Evidence of vitamin D synthesis in insects exposed to UVb light.

Vertebrates obtain the prohormone vitamin D primarily by endogenous cutaneous synthesis under ultraviolet b (UVb) exposure. To date, endogenous synthesis of vitamin D in insects has never been investigated. In an initial experiment, we exposed four insect species which differ in ecology and morphology (migratory locusts, house crickets, yellow mealworms and black soldier fly larvae (BSFL)) to a low irradiance UVb source. In a second experiment we exposed these species to a higher UV irradiance, and in a third we tested the effect of exposure duration on vitamin D concentrations in yellow mealworms. Low irradiance UVb tended to increase vitamin D3 levels in house crickets, vitamin D2 levels in BSFL and vitamin D2 and D3 in yellow mealworms. Higher UVb irradiance increased vitamin D3 levels in all species but BSFL. Both BSFL and migratory locusts had increased vitamin D2 levels. Longer UVb exposure of yellow mealworms increased vitamin D2 and increased vitamin D3 until a plateau was reached at 6400 IU/kg. This study shows that insects can synthesize vitamin D de novo and that the amounts depend on UVb irradiance and exposure duration.

The Genomic Architecture of a Rapid Island Radiation: Recombination Rate Variation, Chromosome Structure, and Genome Assembly of the Hawaiian Cricket Laupala.

Phenotypic evolution and speciation depend on recombination in many ways. Within populations, recombination can promote adaptation by bringing together favorable mutations and decoupling beneficial and deleterious alleles. As populations diverge, crossing over can give rise to maladapted recombinants and impede or reverse diversification. Suppressed recombination due to genomic rearrangements, modifier alleles, and intrinsic chromosomal properties may offer a shield against maladaptive gene flow eroding coadapted gene complexes. Both theoretical and empirical results support this relationship. However, little is known about this relationship in the context of behavioral isolation, where coevolving signals and preferences are the major hybridization barrier. Here we examine the genomic architecture of recently diverged, sexually isolated Hawaiian swordtail crickets (Laupala). We assemble a de novo genome and generate three dense linkage maps from interspecies crosses. In line with expectations based on the species' recent divergence and successful interbreeding in the laboratory, the linkage maps are highly collinear and show no evidence for large-scale chromosomal rearrangements. Next, the maps were used to anchor the assembly to pseudomolecules and estimate recombination rates across the genome to test the hypothesis that loci involved in behavioral isolation (song and preference divergence) are in regions of low interspecific recombination. Contrary to our expectations, the genomic region where a male song and female preference QTL colocalize is not associated with particularly low recombination rates. This study provides important novel genomic resources for an emerging evolutionary genetics model system and suggests that trait-preference coevolution is not necessarily facilitated by locally suppressed recombination.

Artificial light at night prolongs juvenile development time in the black field cricket, Teleogryllus commodus.

A growing body of evidence exists to support a detrimental effect of the presence of artificial light at night (ALAN) on life-history and fitness traits. However, few studies simultaneously investigate multiple traits and the life stages at which changes manifest. We experimentally manipulated ALAN intensities, within those found in the natural environment, to explore the consequences for growth, survival, and reproductive success of the field cricket, Teleogryllus commodus. We reared crickets from egg to adult under a daily light-cycle consisting of 12 hr bright daylight (2,600 lx) followed by either 12 hr darkness (0 lx) or dim-light environments (1, 10, or 100 lx). We found egg hatch, adult survival, and reproductive measures were largely comparable for all treatments. However, juvenile development time (number of days from egg to adult) was on average 10 days (14%) longer and adults were also larger when crickets were exposed to any light at night (1, 10, or 100 lx). Our data demonstrate that chronic lifetime exposure to ALAN can modulate the timing of life-history events and may disrupt phenology to a similar extent as other abiotic factors.

Developmental experience with anthropogenic noise hinders adult mate location in an acoustically signalling invertebrate.

Phenotypic plasticity facilitates survival and reproduction in rapidly changing and novel environments. Traffic noise spectrally overlaps with (i.e. masks) the sounds used by many acoustically signalling organisms to locate and secure mates. To determine if pre-reproductive exposure to noise improves adult performance in noisy environments, we reared field crickets (Teleogryllus oceanicus) in one of three noise environments: masking traffic noise, traffic noise from which frequencies that spectrally overlap with the crickets' song were removed (non-masking), or silence. At reproductive maturity, we tested female mate location ability under one of the same three acoustic conditions. We found that exposure to noise during rearing hindered female location of mates, regardless of the acoustic environment at testing. Females reared in masking noise took 80% longer than females reared in silence to locate a simulated singing male who was less than 1 m away. Impaired mate location ability can be added to a growing list of fitness costs associated with anthropogenic noise, alongside reductions in pairing success, nesting success and offspring survival.

Is radon emission in caves causing deletions in satellite DNA sequences of cave-dwelling crickets?

The most stable isotope of radon, 222Rn, represents the major source of natural radioactivity in confined environments such as mines, caves and houses. In this study, we explored the possible radon-related effects on the genome of Dolichopoda cave crickets (Orthoptera, Rhaphidophoridae) sampled in caves with different concentrations of radon. We analyzed specimens from ten populations belonging to two genetically closely related species, D. geniculata and D. laetitiae, and explored the possible association between the radioactivity dose and the level of genetic polymorphism in a specific family of satellite DNA (pDo500 satDNA). Radon concentration in the analyzed caves ranged from 221 to 26,000 Bq/m3. Specimens coming from caves with the highest radon concentration showed also the highest variability estimates in both species, and the increased sequence heterogeneity at pDo500 satDNA level can be explained as an effect of the mutation pressure induced by radon in cave. We discovered a specific category of nuclear DNA, the highly repetitive satellite DNA, where the effects of the exposure at high levels of radon-related ionizing radiation are detectable, suggesting that the satDNA sequences might be a valuable tool to disclose harmful effects also in other organisms exposed to high levels of radon concentration.

Melatonin: a possible link between the presence of artificial light at night and reductions in biological fitness.

The mechanisms underpinning the ecological impacts of the presence of artificial night lighting remain elusive. One suspected underlying cause is that the presence of light at night (LAN) supresses nocturnal production of melatonin, a key driver of biological rhythm and a potent antioxidant with a proposed role in immune function. Here, we briefly review the evidence for melatonin as the link between LAN and changes in behaviour and physiology. We then present preliminary data supporting the potential for melatonin to act as a recovery agent mitigating the negative effects of LAN in an invertebrate. Adult crickets (Teleogryllus commodus), exposed to constant illumination, were provided with dietary melatonin (concentrations: 0, 10 or 100 µg ml(-1)) in their drinking water. We then compared survival, lifetime fecundity and, over a 4-week period, immune function (haemocyte concentration, lysozyme-like and phenoloxidase (PO) activity). Melatonin supplementation was able only partially to mitigate the detrimental effects of LAN: it did not improve survival or fecundity or PO activity, but it had a largely dose-dependent positive effect on haemocyte concentration and lysozyme-like activity. We discuss the implications of these relationships, as well as the usefulness of invertebrates as model species for future studies that explore the effects of LAN.

The clock gene cycle plays an important role in the circadian clock of the cricket Gryllus bimaculatus.

To dissect the molecular oscillatory mechanism of the circadian clock in the cricket Gryllus bimaculatus, we have cloned a cDNA of the clock gene cycle (Gb'cyc) and analyzed its structure and function. Gb'cyc contains four functional domains, i.e. bHLH, PAS-A, PAS-B and BCTR domains, and is expressed rhythmically in light dark cycles, peaking at mid night. The RNA interference (RNAi) of Clock (Gb'Clk) and period (Gb'per) reduced the Gb'cyc mRNA levels and abolished the rhythmic expression, suggesting that the rhythmic expression of Gb'cyc is regulated by a mechanism including Gb'Clk and Gb'per. These features are more similar to those of mammalian orthologue of cyc (Bmal1) than those of Drosophila cyc. A single treatment with double-stranded RNA (dsRNA) of Gb'cyc effectively knocked down the Gb'cyc mRNA level and abolished its rhythmic expression. The cyc RNAi failed to disrupt the locomotor rhythm, but lengthened its free-running period in constant darkness (DD). It is thus likely that Gb'cyc is involved in the circadian clock machinery of the cricket. The cyc RNAi crickets showed a rhythmic expression of Gb'per and timeless (Gb'tim) in the optic lobe in DD, explaining the persistence of the locomotor rhythm. Surprisingly, cyc RNAi revealed a rhythmic expression of Gb'Clk in DD which is otherwise rather constitutively expressed in the optic lobe. These facts suggest that the cricket might have a unique clock oscillatory mechanism in which both Gb'cyc and Gb'Clk are rhythmically controlled and that under abundant expression of Gb'cyc the rhythmic expression of Gb'Clk may be concealed.

Functional analysis of the circadian clock gene period by RNA interference in nymphal crickets Gryllus bimaculatus.

The circadian clock gene period (Gryllus bimaculatus period, Gb'per) plays a core role in circadian rhythm generation in adults of the cricket Gryllus bimaculatus. We examined the role of Gb'per in nymphal crickets that show a diurnal rhythm rather than the nocturnal rhythm of the adults. As in the adult optic lobes, Gb'per mRNA levels in the head of the third instar nymphs showed daily cycling in light-dark cycles with a peak at mid night, and the rhythm persisted in constant darkness. Injection of Gb'per double-stranded RNA (dsRNA) into the abdomen of third instar nymphs knocked-down the mRNA levels to 25% of that in control animals. Most Gb'per dsRNA injected nymphs lost their circadian locomotor activity rhythm, while those injected with DsRed2 dsRNA as a negative control clearly maintained the rhythm. These results suggest that nymphs and adults share a common endogenous clock mechanism involving the clock gene Gb'per.

Haze, clouds and limited sky visibility: polarotactic orientation of crickets under difficult stimulus conditions.

Field crickets (Gryllus campestris L.) are able to detect the orientation of the electric vector (e-vector) of linearly polarized light. They presumably use this sense to exploit the celestial polarization pattern for course control or navigation. Polarization vision in crickets can be tested by eliciting a spontaneous polarotactic response. Previously, wide and 100% polarized stimuli were employed to induce this behavior. However, field crickets live on meadows where the observation of the sky is strongly limited by surrounding vegetation. Moreover, degrees of polarization (d) in the natural sky are much lower than 100%. We have therefore investigated thresholds for the behavioral response to polarized light under conditions mimicking those experienced by the insects in the field. We show that crickets are able to rely on polarized stimuli of just 1 degrees diameter. We also provide evidence that they exploit polarization down to an (average) polarization level of less than 7%, irrespective of whether the stimulus is homogeneous, such as under haze, or patched, such as a sky spotted by clouds. Our data demonstrate that crickets can rely on skylight polarization even under unfavorable celestial conditions, emphasizing the significance of polarized skylight orientation for insects.

Context-dependent olfactory learning in an insect.

We studied the capability of the cricket Gryllus bimaculatus to select one of a pair of odors and to avoid the other in one context and to do the opposite in another context. One group of crickets was trained to associate one of a pair of odors (conditioned stimulus, CS1) with water reward (appetitive unconditioned stimulus, US+) and another odor (CS2) with saline solution (aversive US, US-) under illumination and to associate CS1 with US- and CS2 with US+ in the dark. Another group of crickets received training of the opposite stimulus arrangement. At 1 d after the training for 3 d, the former group significantly preferred CS1 over CS2 under illumination but preferred CS2 over CS1 in the dark, and the latter group exhibited the opposite odor preference. The results of control experiments showed that the background light condition had no significant effects on memory formation or retrieval unless it was explicitly associated with US during training. Thus, the visual context affected learning performance only when crickets were requested to use it to disambiguate the meaning of CSs and to predict USs.

5-HT(7)-like receptors mediate serotonergic modulation of photo-responsiveness of the medulla bilateral neurons in the cricket, Gryllus bimaculatus.

Serotonin (5-HT) suppresses the photo-responsiveness of medulla bilateral neurons (MBNs) that are involved in the coupling mechanism of the bilaterally paired optic lobe circadian pacemakers in the cricket, Gryllus bimaculatus. We found that forskolin, a highly specific activator of adenylate cyclase, mimicked the effects of serotonin on the MBNs. This fact suggests the involvement of cyclic 3', 5'-adenosine monophosphate (cAMP) in mediating the action of serotonin. We therefore tested the effects of various 5-HT receptor agonists and antagonists that are coupled to adenylate cyclase to specify the receptor involved. Application of 8-OH-DPAT that has affinity for both 5-HT(1A) and 5-HT(7) receptors suppressed the photo-responsiveness, like forskolin. The inhibitory effect of 8-OH-DPAT was effectively blocked by clozapine, a high affinity 5-HT(7) receptor antagonists with a very low affinity for 5-HT(2). Ketanserin, a selective 5-HT(2) antagonist, and NAN-190, a 5-HT(1A) antagonist, did not block it. These results suggest that serotonergic suppression of the photo-responsiveness of the MBNs is mediated by 5-HT(7)-like receptor subtypes.

Serotonin sets the day state in the neurons that control coupling between the optic lobe circadian pacemakers in the cricket Gryllus bimaculatus.

The bilaterally paired optic lobe circadian pacemakers of the cricket Gryllus bimaculatus mutually exchange photic and circadian information to keep their activity synchronized. The information is mediated by a neural pathway, consisting of the so-called medulla bilateral neurons, connecting the medulla areas of the two optic lobes. We investigated the effects of serotonin on the neural activity in this coupling pathway. Spontaneous and light-induced electrical activity of the neurons in the coupling pathway showed daily variations, being more intense during the night than the day. Microinjection of serotonin or a serotonin-receptor agonist, quipazine, into the optic lobe caused a dose- and time-dependent inhibition of spontaneous and light-induced responses, mimicking the day state. The amount of suppression was greater and the recovery from the suppression occurred faster during the night. Application of metergoline, a non-selective serotonin-receptor antagonist, increased spontaneous activity and light-evoked responses during both the day and the night, with higher effect during the day. In addition, metergoline effectively attenuated the effects of serotonin. These facts suggest that in the cricket's optic lobe, serotonin is released during the daytime and sets the day state in the neurons regulating coupling between the bilaterally paired optic lobe circadian pacemakers.

Selective photoreceptor damage in four species of insects induced by experimental exposures to UV-irradiation.

Damage to photoreceptive cells of insect compound eyes exposed to abnormally high doses of UV-radiation of 350nm peak wavelength manifests itself in at least two different ways. In the butterflies Papilio xuthus and Pieris napi from Japan and northern Finland, respectively, only the cell bodies of retinula cells 1 and 2, (identified as short wavelength receptors), but not their corresponding rhabdomeres, exhibit damage with apoptotic features. In the eye of UV-irradiated adult crickets, however, cell bodies and cytoplasm remain normal, while the rhabdomeres of cells 7 and 8 exhibit signs of severe membrane disruptions. No signs of damage whatsoever occurred in the eyes of northern Finnish bumblebees exposed to UV. It is suggested that metabolic shortfalls in the UV-sensitive cells of the butterfly eyes result in cellular shut-down, but that in the cricket receptors UV-induced changes of the membrane lipids dominate, leading to membrane instability without concomittant cell death. The strong resistance of the bumblebee eye to UV-induced damage requires further investigation, but since preconditioning to light can reduce photic damage in the rat eye, the 24h daylight experienced by northern Finnish bumblebees during the summer season could be involved.