an attractive option because mating studies have confirmed the absence of mating barriers between codling moth populations from geographically different areas. To assess the feasibility of long-distance transportation of codling moths, pupae and adult moths were transported in 2004 from Canada to South Africa in four shipments by using normal commercial transport routes. The total transport time remained below 67 h in three of the consignments, but it was 89 h in the fourth consignment. Temperature in the shipping boxes was fairly constant and remained between -0.61 and 0.16 degrees C for 76.8-85.7% of the time. The data presented indicate that transporting codling moths as adults and pupae from Canada to South Africa had little effect on moth emergence, longevity, and ability to mate, as assessed in the laboratory. These results provide support to the suggestion that the STT for codling moth in pome fruit production areas might be evaluated and implemented by the importation of irradiated moths from rearing facilities in a different country or hemisphere.     

Effect of rearing strategy and gamma radiation on fecundity and fertility of codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae)

The codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) is a serious pest of pome fruit worldwide. In an effort to reduce the use of pesticides to control this pest, the sterile insect technique (SIT) is used or considered for use as a component of area‐wide integrated pest management programmes. Rearing codling moths through diapause has been shown to improve the competitiveness of sterile moths released in orchards, and provides management alternatives that would allow mass‐rearing facilities to increase their yearly production of sterile moths. Because radiosensitivity in insects can be influenced by numerous biological factors, laboratory tests were conducted to examine whether the response to increasing doses of radiation, as expressed in the fecundity and fertility of cohorts of moths, is similar for adult moths mass‐reared through diapause or through standard (non‐diapause) production protocols. Our data revealed that the effect of increasing doses of radiation on fecundity and fertility of codling moths reared through both rearing strategies was similar. In the case of fertility, this is a particularly important finding for the expanded application of codling moth SIT. If mass‐rearing facilities use year‐round diapause rearing, the dose required to treat the insects prior to release will be similar to that used when codling moths are reared through standard production protocols.     

Ultrastructural evidence for secretion from the epithelium of ampulla ductus deferentis of the fan-throated lizard Sitana ponticeriana Cuvier

Among reptiles, an ampulla ductus deferentis has been reported only in Squamata. Fairly detailed studies are available only for two species, the lizard Calotes versicolor (Fam: Agamidae) and the snake Seminatrix pygaea (Fam: Colubridae). The light microscopic study on C. versicolor revealed the ampulla to be a prominent organ, whereas the light and transmission electron microscopic study in S. pygaea revealed it to be discernable only in histological preparations. Further, the epithelium of the ductal portion of vas deferens as well as the ampulla of C. versicolor appears to contribute to the seminal plasma and can also phagocytose dead sperm, whereas in S. pygaea neither of these roles has been established. Thus, we hypothesize that there may be variations in the anatomy, histology, and the role of the vas deferens in general, and the ampulla in particular, of the squamate reptiles. In this study, the ductus deferens of the small fan-throated lizard Sitana ponticeriana (Fam: Agamidae) was subjected to light and transmission electron microscopic analysis. In this lizard the ampulla is more prominent than in C. versicolor. The epithelium of the ductal portion of vas deferens consists of principal cells (with features reflecting roles in endocytosis and phagocytosis of dead sperm), dark cells (which are absent in the epithelium of the ductal portion of vas deferens of snakes), and basal cells. The ampulla of S. ponticeriana is differentiated into storage and glandular portions. The epithelium of the storage portion is like that in the ductal portion of the vas deferens, whereas that of the glandular portion, consisting of dark and light principal cells and foamy cells, is tall and forms into smooth villous folds. All three cell types show evidence for a role in secretion, in all likelihood different from each other, for release into the lumen to contribute to seminal plasma. These cells do not provide evidence of a role in phagocytosis of dead sperm. It appears that within the Squamata, the ductal ampulla differs in structure as well as function. We suggest that the ductal ampulla of agamid lizards is a composite gland of the ampulla ductus deferentis and seminal vesicles of mammals.     

Abdominally entrained periodicities of testis and vas deferens activity in the Mediterranean flour moth

In a light-dark (LD) regimen, sperm, first apyrene and then eupyrene, start moving out of the fused testes of the Mediterranean flour moth, Anagasta kuehniella, toward the beginning of the scotophase. At 27° ± 2°C, the sperm mass remains in the proximal part of the vasa deferentia for 10 to 12 hr and then passes rapidly into the seminal vesicles, remains in these organs for about 5 hr, and is then transported to the ductus ejaculatoris duplex where it becomes available for ejaculation. The phases of sperm movement appear to be closely related to sperm development, and the reproductive activity of the moths. In isolated abdomens there is a significant reduction in the amount of sperm released from the testes, but normal periodicity of sperm release and movement continues in either LD or continuous dark (DD) regimens, and rapid phase shifting occurs when a LD regimen is reversed. All stages of sperm movement are disrupted in continuous light (LL), but normal periodicity is usually resumed when isolated abdomens of the LL moths are placed in LD or DD regimens. Normal periodicity also occurs in moths paralyzed with tetrodotoxin or procaine. Removal of any one of the four abdominal ganglia from LL moths does not prevent increased sperm release when the moths are placed in LD, though with each ganglion there is some disruption of the normal pattern of movement down the vasa deferentia. It is thought that the testes and vasa deferentia down to at least the seminal vesicles represent a semiautonomous complex in which periodicity is maintained by endogenous circadian activity in cells of the testes (and possibly the vasa deferentia) or more probably in a peripheral control center.     

Control of Cydia pomonella L. and Cydia molesta (Busck) (Lepidoptera Tortricidae) in pome-fruit orchards with Ecodian sex pheromone dispensers

A mating disruption approach using high densities of pheromone dispensers, has been recently proposed for codling moth, Cydia pomonella (L.), and oriental fruit moth, Cydia molesta (Busck.), (Lepidoptera Tortricidae), control. Ecodian Star dispensers, made of low-cost biodegradable material and easy to apply, were formulated with 10 mg of codlemone (E8,E10-12OH) and 10 mg of grapamone (Z8-12OH) and placed at a rate of 1,400-2,000 dispensers/ha. The pheromone release rates from new and field aged dispensers were evaluated by hexane extraction of the residual attractant (indirectly) and gas-chromatographic analysis. The release rate of field-aged dispensers decreased over time with a good linearity; they released a significant amount of synthetic sex pheromones over the entire season. Dispensers elicited close-range approaches of codling moth males in wind tunnel irrespective of their age. Field trials carried out from 2003 to 2004 confirmed the efficacy of Ecodian Star dispensers for codling moth and oriental fruit moth control, regardless the size of the treated area. Our results demonstrate that Ecodian dispensers achieved a good level of activity and longevity over the season. The potential of this strategy for the control of the moths is discussed.     

Effect of radiation on fecundity and fertility of codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) from South Africa

Codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) is the key pest of pome fruit in South Africa, and it’s control in apple and pear orchards relies on the application of insecticides and pheromone‐mediated mating disruption. Development of resistance to insecticides and placement of restrictions on the use of certain insecticides has made control of codling moth in South Africa increasingly problematic. The use of the sterile insect technique (SIT) as a control tactic for codling moth is under investigation as a potential addition to the current control strategy. We investigated the radiosensitivity of a laboratory strain of codling moth that was established from moths collected from commercial and organic orchards in the Western Cape, South Africa. Fecundity and fertility of this strain following radiation were consistent with values for the codling moth strain in the Canadian rearing facility in British Columbia. For both strains, the female codling moth was considerably more radiosensitive than the male. At a radiation dose of 100 Gy or higher, treated females were 100% sterile. The fertility of the South African strain was higher (86.3%) than for the Canadian strain (71.9%). This difference in fertility between the two strains was maintained when the dose of radiation was 100 Gy. However, the level of fertility was very similar between the two strains for doses ≥150 Gy. Therefore, based upon previously published work and the data from this study, an operational dose of 150 Gy is recommended for future codling moth SIT programmes in South Africa.     

A flight cylinder bioassay as a simple,effective quality control test for Cydia pomonella

Assessment of quality of the sterile male insects that are being mass‐reared for release in area‐wide integrated pest management programmes that include a sterile insect technique component is crucial for the success of these programmes. Routine monitoring of sterile male quality needs to be carried out both in the mass‐rearing facility and in the field. Simple bioassays that can be conducted in the laboratory and that would be surrogates for laborious field tests would be a very cost‐effective way of monitoring sterile male field performance. Simple flight cylinders were used to assess whether these could detect differences in quality of male codling moth Cydia pomonella. The number of male and female codling moths that flew out of the cylinders was influenced by cylinder diameter, cylinder height and number of hours following the initiation of the test. The flight cylinder bioassay was capable of detecting differences in quality of codling moths induced by irradiation when moths were shipped, but no differences were found in flight ability when the moths were not transported. The tests also confirmed that handling and shipment reduced quality more for irradiated than for non‐treated codling moth, and that insect quality was significantly influenced by larval rearing protocols. The flight cylinder bioassay was therefore successful in detecting differences in codling moth quality induced by various treatments that had been identified previously by more complex laboratory bioassays and field trials. Treatment differences were most likely detected when flight cylinders were 16 cm high.     

Overwintering of codling moth (Lepidoptera: Tortricidae) larvae in apple harvest bins and subsequent moth emergence.

Codling moths, Cydia pomonella (L.), have long been suspected of emerging from stacks of harvest bins in the spring and causing damage to nearby apple and pear orchards. With increased use of mating disruption for codling moth control, outside sources of infestation have become more of a concern for growers using pheromone based mating disruption systems. Studies were designed to provide information on bins as a source of codling moth and the pattern of codling moth emergence from stacks of bins. In these studies, codling moth larvae colonized wood harvest bins at a much higher frequency than harvest bins made of injection molded plastic (189 moths emerged from wood compared with five from plastic). There was no statistical difference in the number of moths infesting bins that had been filled with infested fruit compared with bins left empty at harvest. This suggests that codling moth enter the bins during the time that the bins are in the orchard before harvest. Emergence of laboratory reared adult codling moth from wood bins placed in stacks was found to be prolonged compared with field populations. Temperature differences within the bin stacks accounted for this attenuated emergence pattern. Covering bin stacks with clear plastic accelerated codling moth development in the upper levels of the stack. Codling moth emergence patterns from plastic-covered stacks more closely coincided with male flight in field populations. This information could be important in developing a technique for neutralizing codling moth-infested bins, and in understanding how infested bins may influence pest management in fruit orchards that are located near bin piles. Implications for control of codling moth in conventional orchards and in those using mating disruption as the principal component of an integrated pest management system include increased numbers of treatments directed at areas affected by infested bins.     

Circadian mating activity and effect of pheromone pre-exposure on pheromone response rhythms in the moth Spodoptera littoralis

Mating in moths is generally mediated by female-produced sex pheromones. Mating activity, female pheromone production/release and male pheromone responsiveness all show diurnal variations in many species. We found that the response of the male Egyptian cotton leafworm, Spodoptera littoralis, to sex pheromone gland extracts showed a diel rhythm in olfactometer tests, and the variation was persistent for at least 1 day in constant darkness. High male response to sex pheromone was correlated in time with high mating and locomotor activity. Male S. littoralis, maintained in constant darkness and exposed to pheromone gland extracts on a daily basis, showed an induced temporal variation in response after several days, in contrast to unexposed males. This suggests that in the absence of other external zeitgebers, exposure to sex pheromone may function to synchronise circadian behavioural rhythms in male moths. The daily rhythm in mating activity in S. littoralis is also shown to be persistent for at least 2 days in constant darkness. Pairs mated significantly less when either the male or female had been raised in a light:dark cycle 10 h out of phase, indicating that the proposed circadian rhythm in mating activity is composed of rhythmic mating preference/ability in both sexes.     

Movement of spermatozoa in the reproductive tract of adult male Spodoptera litura: daily rhythm of sperm descent and the effect of light regime on male reproduction

Sperm production and movement from the fused testes into the male reproductive tract of the common cutworm Spodoptera litura were studied in insects maintained in a 12 h:12 h light dark (LD) regime. Two types of sperm bundles, eupyrene (nucleated) and apyrene (anucleate) were present in the adult testes. Eupyrene bundles constituted about 25% of the total. Descent of spermatozoa from the testes into the upper vas deferens (UVD) first occurred about 24-30 h before adult eclosion. On entering the reproductive tract, eupyrene spermatozoa remained in bundles while apyrene bundles became dissociated before they reached the UVD. Downward movement of both eupyrene and apyrene spermatozoa within the male tract occurred in a daily rhythm. Sperm descent from the testes into the UVD occurred during the early scotophase, followed by their further descent into the seminal vesicle (SV) during the photophase. Spermatozoa remained in the SV for only a short duration, whence sperm quickly passed through the lower vas deferens into the duplex, which acted as the main sperm storage organ until mating was initiated. During mating 80% of sperm left the duplex, but mating did not influence the number of sperm bundles that subsequently descended into the duplex or the rate of their descent. There was no evidence of sperm reflux. Rearing in constant light (LL) and in constant dark (DD) reduced the number of eupyrene sperm present in the testes of adults that emerged in LL and DD compared to controls (LD), although there was no significant effect on the number of apyrene sperm in the testes. The rhythmic pattern of sperm descent was suppressed in both LL and DD regimes, and the number of sperm in the duplex was adversely affected, with a marked impact in LL reared insects. Male longevity, mating behaviour, oviposition and fertility were found to be more severely affected in LL than in DD.