Competitive history shapes rapid evolution in a seasonal climate.

Eco-evolutionary dynamics will play a critical role in determining species' fates as climatic conditions change. Unfortunately, we have little understanding of how rapid evolutionary responses to climate play out when species are embedded in the competitive communities that they inhabit in nature. We tested the effects of rapid evolution in response to interspecific competition on subsequent ecological and evolutionary trajectories in a seasonally changing climate using a field-based evolution experiment with Drosophila melanogaster Populations of D. melanogaster were either exposed, or not exposed, to interspecific competition with an invasive competitor, Zaprionus indianus, over the summer. We then quantified these populations' ecological trajectories (abundances) and evolutionary trajectories (heritable phenotypic change) when exposed to a cooling fall climate. We found that competition with Z. indianus in the summer affected the subsequent evolutionary trajectory of D. melanogaster populations in the fall, after all interspecific competition had ceased. Specifically, flies with a history of interspecific competition evolved under fall conditions to be larger and have lower cold fecundity and faster development than flies without a history of interspecific competition. Surprisingly, this divergent fall evolutionary trajectory occurred in the absence of any detectible effect of the summer competitive environment on phenotypic evolution over the summer or population dynamics in the fall. This study demonstrates that competitive interactions can leave a legacy that shapes evolutionary responses to climate even after competition has ceased, and more broadly, that evolution in response to one selective pressure can fundamentally alter evolution in response to subsequent agents of selection.

Survival of Phortica variegata experimentally and naturally infected with Thelazia callipaeda.

Knowledge of the effects that Thelazia callipaeda (Spirurida, Thelaziidae) infection has on the survival of its vector Phortica variegata (Drosophilidae, Steganinae) is scarce. The present study aimed to: (a) assess the rate of infection between experimentally infected (EI) and not experimentally infected (NEI) flies and (b) determine how T. callipaeda infection may affect the survival of P. variegata. In addition, fat composition was evaluated in flies that died during overwintering. Molecular analysis showed that T. callipaeda prevalence in flies that died before experimental infection, plus those from the NEI group, is 0.75% (i.e. 11 out of 1462 individuals). The EI group showed a significantly higher positivity to T. callipaeda (i.e. 51 out of 682 individuals; 7.48%) compared with the NEI group (i.e. 9 out of 750 individuals; 1.2%). Thelazia callipaeda DNA was detected until 147 days after experimental infection. This demonstrates that larvae of this eyeworm may survive in the fly for a significant period of the winter. Fat composition analysis showed that flies produced more unsaturated than saturated fatty acids during diapause, probably because unsaturated fatty acids remain in a liquid state at lower temperatures, providing anti-freeze properties to survive winter.

Can insect assemblages tell us something about the urban environment health?

If we consider Drosophilidae, the answer to the question above is yes. Many research groups in Brazil and abroad have been showing that assemblages of flies of this family can reflect environmental alteration levels caused by urbanization, and/or by other human disturbances. I will present here a summary of our findings in Drosophilidae assemblages reflecting different degrees of environmental perturbation. These studies were done by graduate students of two post graduate programs of UFRGS, under my supervision, along several decades. I will also present the results stemming from the effort of other Brazilian Drosophilid study groups while identifying the members of those assemblages in different Biomes. As a result of those field studies, several biological invasions were detected and many new important biological problems arose prone to be investigated by genetic, molecular biology and other related approaches.

Two Drosophilids exhibit distinct EGF pathway patterns in oogenesis.

Deciphering the evolution of morphological structures is a remaining challenge in the field of developmental biology. The respiratory structures of insect eggshells, called the dorsal appendages, provide an outstanding system for exploring these processes since considerable information is known about their patterning and morphogenesis in Drosophila melanogaster and dorsal appendage number and morphology vary widely across Drosophilid species. We investigated the patterning differences that might facilitate morphogenetic differences between D. melanogaster, which produces two oar-like structures first by wrapping and then elongating the tubes via cell intercalation and cell crawling, and Scaptodrosophila lebanonensis, which produces a variable number of appendages simply by cell intercalation and crawling. Analyses of BMP pathway components thickveins and P-Mad demonstrate that anterior patterning is conserved between these species. In contrast, EGF signaling exhibits significant differences. Transcripts for the ligand encoded by gurken localize similarly in the two species, but this morphogen creates a single dorsolateral primordium in S. lebanonensis as defined by activated MAP kinase and the downstream marker broad. Expression patterns of pointed, argos, and Capicua, early steps in the EGF pathway, exhibit a heterochronic shift in S. lebanonensis relative to those seen in D. melanogaster. We demonstrate that the S. lebanonensis Gurken homolog is active in D. melanogaster but is insufficient to alter downstream patterning responses, indicating that Gurken-EGF receptor interactions do not distinguish the two species' patterning. Altogether, these results differentiate EGF signaling patterns between species and shed light on how changes to the regulation of patterning genes may contribute to different tube-forming mechanisms.

Thermal analysis of ice and glass transitions in insects that do and do not survive freezing.

Some insects rely on the strategy of freeze tolerance for winter survival. During freezing, extracellular body water transitions from the liquid to the solid phase and cells undergo freeze-induced dehydration. Here, we present results of a thermal analysis (from differential scanning calorimetry) of ice fraction dynamics during gradual cooling after inoculative freezing in variously acclimated larvae of two drosophilid flies, Drosophila melanogaster and Chymomyza costata Although the species and variants ranged broadly between 0 and close to 100% survival of freezing, there were relatively small differences in ice fraction dynamics. For instance, the maximum ice fraction (IFmax) ranged between 67.9% and 77.7% total body water (TBW). Chymomyza costata larvae showed statistically significant phenotypic shifts in parameters of ice fraction dynamics (melting point and IFmax) upon entry into diapause, cold acclimation and feeding on a proline-augmented diet. These differences were mostly driven by colligative effects of accumulated proline (ranging between 6 and 487 mmol kg-1 TBW) and other metabolites. Our data suggest that these colligative effects per se do not represent a sufficient mechanistic explanation for high freeze tolerance observed in diapausing, cold-acclimated C. costata larvae. Instead, we hypothesize that accumulated proline exerts its protective role via a combination of mechanisms. Specifically, we found a tight association between proline-induced stimulation of glass transition in partially frozen body liquids (vitrification) and survival of cryopreservation in liquid nitrogen.

Heat- and humidity-induced plastic changes in body lipids and starvation resistance in the tropical fly Zaprionus indianus during wet and dry seasons.

Insects in tropical wet or dry seasons are likely to cope with starvation stress through plastic changes (developmental as well as adult acclimation) in energy metabolites. Control and experimental groups of Zaprionus indianus flies were reared under wet or dry conditions, but adults were acclimated at different thermal or humidity conditions. Adult flies of the control group were acclimated at 27°C and low (50%) or high (60%) relative humidity (RH). For experimental groups, adult flies were acclimated at 32°C for 1 to 6 days and under low (40%) or high (70%) RH. For humidity acclimation, adult flies were acclimated at 27°C but under low (40%) or high (70%) RH for 1 to 6 days. Plastic changes in experimental groups as compared with the control group (developmental as well as adult acclimation) revealed significant accumulation of body lipids owing to thermal or humidity acclimation of wet season flies, but low humidity acclimation did not change the level of body lipids in dry season flies. Starvation resistance and body lipids were higher in the males of dry season flies but in the females of wet season flies. Adults acclimated under different thermal or humidity conditions exhibited changes in the rate of utilization of body lipids, carbohydrates and proteins. Adult acclimation of wet or dry season flies revealed plastic changes in mean daily fecundity; and a reduction in fecundity under starvation. Thus, thermal or humidity acclimation of adults revealed plastic changes in energy metabolites to support starvation resistance of wet or dry season flies.

Insect fat body cell morphology and response to cold stress is modulated by acclimation.

Mechanistic understanding about the nature of cellular cryoinjury and mechanisms by which some animals survive freezing while others do not is currently lacking. Here, we exploited the broadly manipulable freeze tolerance of larval malt flies (Chymomyza costata) to uncover cell and tissue morphological changes associated with freeze mortality. Diapause induction, cold acclimation and dietary proline supplementation generate malt fly variants ranging from weakly to extremely freeze tolerant. Using confocal microscopy and immunostaining of the fat body, Malpighian tubules and anterior midgut, we described tissue and cytoskeletal (F-actin and α-tubulin) morphologies among these variants after exposure to various cold stresses (from chilling at -5°C to extreme freezing at -196°C), and upon recovery from cold exposure. Fat body tissue appeared to be the most susceptible to cryoinjury: freezing caused coalescence of lipid droplets, loss of α-tubulin structure and apparent aggregation of F-actin. A combination of diapause and cold acclimation substantially lowered the temperature at which these morphological disruptions occurred. Larvae that recovered from a freezing challenge repaired F-actin aggregation but not lipid droplet coalescence or α-tubulin structure. Our observations indicate that lipid coalescence and damage to α-tubulin are non-lethal forms of freeze injury, and suggest that repair or removal (rather than protection) of actin proteins is a potential mechanism of acquired freeze tolerance.

Evolution of herbivory in Drosophilidae linked to loss of behaviors, antennal responses, odorant receptors, and ancestral diet.

Herbivory is a key innovation in insects, yet has only evolved in one-third of living orders. The evolution of herbivory likely involves major behavioral changes mediated by remodeling of canonical chemosensory modules. Herbivorous flies in the genus Scaptomyza (Drosophilidae) are compelling species in which to study the genomic architecture linked to the transition to herbivory because they recently evolved from microbe-feeding ancestors and are closely related to Drosophila melanogaster. We found that Scaptomyza flava, a leaf-mining specialist on plants in the family (Brassicaceae), was not attracted to yeast volatiles in a four-field olfactometer assay, whereas D. melanogaster was strongly attracted to these volatiles. Yeast-associated volatiles, especially short-chain aliphatic esters, elicited strong antennal responses in D. melanogaster, but weak antennal responses in electroantennographic recordings from S. flava. We sequenced the genome of S. flava and characterized this species' odorant receptor repertoire. Orthologs of odorant receptors, which detect yeast volatiles in D. melanogaster and mediate critical host-choice behavior, were deleted or pseudogenized in the genome of S. flava. These genes were lost step-wise during the evolution of Scaptomyza. Additionally, Scaptomyza has experienced gene duplication and likely positive selection in paralogs of Or67b in D. melanogaster. Olfactory sensory neurons expressing Or67b are sensitive to green-leaf volatiles. Major trophic shifts in insects are associated with chemoreceptor gene loss as recently evolved ecologies shape sensory repertoires.

Cultivable microbiome and its resistance to antimicrobials isolated from Zaprionus indianus.

The objective of this study was to identify Z. indianus in PEJC and PESCAN. Listed are the following methods: to isolate bacteria from the integument of the Z. indianus species collected and to check the resistance of microorganisms to antibiotics. Collections of Z. indianus were performed in four seasons of the two parks. The results obtained suggest that the low amount of Z. indianus collected can be justified by environmental factors such as high average temperature and low average humidity. It is noted that there is a predominance of bacteria of the Enterobacteriaceae family found in both the PEJC and the PESCAN. The antibiogram performed for the isolated PEJC bacteria shows statistical significance when comparing the edge and inside values of the park. Studies with fungi were also carried out, and it was evidenced that Trichophyton spp. was the genus that most inhabited the two environments studied. The ability of fluconazole and ketoconazole to inhibit fungal growth was also investigated, and considering the concentration tested may suggest that they have good action spectra. Plasmid profile data show that 60% of antibiotic-resistant bacteria have plasmids. The values found show that Z. indianus can act as vectors of microorganisms that affect the healthy animals and humans and that these organisms may be influenced by seasons.

Mutualism exploitation: predatory drosophilid larvae sugar-trap ants and jeopardize facultative ant-plant mutualism.

An open question in the evolutionary ecology of ant-plant facultative mutualism is how other members of the associated community can affect the interaction to a point where reciprocal benefits are disrupted. While visiting Qualea grandiflora shrubs to collect sugary rewards at extrafloral nectaries, tropical savanna ants deter herbivores and reduce leaf damage. Here we show that larvae of the fly Rhinoleucophenga myrmecophaga, which develop on extrafloral nectaries, lure potentially mutualistic, nectar-feeding ants and prey on them. Foraging ants spend less time on fly-infested foliage. Field experiments showed that predation (or the threat of predation) on ants by fly larvae produces cascading effects through three trophic levels, resulting in fewer protective ants on leaves, increased numbers of chewing herbivores, and greater leaf damage. These results reveal an undocumented mode of mutualism exploitation by an opportunistic predator at a plant-provided food source, jeopardizing ant-derived protection services to the plant. Our study documents a rather unusual case of predation of adult ants by a dipteran species and demonstrates a top-down trophic cascade within a generalized ant-plant mutualism.

Evolution in an ancient detoxification pathway is coupled with a transition to herbivory in the drosophilidae.

Chemically defended plant tissues present formidable barriers to herbivores. Although mechanisms to resist plant defenses have been identified in ancient herbivorous lineages, adaptations to overcome plant defenses during transitions to herbivory remain relatively unexplored. The fly genus Scaptomyza is nested within the genus Drosophila and includes species that feed on the living tissue of mustard plants (Brassicaceae), yet this lineage is derived from microbe-feeding ancestors. We found that mustard-feeding Scaptomyza species and microbe-feeding Drosophila melanogaster detoxify mustard oils, the primary chemical defenses in the Brassicaceae, using the widely conserved mercapturic acid pathway. This detoxification strategy differs from other specialist herbivores of mustard plants, which possess derived mechanisms to obviate mustard oil formation. To investigate whether mustard feeding is coupled with evolution in the mercapturic acid pathway, we profiled functional and molecular evolutionary changes in the enzyme glutathione S-transferase D1 (GSTD1), which catalyzes the first step of the mercapturic acid pathway and is induced by mustard defense products in Scaptomyza. GSTD1 acquired elevated activity against mustard oils in one mustard-feeding Scaptomyza species in which GstD1 was duplicated. Structural analysis and mutagenesis revealed that substitutions at conserved residues within and near the substrate-binding cleft account for most of this increase in activity against mustard oils. Functional evolution of GSTD1 was coupled with signatures of episodic positive selection in GstD1 after the evolution of herbivory. Overall, we found that preexisting functions of generalized detoxification systems, and their refinement by natural selection, could play a central role in the evolution of herbivory.

Grape Juice as a Bait for Anastrepha suspensa (Diptera: Tephritidae) and Zaprionus indianus (Diptera: Drosophilidae).

In field tests conducted in south Florida to test grape juice as a bait for the Caribbean fruit fly, Anastrepha suspensa Loew, high numbers of Zaprionus indianus Gupta were captured in traps with aqueous grape juice. These experiments included comparisons of grape juice bait with established A. suspensa protein-based baits (ammonium acetate + putrescine lures, or torula yeast) or wine, a bait found previously to be attractive to Z. indianus. Effects of different preservatives (polypropylene glycol, polyethylene glycol, proxel, or sodium tetraborate) and bait age were also tested. Traps with grape juice baits captured more A. suspensa than unbaited traps, but more were captured in traps with grape juice plus preservative baits and the highest numbers were captured in traps containing the established protein-based baits. In contrast, grape juice baits without preservative that were prepared on the day of deployment (0 d) or that were aged for 3-4 d in the laboratory captured the highest numbers of Z. indianus, while solutions that were aged in the laboratory for 6 or 9 d captured fewer. Although these studies found that aqueous grape juice is a poor bait for A. suspensa, we found that actively fermenting aqueous grape juice may be an effective bait for Z. indianus.

Hyperprolinemic larvae of the drosophilid fly, Chymomyza costata, survive cryopreservation in liquid nitrogen.

The larva of the drosophilid fly, Chymomyza costata, is probably the most complex metazoan organism that can survive submergence in liquid nitrogen (-196 °C) in a fully hydrated state. We examined the associations between the physiological and biochemical parameters of differently acclimated larvae and their freeze tolerance. Entering diapause is an essential and sufficient prerequisite for attaining high levels of survival in liquid nitrogen (23% survival to adult stage), although cold acclimation further improves this capacity (62% survival). Profiling of 61 different metabolites identified proline as a prominent compound whose concentration increased from 20 to 147 mM during diapause transition and subsequent cold acclimation. This study provides direct evidence for the essential role of proline in high freeze tolerance. We increased the levels of proline in the larval tissues by feeding larvae proline-augmented diets and found that this simple treatment dramatically improved their freeze tolerance. Cell and tissue survival following exposure to liquid nitrogen was evident in proline-fed nondiapause larvae, and survival to adult stage increased from 0% to 36% in proline-fed diapause-destined larvae. A significant statistical correlation was found between the whole-body concentration of proline, either natural or artificial, and survival to the adult stage in liquid nitrogen for diapause larvae. Differential scanning calorimetry analysis suggested that high proline levels, in combination with a relatively low content of osmotically active water and freeze dehydration, increased the propensity of the remaining unfrozen water to undergo a glass-like transition (vitrification) and thus facilitated the prevention of cryoinjury.

First report of Zaprionus indianus (Diptera: Drosophilidae) in commercial fruits and vegetables in Pennsylvania.

Zaprionus indianus (Gupta) (Diptera: Drosophilidae), an invasive vinegar fly, was found for the first time in Adams County, Pennsylvania, in 2011. It was found in a commercial tart cherry orchard using apple cider vinegar (ACV) traps that were monitoring another invasive vinegar fly, the spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae). Coincidentally, the first record of D. suzukii found in Pennsylvania was also found in this same cherry orchard only 3 months earlier as part of a spotted wing drosophila survey effort in raspberry, blackberry, grape, and tart cherry in Adams County. These same crops plus blueberry and tomato were monitored again in 2012. In this article, adult Z. indianus captures in ACV traps and other traps deployed in the aforementioned crops during 2012 season are presented and the economic importance of Z. indianus is discussed.

Development of Novel Management Tools for Phortica variegata (Diptera: Drosophilidae), Vector of the Oriental Eyeworm, Thelazia callipaeda (Spirurida: Thelaziidae), in Europe.

Lachryphagous males of Phortica variegata (Fallén, 1823) are gaining increasing attention in Europe, as they act as vectors of the nematode Thelazia callipaeda Railliet & Henry, 1910, causal agent of thelaziosis, an emergent zoonotic disease. Currently, there are no effective control strategies against the vector, and surveillance and monitoring rely on time-consuming and nonselective sampling methods. Our aim was to improve the knowledge about the population dynamics and the chemical ecology of the species. A total of 5,726 P. variegata flies (96.4% males and 3.6% females, mostly gravid) were collected in field experiments during June-September of 2020 in an oak forest in northern Spain. Our results indicate that 1) by means of sweep netting a significantly higher number of captures were found both around the collector´s body and in the air than at ground level; 2) a positive relationship was detected between the abundance of Phortica flies and temperature, with two significant peaks of abundance at 24 and 33°C; 3) the blend of red wine and cider vinegar was the most attractive bait; 4) yellow traps captured fewer flies compared to black and transparent traps; and 5) a significant reduction toward vinegar and wine was detected in presence of the phenolic monoterpenoid carvacrol. In addition, all the males (n = 690) analyzed by both molecular detection and dissection resulted negative for the presence of T. callipaeda larvae. Overall, these findings provide a better understanding of the vector in terms of monitoring and management strategies.

Mining the plant-herbivore interface with a leafmining Drosophila of Arabidopsis.

Experimental infections of Arabidopsis thaliana (Arabidopsis) with genomically characterized plant pathogens such as Pseudomonas syringae have facilitated the dissection of canonical eukaryotic defence pathways and parasite virulence factors. Plants are also attacked by herbivorous insects, and the development of an ecologically relevant genetic model herbivore that feeds on Arabidopsis will enable the parallel dissection of host defence and reciprocal resistance pathways such as those involved in xenobiotic metabolism. An ideal candidate is Scaptomyza flava, a drosophilid fly whose leafmining larvae are true herbivores that can be found in nature feeding on Arabidopsis and other crucifers. Here, we describe the life cycle of S. flava on Arabidopsis and use multiple approaches to characterize the response of Arabidopsis to S. flava attack. Oviposition choice tests and growth performance assays on different Arabidopsis ecotypes, defence-related mutants, and hormone and chitin-treated plants revealed significant differences in host preference and variation in larval performance across Arabidopsis accessions. The jasmonate and glucosinolate pathways in Arabidopsis are important in mediating quantitative resistance against S. flava, and priming with jasmonate or chitin resulted in increased resistance. Expression of xenobiotic detoxification genes was reduced in S. flava larvae reared on Arabidopsis jasmonate signalling mutants and increased in plants pretreated with chitin. These results and future research directions are discussed in the context of developing a genetic model system to analyse insect-plant interactions.

Contrasting levels of genotype by environment interaction for life history and morphological traits in invasive populations of Zaprionus indianus (Diptera: Drosophilidae).

It has been demonstrated that phenotypic plasticity and genotype by environment interaction are important for coping with new and heterogeneous environments during invasions. Zaprionus indianus Gupta (Diptera: Drosophilidae) is an Afrotropical invasive fly species introduced to the South American continent in 1999. This species is generalist and polyphagous, since it develops and feeds in several different fruit species. These characteristics of Z. indianus suggest that phenotypic plasticity and genotype by environment interaction may be important in this species invasion process. In this sense, our aim was to investigate the role of genetic variation for phenotypic plasticity (genotype by environment interaction) in Z. indianus invasion of the South American continent. Specifically, we quantified quantitative genetic variation and genotype by environment interactions of morphological and life history traits in different developmental environments, that is, host fruits. This was done in different populations in the invasive range of Z. indianus in Argentina. Results showed that Z. indianus populations have considerable amounts of quantitative genetic variation. Also, genotype by environment interactions was detected for the different traits analyzed in response to the different developmental environments. Interestingly, the amounts and patterns of these parameters differed between populations. We interpreted these results as the existence of differences in evolutionary potential between populations that have an important role in the short- and long-term success of the Z. indianus invasion process.

Temperature is a common climatic descriptor of lachryphagous activity period in Phortica variegata (Diptera: Drosophilidae) from multiple geographical locations.

BACKGROUND: The drosophilid Phortica variegata is known as vector of Thelazia callipaeda, the oriental eyeworm native to Asia that has become an emergent zoonotic agent in several European regions. Unlike almost all other arthropod vectors of pathogens, only P. variegata males feed of lachrymal secretions of animals, ingesting first-stage larvae (L1) of the worm living in the orbital cavities of the host, and allowing with the same behaviour the introduction of infective L3. Despite the increased detection of T. callipaeda in many European countries, information about the length of the lachryphagous activity period of P. variegata and a deep knowledge of the environmental and climatic variables involved are still limited. METHODS: We herein present the results of a multicentre study involving five sites from four different countries (Italy, Spain, UK and USA) where canine thelaziosis is endemic and/or where it has already been ascertained the presence of P. variegata. Field data have been obtained on a fortnightly basis from mid-April to the end of November 2018 from a contemporary standardized sampling (same sampling effort and time of collection in all sites) of lachryphagous flies collected around the eyes of a human bait using an entomological net. These data have been associated to data collection of local climatic variables (day length, temperature, wind speed, barometric pressure and relative humidity). RESULTS: Overall, a total of 4862 P. variegata flies (4637 males and 224 females) were collected, with high differences in densities among the different sampling sites. Significant positive correlations were found between P. variegata male density and temperature and wind speed, while negative correlations were observed for barometric pressure and relative humidity. However, the above significant differences are confirmed in each sampling site separately only for the temperature. CONCLUSIONS: This multicentre study highlights that temperature is the major common environmental driver in describing the lachryphagous activity of P. variegata in Europe and USA and, therefore, the transmission risk of thelaziosis.

Photoperiodic induction of diapause requires regulated transcription of timeless in the larval brain of Chymomyza costata.

Photoperiodic signal stimulates induction of larval diapause in Chymomyza costata. Larvae of NPD strain (npd-mutants) do not respond to photoperiod. Our previous results indicated that the locus npd could code for the timeless gene and its product might represent a molecular link between circadian and photoperiodic clock systems. Here we present results of tim mRNA (real time-PCR) and TIM protein (immunohistochemistry) analyses in the larval brain. TIM protein was localized in 2 neurons of each brain hemisphere of the 4-d-old 3rd instar wild-type larvae. In a marked contrast, no TIM neurons were detected in the brain of 4-day-old 3rd instar npd -mutant larvae and the level of tim transcripts was approximately 10-fold lower in the NPD than in the wild-type strain. Daily changes in tim expression and TIM presence appeared to be under photoperiodic control in the wild-type larvae. Clear daily oscillations of tim transcription were observed during the development of 3rd instars under the short-day conditions. Daily oscillations were less apparent under the long-day conditions, where a gradual increase of tim transcript abundance appeared as a prevailing trend. Analysis of the genomic structure of tim gene revealed that npd-mutants carry a 1855 bp-long deletion in the 5'-UTR region. This deletion removed the start of transcription and promoter regulatory motifs E-box and TER-box. The authors hypothesize that this mutation was responsible for dramatic reduction of tim transcription rates, disruption of circadian clock function, and disruption of photoperiodic calendar function in npd-mutant larvae of C. costata.