Juvenile hormone identification in the cabbage bug Eurydema rugosa.

Juvenile hormone (JH) plays a pivotal role in almost every aspect of insect development and reproduction. The chemical structure of the JH in heteropteran species has long remained elusive until methyl (2R,3S,10R)-2,3;10,11-bisepoxyfarnesoate, commonly named as juvenile hormone III skipped bisepoxide (JHSB3), was isolated from Plautia stali (Hemiptera: Heteroptera: Pentatomidae). Recently, several groups reported the presence of JHSB3 in other heteropteran species. However, most of the studies paid no attention to the determination of the relative and absolute structure of the JH. In this study, we investigated the JH of the cabbage bug Eurydema rugosa (Hemiptera: Heteroptera: Pentatomidae), known as a pest for wild and cultivated crucifers. JHSB3 was detected in the hexane extract from the corpus allatum (CA) product using a chiral ultraperformance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) which can inform the absolute stereochemistry of the JH. Its stereoisomers were not detected. Topical application of the synthetic JHSB3 to the last instar nymphs inhibited their metamorphosis and induced nymphal-type colouration of the dorsal abdomen in a dose-dependent manner. Additionally, the topical application of JHSB3 effectively terminated summer and winter diapauses in females. These results indicate that the JH of E. rugosa is JHSB3. Although individuals in summer and winter diapauses are physiologically distinct in E. rugosa, the results suggest that the physiological differences between these diapauses are based, not on the responsiveness to JH, but on the processes governing activation of the CA or on its upstream cascades.

Circadian clock outputs regulating insect photoperiodism: A potential role for glutamate transporter.

Many temperate ectotherms survive winter by entering diapause - a state of developmental (or reproductive) suppression or arrest - in response to short autumnal day lengths. Day lengths are assessed by the circadian clock, the biological time-keeping system that governs biological rhythms with a period of approximately 24 h. However, clock output molecules controlling this photoperiodic response are largely unknown for many insects. To identify these molecules in Hemiptera, we performed RNAi knockdowns of several candidate genes in the bean bug Riptortus pedestris to determine whether their silencing affects photoperiodic regulation of ovarian development (reproductive diapause). Knockdown of diuretic hormone 31, short neuropeptide F, neuropeptide F, ion transport peptide, neuropeptide-like precursor 1, and choline acetyltransferase had no effect on ovarian development and were therefore ruled out as regulators of the photoperiodic response. However, knockdown of vesicular glutamate transporter promoted ovarian development under diapause-inducing short days, and this is the first report of the functional involvement of glutamate signalling in insect photoperiodism. Improved knockdown of this transporter (or receptor) and RNAi of other genes involved in glutamate signal transduction is required to verify its role as an output of the circadian clock.

Photoperiod controls egg laying and caudodorsal cell hormone expression but not gonadal development in the pond snail Lymnaea stagnalis.

Photoperiod is a reliable cue to regulate growth and reproduction for seasonal adaptation. Although photoperiodism has been well studied in Chordata and Arthropoda, less is known about Mollusca. We examined photoperiodic effects on egg laying, body size, gonad-somatic index, oocyte size and relative amounts of caudodorsal cell hormone mRNA in individual rearing conditions in the pond snail Lymnaea stagnalis. Twenty-five weeks after hatching, the percentages of egg-laying snails under a photoperiod of 12 h light and 12 h darkness (12L:12D) were significantly smaller than those under longer days. The total numbers of eggs and egg masses under 12L:12D were significantly smaller than those under longer days. Significant differences between 16L:8D and 12L:12D were not observed in the soft body and ovotestis weight, and the gonad-somatic index. Photoperiodic effects were also not observed in oocyte diameters twenty-two weeks after hatching. Twenty-seven weeks after hatching amounts of caudodorsal cell hormone mRNA were significantly lower in the cerebral ganglia with commissure under 12L:12D than 16L:8D. L. stagnalis exhibited a clear photoperiodic response in egg laying and the amount of caudodorsal cell hormone mRNA, but not in gonadal development. Under 12L:12D suppression of caudodorsal cell hormone expression might suppress egg laying.

Expression of aquaporins in response to distinct dehydration stresses that confer stress tolerance in the Antarctic midge Belgica antarctica.

Larvae of the Antarctic midge Belgica antarctica Jacobs (Diptera: Chironomidae) are highly tolerant of diverse environmental stresses, including freezing, severe desiccation, and osmotic extremes. Furthermore, dehydration confers subsequent desiccation and freeze tolerance. While a role for aquaporins-channels for water and other solutes-has been proposed in these dehydration processes, the types of aquaporins involved in dehydration-driven stress tolerance remain unknown. In the present study, we investigated expression of six aquaporins (Drip, Prip, Eglp1, Eglp2, Aqp12L, and Bib) in larvae of B. antarctica subjected to three different dehydration conditions: desiccation, cryoprotective dehydration, and osmotic dehydration. The expression of Drip and Prip was up-regulated under desiccation and cryoprotective dehydration, suggesting a role for these aquaporins in efficient water loss under these dehydration conditions. Conversely, expression of Drip and Prip was down-regulated under osmotic dehydration, suggesting that their expression is suppressed in larvae to combat dehydration. Larval water content was similarly decreased under all three dehydration conditions. Differences in responses of the aquaporins to the three forms of dehydration suggests distinct water management strategies associated with different forms of dehydration stress.

Juvenile hormone III skipped bisepoxide, not its stereoisomers, as a juvenile hormone of the bean bug Riptortus pedestris.

Juvenile hormone (JH) plays a pivotal role in many aspects of insect physiology. Although its presence was first reported in a blood-sucking bug belonging to the suborder Heteroptera (true bugs), JH species in the group has long been controversial. Although some recent studies proposed a putative JH molecular species in several Heteropteran species, it is not conclusive because physicochemical analyses were insufficient in most cases. Here, we studied this issue with an ultraperformance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) equipped with C18 and chiral columns in the bean bug Riptortus pedestris (Heteroptera, Alydidae), in which the JH species has long been controversial. Although a recent study describes JHSB3 as the major JH of this species, that finding was not conclusive because its chirality has not been clarified. In the present study, we detected methyl (2R,3S,10R)-2,3;10,11-bisepoxyfarnesoate, commonly named juvenile hormone III skipped bisepoxide (JHSB3), in the culture media of the corpora cardiaca-corpus allatum (CC-CA) complex and in the hemolymph of this species by a chiral ultraperformance liquid chromatography- tandem mass spectrometer (UPLC-MS/MS). Other JHSB3 stereoisomers were not detected. Topical application of JHSB3 effectively averted diapause. These results indicate that JHSB3 is the major JH of R. pedestris. The present study further revealed that JHSB3 and its (2R,3S,10S) isomer are more potent than (2S,3R,10R) and (2S,3R,10S) isomers, which suggests that there is a significance to the configuration of the 2,3-epoxide moiety in JH action. We further found a supplemental significance to the configuration of the 10-position.

Northward expansion of the bivoltine life cycle of the cricket over the last four decades.

Recent climate warming has affected some life-history traits of insects, including voltinism and body size. The magnitude of changes in these traits may differ latitudinally within a species because of the differing lengths of season available for growth. The present study aims to estimate the change in voltinism of the lawn ground cricket, Polionemobius mikado (Shiraki) (Orthoptera: Trigonidiidae), over the last four decades by comparing the body size between adults collected from a wide range of latitudes in Japan in recent years (2015-2017) and those collected four decades ago (1969-1976). The body size of adults collected in recent years showed a latitudinal saw-tooth cline, in the same way as body size did four decades ago, and the cline shifted northward over the last four decades: In 2015-2017, the body size decreased slightly with increasing latitude from 31°N to 36°N, and then increased to 40°N, and again decreased from 40°N to 44°N. Comparison of the body size between recent years and four decades ago revealed that the body size has decreased significantly at the middle latitudes (36-40°N), suggesting that the proportion of smaller bivoltine individuals there has increased over the last four decades. The sum of effective temperatures for postdiapause embryonic development at around 36°N in recent years was comparable to that at 31-35°N four decades ago, at which P. mikado populations were bivoltine. Taken together, these findings suggested that the latitudinal range suitable for the bivoltine life cycle of P. mikado has expanded northward over the last four decades because of climate warming. This is the first report that shows that a decrease in body size can be caused by climate warming via an increase in voltinism.

Both the anterior and posterior eyes function as photoreceptors for photoperiodic termination of diapause in the two-spotted spider mite.

Photoreceptors involved in photoperiodism in insects and mites can be either the retinal photoreceptors in the visual system or nonvisual extraretinal photoreceptors. Mites with no eyes have a clear photoperiodic response, suggesting the involvement of extraretinal photoreceptors in mite photoperiodism. In mites equipped with eyes, however, it is not known whether the retinal or extraretinal photoreceptors are involved in photoperiodism. The two-spotted spider mite Tetranychus urticae possesses two pairs of eyes. Adult females of this species terminate diapause in response to long days. To investigate whether the eyes function as photoperiodic photoreceptors in T. urticae, their eyes were ablated using a laser ablation system. Mites with their eyes intact terminated diapause under long days after low temperature exposure, whereas they remained in diapause under short days. Under constant darkness, they did not terminate diapause. When all eyes were removed, the mites remained in diapause even when they were maintained under long days. In contrast, the mites showed clear photoperiodic response when only the anterior or posterior eyes were removed. These results indicate that both the anterior and posterior eyes function as photoreceptors in photoperiodic termination of diapause in T. urticae.

Involvement of the brain region containing pigment-dispersing factor-immunoreactive neurons in the photoperiodic response of the bean bug, Riptortus pedestris.

The concept of insect photoperiodism based on a circadian clock has been supported by many studies demonstrating that the behavioural circadian rhythm and the photoperiodic response are driven by the same circadian clock genes. However, the neuronal mechanism of the circadian clock underlying photoperiodism is poorly understood. To examine whether circadian rhythm and photoperiodism share a neuronal mechanism, we focused on the neurons that express neuropeptide pigment-dispersing factor (PDF) in the bean bug, Riptortus pedestris. PDF has been identified as an important regulator of the insect circadian rhythm and is expressed in circadian clock neurons of various insect species. In R. pedestris, PDF immunoreactivity was detected in some clusters of cells and their fibres in the optic lobe and the protocerebrum. cDNA encoding a PDF precursor protein was highly conserved between R. pedestris and many other insects. Differences between day and night were not observed in the immunolabelling intensity in cell bodies of PDF-immunoreactive neurons and pdf mRNA expression levels in the head. Surgical removal of the region containing PDF-immunoreactive cell bodies at the medulla disrupted the photoperiodic regulation of diapause. However, gene suppression of pdf by RNA interference did not affect the photoperiodic response. These results suggest that the region containing PDF-immunoreactive somata is important for the photoperiodic response in R. pedestris, but pdf mRNA expression is probably not required for the response.

Desiccation and heat tolerance of eggs of the Asian tadpole shrimp, Triops granarius.

Triops granarius (Lucas) is a freshwater crustacean that is distributed from South Africa to the Eurasian continent. This species lives in temporary water pools and is exposed to desiccation and extreme temperatures after the water dries up in its habitat. To withstand this severe environment, Triops eggs enter anhydrobiosis when dehydrated. To clarify the physiological characteristics of T. granarius anhydrobiosis, we examined hatching rates after rehydration of eggs that were dehydrated at several humidity levels for 10 or 100 days. Lower humidity produced higher hatching rates when dehydration was continued for 100 days. These results suggest that drying at low humidity is required for long-term anhydrobiosis of T. granarius eggs. The eggs survived desiccation when dehydrated at the blastula, gastrula, and early organogenesis stages. The most dehydration-tolerant stage was early organogenesis. Non-dehydrated eggs hatched after temperature treatments of up to 50°C for 1 h, but did not hatch after exposure to 60°C for 1 h in air and under water. Similar results were obtained for dehydrated eggs exposed to high temperatures under water. In contrast, dehydrated eggs hatched after 1 h at 80°C in air but did not after 1 h at 90°C in air. Our results show that Triops eggs exhibit tolerance for desiccation and high temperature in a dried state, once they have entered anhydrobiosis.

RNAi of the circadian clock gene period disrupts the circadian rhythm but not the circatidal rhythm in the mangrove cricket.

The clock mechanism for circatidal rhythm has long been controversial, and its molecular basis is completely unknown. The mangrove cricket, Apteronemobius asahinai, shows two rhythms simultaneously in its locomotor activity: a circatidal rhythm producing active and inactive phases as well as a circadian rhythm modifying the activity intensity of circatidal active phases. The role of the clock gene period (per), one of the key components of the circadian clock in insects, was investigated in the circadian and circatidal rhythms of A. asahinai using RNAi. After injection of double-stranded RNA of per, most crickets did not show the circadian modulation of activity but the circatidal rhythm persisted without a significant difference in the period from controls. Thus, per is functionally involved in the circadian rhythm but plays no role, or a less important role, in the circatidal rhythm. We conclude that the circatidal rhythm in A. asahinai is controlled by a circatidal clock whose molecular mechanism is different from that of the circadian clock.

Juvenile hormone as a causal factor for maternal regulation of diapause in a wasp.

Most temperate multivoltine insects enter diapause, a hormonally controlled developmental suspension, in response to seasonal photoperiodic and/or thermal cues. Some insect species exhibit maternal regulation of diapause in which developmental trajectories of the offspring are determined by mothers in response to environmental cues that the mother received. Although maternally regulated diapause is common among insects, the maternal endocrinological mechanisms are largely veiled. To approach this issue, we used the jewel wasp Nasonia vitripennis, which produces non-diapause-destined offspring under long days and diapause-destined offspring under short days or low temperatures. Comparative transcriptomics of these wasps revealed possible involvement of the juvenile hormone (JH) biosynthetic cascade in maternal diapause regulation. The expression of juvenile hormone acid O-methyltransferase (jhamt) was typically downregulated in short-day wasps, and this was reflected by a reduction in haemolymph JH concentrations. RNAi targeted at jhamt reduced haemolymph JH concentration and induced wasps to produce diapause-destined offspring even under long days. In addition, topical application of JH suppressed the production of diapause-destined offspring under short days or low temperatures. These results indicate that diapause in N. vitripennis is determined by maternal jhamt expression and haemolymph JH concentration in response to day length. We therefore report a novel role for JH in insect seasonality.

Photoperiodic response requires mammalian-type cryptochrome in the bean bug Riptortus pedestris.

The hypothesis that a circadian clock comprised of circadian clock genes is causally involved in insect photoperiodism has been supported by several studies. However, there remains a possibility that the effects of the circadian clock genes on photoperiodism are exerted through pleiotropic (non-circadian) functions provided by each gene independently from its role in the circadian clock. In the present study, we investigated the involvement of the circadian clock gene mammalian-type cryptochrome (cry-m) in photoperiodic regulation of ovarian development in the bean bug Riptortus pedestris by using RNA interference (RNAi). Injection of cry-m double-stranded RNA (dsRNA) induced expression of period (per), whereas did not affect expression of cycle (cyc), showing that CRY-m functions as a negative element on CYC-mediated transcription in the circadian clock. If the circadian clock is indeed involved in photoperiodism, a phenotype produced by RNAi of cry-m will be the same as that produced by RNAi of per, another negative element. The intact insects and insects injected with control dsRNA were found to enter diapause when kept under short-day conditions after adult emergence, while they developed ovaries when kept under long-day conditions after adult emergence. However, cry-m RNAi significantly increased the incidence of reproductive individuals under diapause-inducing short-day conditions, as per RNAi did, in accordance with our expectation.

Peripheral circadian clock for the cuticle deposition rhythm in Drosophila melanogaster.

Insect endocuticle thickens after adult emergence by daily alternating deposition of two chitin layers with different orientation. Although the cuticle deposition rhythm is known to be controlled by a circadian clock in many insects, the site of the driving clock, the photoreceptor for entrainment, and the oscillatory mechanism remain elusive. Here, we show that the cuticle deposition rhythm is regulated by a peripheral oscillator in the epidermis in Drosophila melanogaster. Free-running and entrainment experiments in vitro reveal that the oscillator for the cuticle deposition rhythm is independent of the central clock in the brain driving the locomotor rhythms. The cuticle deposition rhythm is absent in null and dominant-negative mutants of clock genes (i.e., period, timeless, cycle, and Clock), indicating that this oscillator is composed of the same clock genes as the central clock. Entrainment experiments with monochromatic light-dark cycles and cry(b) flies reveal that a blue light-absorbing photoreceptor, cryptochrome (CRY), acts as a photoreceptor pigment for the entrainment of the cuticle deposition rhythm. Unlike other peripheral rhythms in D. melanogaster, the cuticle deposition rhythm persisted in cry(b) and cry(OUT) mutant flies, indicating that CRY does not play a core role in the rhythm generation in the epidermal oscillator.

Photoperiodic diapause under the control of circadian clock genes in an insect.

BACKGROUND: Most organisms have evolved a circadian clock in order to anticipate daily environmental changes and many of these organisms are also capable of sophisticated measurement of daylength (photoperiodism) that is used to regulate seasonal events such as diapause, migration and polymorphism. It has been generally accepted that the same elements are involved in both circadian (daily) and seasonal (annual) rhythms because both rely upon daily light-dark cycles. However, as reasonable as this sounds, there remains no conclusive evidence of such a molecular machinery in insects. We have approached this issue by using RNA interference (RNAi) in Riptortus pedestris. RESULTS: The cuticle deposition rhythm exhibited the major properties of circadian rhythms, indicating that the rhythm is regulated by a circadian clock. RNAi directed against the circadian clock genes of period and cycle, which are negative and positive regulators in the circadian clock, respectively, disrupted the cuticle deposition rhythm and distinct cuticle layers were produced by these RNAi. Simultaneously, period RNAi caused the insect to avert diapause under a diapause-inducing photoperiod whereas cycle RNAi induced diapause under a diapause-averting photoperiod. The expression patterns of juvenile hormone-regulated genes and the application of juvenile hormone analogue suggested that neither ovarian development itself nor a downstream cascade of juvenile hormone secretion, were disturbed by period and cycle RNAi. CONCLUSIONS: This study revealed that the circadian clock genes are crucial not only for daily rhythms but also for photoperiodic diapause. RNAi directed against period and cycle had opposite effects not only in the circadian cuticle deposition rhythm but also in the photoperiodic diapause. These RNAi also had opposite effects on juvenile hormone-regulated gene expression. It is still possible that the circadian clock genes pleiotropically affect ovarian development but, based on these results, we suggest that the circadian clock operated by the circadian clock genes, period and cycle, governs seasonal timing as well as the daily rhythms.

Urban warming and artificial light alter dormancy in the flesh fly.

Seasonal changes in temperature and day length are distinct between rural and urban areas due to urban warming and the presence of artificial light at night. Many studies have focused on the impacts of these ubiquitous signatures on daily biological events, but empirical studies on their impacts on insect seasonality are limited. In the present study, we used the flesh fly Sarcophaga similis as a model insect to determine the impacts of urbanization on the incidence and timing of diapause (dormancy), not only in the laboratory but also in rural and urban conditions. In the laboratory, diapause entry was affected by night-time light levels as low as 0.01 lux. We placed fly cages on outdoor shelves in urban and rural areas to determine the timing of diapause entry; it was retarded by approximately four weeks in urban areas relative to that in rural areas. Moreover, almost all flies in the site facing an urban residential area failed to enter diapause, even by late autumn. Although an autumnal low temperature in the urban area would mitigate the negative effect of artificial light at night, strong light pollution seriously disrupts the flesh fly seasonal adaptation.

Juvenile hormone III skipped bisepoxide is widespread in true bugs (Hemiptera: Heteroptera).

Juvenile hormone (JH) plays important roles in almost every aspect of insect development and reproduction. JHs are a group of acyclic sesquiterpenoids, and their farnesol backbone has been chemically modified to generate a homologous series of hormones in some insect lineages. JH III (methyl farnesoate, 10,11-epoxide) is the most common JH in insects, but Lepidoptera (butterflies and moths) and 'higher' Diptera (suborder: Brachycera; flies) have developed their own unique JHs. Although JH was first proposed in the hemipteran suborder Heteroptera (true bugs), the chemical identity of the heteropteran JH was only recently determined. Furthermore, recent studies revealed the presence of a novel JH, JH III skipped bisepoxide (JHSB3), in some heteropterans, but its taxonomic distribution remains largely unknown. In the present study, we investigated JHSB3 production in 31 heteropteran species, covering almost all heteropteran lineages, through ultra-performance liquid chromatography coupled with tandem mass spectrometry. We found that all of the focal species produced JHSB3, indicating that JHSB3 is widespread in heteropteran bugs and the evolutionary occurrence of JHSB3 ascends to the common ancestor of Heteroptera.

Molecular characterization of visual pigments in Branchiopoda and the evolution of opsins in Arthropoda.

Studies on color vision in invertebrates have focused primarily on insect visual pigments, with little attention given to crustacean visual pigments. None of the blue-green-, blue-, or ultraviolet (UV)-sensitive-opsins have been identified in crustaceans. In addition, the discussion of visual pigments has been limited to long-wavelength-sensitive opsins in Pancrustacea. Here, we focused on Branchiopoda (Crustacea), which is a sister group of Hexapoda including insects. In the tadpole shrimp Triops granarius, the visual pigment chromophore was retinal. Multiple opsins were isolated from each of three branchiopod species, T. granarius, Triops longicaudatus, and the fairy shrimp Branchinella kugenumaensis (five, five, and four opsins from these species, respectively). Phylogenetic analyses and the presence of a lysine residue corresponding to position 90 in bovine rhodopsin suggested that three of the branchiopod opsins comprise UV-sensitive pigments. In addition, the phylogenetic relationships between insect and branchiopod UV-sensitive opsins revealed that the divergence of blue- and UV-sensitive pigments predates the Branchiopoda and Insecta divergence. The other branchiopod opsins show distant relationships to other known insect opsins and form novel clusters. The present results strongly suggest that the ancestral arthropod of the Chelicerata-Pancrustacea lineages possessed at least four types of opsins. The ancestors of Pancrustacea and the Insecta-Branchiopoda lineages possessed at least five and six types of opsins, respectively. Our results suggest that in the evolutionary process associated with each lineage, several opsins appeared and diversified with repeated gene duplication, of which some have been lost in some taxa.

Fatty acids of membrane phospholipids in Drosophila melanogaster lines showing rapid and slow recovery from chill coma.

We investigated the fatty acid compositions of phospholipids in Drosophila melanogaster lines showing rapid (CR), intermediate (CTL), or slow (CS) recovery from chill coma, which were established by artificial selection or by free recombination without selection. Compared to CTL, CS showed a low composition of dienoic acids and a small number of double bonds in the fatty acids. The ratio of unsaturated fatty acids and saturated fatty acids (UFAs/SFAs) was significantly lower in CS than in CTL. CR had higher monoenoic acid composition and lower dienoic acid composition than CTL. In addition, the amount of SFAs was lower and therefore the UFAs/SFAs ratio considerably higher in CR than in CTL. These changes in phospholipid fatty acids probably contributed to losing and maintaining the homeoviscosity of the cellular membranes in CS and CR, respectively, at low temperature and therefore produced their distinct phenotypes in recovery from chill coma.

Effect of photoperiod on clock gene expression and subcellular distribution of PERIOD in the circadian clock neurons of the blow fly Protophormia terraenovae.

We examined the effect of photoperiod on the expression of circadian clock genes period (per) and timeless (tim), using quantitative real-time polymerase chain reaction (PCR), and the effect of photoperiod on subcellular distribution of PERIOD (PER), using immunocytochemistry, in the blow fly, Protophormia terraenovae. Under both short-day and long-day conditions, the mRNA levels of per and tim in the brain oscillated, and their peaks and troughs occurred around lights-off and lights-on, respectively. The oscillations persisted even under constant darkness. In the large ventral lateral neurons (l-LN(v)s), small ventral lateral neurons (s-LN(v)s), dorsal lateral neurons (LN(d)s), and medial dorsal neurons (DN(m)s), the subcellular distribution of PER-immunoreactivity changed with time. The number of cells with PER-immunoreactivity in the nucleus was highest 12 h after lights-off and lowest 12 h after lights-on, regardless of photoperiod, suggesting that PER nuclear translocation entrains to photoperiod. When temporal changes in the nuclear localization of PER were compared, the neurons could be classified into 2 groups: the l-LN(v)s were similar to the s-LN(v)s, and the LN(d)s were similar to DN(m)s. In LN(d)s and DN(m)s, decreasing rates of the number of cells with PER immunoreactivity in the nucleus per brain from the maximum were large as compared with those in l-LN(v)s and s-LN(v)s under short-day conditions. These results suggest that photoperiodic information is reflected in the expression patterns of circadian clock genes per and tim and in the subcellular distribution of PER. This observation suggests that the 2 different groups of clock neurons respond to photoperiod in slightly different manners.