The phospholipid flippase ATP9A enhances macropinocytosis to promote nutrient starvation tolerance in hepatocellular carcinoma.

Macropinocytosis is an effective strategy to mitigate nutrient starvation. It can fuel cancer cell growth in nutrient-limited conditions. However, whether and how macropinocytosis contributes to the rapid proliferation of hepatocellular carcinoma cells, which frequently experience an inadequate nutrient supply, remains unclear. Here, we demonstrated that nutrient starvation strongly induced macropinocytosis in some hepatocellular carcinoma cells. It allowed the cells to acquire extracellular nutrients and supported their energy supply to maintain rapid proliferation. Furthermore, we found that the phospholipid flippase ATP9A was critical for regulating macropinocytosis in hepatocellular carcinoma cells and that high ATP9A levels predicted a poor outcome for patients with hepatocellular carcinoma. ATP9A interacted with ATP6V1A and facilitated its transport to the plasma membrane, which promoted plasma membrane cholesterol accumulation and drove RAC1-dependent macropinocytosis. Macropinocytosis inhibitors significantly suppressed the energy supply and proliferation of hepatocellular carcinoma cells characterised by high ATP9A expression under nutrient-limited conditions. These results have revealed a novel mechanism that overcomes nutrient starvation in hepatocellular carcinoma cells and have identified the key regulator of macropinocytosis in hepatocellular carcinoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Storage protein SfSP8 mediates larval starvation tolerance of Spodoptera frugiperda.

BACKGROUND: Energy homeostasis is vital for insects to survive food shortages. This study investigated the starvation tolerance of Spodoptera frugiperda, which invaded China in 2019, focusing on its storage protein family, crucial for energy balance. 10 storage protein family members were identified and their expression patterns at different development stages and under different starvation stress were analyzed. METHODS AND RESULTS: We used qPCR to evaluate the expression levels of storage protein family members under various larval instars and starvation conditions. We discovered that, among above 10 members, only 2 storage proteins, SfSP8 and SfSP7 showed significant upregulation in response to starvation stress. Notably, SfSP8 upregulated markedly after 24 h of fasting, whereas SfSP7 exhibited a delayed response, with significant upregulation observed only after 72 h of starvation. Then we significantly reduced the starvation tolerance of larvae through RNAi-mediated knockdown of SfSP8 and also altered the starvation response of SfSP7 from a late to an early activation pattern. Finally, we constructed transgenic Drosophila melanogaster with heterologous overexpressing SfSP8 revealed that the starvation tolerance of the transgenic line was significantly stronger than that of wild-type lines. CONCLUSIONS: SfSP8 was the core storage protein member that mediated the starvation tolerance of larvae of S. frugiperda. Our study on the novel function of storage proteins in mediating larval starvation tolerance of S. frugiperda is conducive to understanding the strong colonization of this terrible invasive pest.

Negative effects of fluctuating temperatures around the optimal temperature on reproduction and survival of the red flour beetle.

Whereas the vast majority of animals in nature experience daily or seasonal thermal fluctuations, most laboratory experiments use constant temperatures. We examined the effect of fluctuating temperatures on reproduction and survival under starvation, two important components of fitness. We used the red flour beetle as a model organism, which is a significant pest in grain mills around the world. Fluctuations around the optimal temperature were always negative for the adult survival under starvation. The effect of thermal fluctuations on the number of offspring reaching adulthood was negative as well but increased with the extent of exposure. It was the strongest when the adult parents were kept and the offspring were raised under fluctuating temperatures. However, the later the offspring were exposed to fluctuations during their development, the weaker the effect of fluctuating temperatures was. Moreover, raising the parents under fluctuating temperatures but keeping them after pupation at constant temperatures fully alleviated the negative effects of fluctuations on the offspring. Finally, we demonstrate that keeping the parents a few days under fluctuating temperatures is required to induce negative effects on the number of offspring reaching adulthood. Our study disentangles between the effects of thermal fluctuations experienced during the parental and offspring stage thus contributing to the ongoing research of insects under fluctuating temperatures.

Allelic polymorphism at foxo contributes to local adaptation in Drosophila melanogaster.

The insulin/insulin-like growth factor signalling pathway has been hypothesized as a major determinant of life-history profiles that vary adaptively in natural populations. In Drosophila melanogaster, multiple components of this pathway vary predictably with latitude; this includes foxo, a conserved gene that regulates insulin signalling and has pleiotropic effects on a variety of fitness-associated traits. We hypothesized that allelic variation at foxo contributes to genetic variance for size-related traits that vary adaptively with latitude. We first examined patterns of variation among natural populations along a latitudinal transect in the eastern United States and show that thorax length, wing area, wing loading, and starvation tolerance exhibit significant latitudinal clines for both males and females but that development time does not vary predictably with latitude. We then generated recombinant outbred populations and show that naturally occurring allelic variation at foxo, which exhibits stronger clinality than expected, is associated with the same traits that vary with latitude in the natural populations. Our results suggest that allelic variation at foxo contributes to adaptive patterns of life-history variation in natural populations of this genetic model.

Molecular characterization of insulin-like peptides in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

Insulin-like peptides (ILPs) including insulin, insulin-like growth factor (IGF) and relaxin are evolutionarily conserved hormones in metazoans, and they are involved in diverse physiological processes. The migratory brown planthopper (BPH), Nilaparvata lugens, encodes four ILP genes (Nlilp1, Nlilp2, Nlilp3 and Nlilp4) but their physiological roles are largely unknown. Sequence analysis showed that NlILP1 contained a relaxin-specific G protein-coupled receptor-binding motif and a variant motif of cysteine residues, and NlILP2 and NlILP4 resembled vertebrate IGFs. RNA interference (RNAi)-mediated gene silencing showed that depletion of each of Nlilp1, 2 and 3 significantly delayed the developmental duration of nymphs, and this effect could be exacerbated by double or triple gene depletion. Depletion of Nlilp1, Nlilp2 or Nlilp3 induces the accumulation of glucose, trehalose and glycogen, which is contradictory to depletion of the insulin receptor (NlInR1) in the BPH. Depletion of Nlilp1 significantly enhanced starvation resistance in both females and males although its extent was smaller than NlInR1 depletion. A parental RNAi assay showed that depletion of each of Nlilp1-4 dramatically impaired female fecundity. These findings indicate that NlILP1-4 have redundant and distinct roles in physiological processes in the BPH, thereby enhancing our understanding of the contribution of each NlILP to the ecological success of this species in natural habitats.

RNA-seq analysis provides insight into molecular adaptations of Andrias davidianus.

The Chinese giant salamander Andrias davidianus is regarded as an ideal model for studying local adaptations, such as longevity, tolerance to starvation, and cutaneous respiration. Transcriptome analysis is useful for studying the large and complex genomes of amphibians. Based on the coding gene set of adult A. davidianus, dozens of A. davidianus-specific genes were identified and three signaling pathway (JAK-STAT, HIF-1, and FoxO) genes were expanded as compared with other amphibians. The results of the pathway analysis of A. davidianus-specific genes indicated that the molecular adaptation of A. davidianus may have required a more rapid evolution of the immune system. Additionally, for the first time, the gene expressions in different parts of the skin tissue were compared. The results of the comparison analysis demonstrated that lateral skin could be more focused on mucus secretion, dorsal skin on immunity and melanogenesis, and abdominal skin on water and salt metabolism. This study provides the first insight into studying longevity and starvation tolerance in A. davidianus, and offers a basis for further investigation of the molecular mechanisms of adaptations in amphibians.

Offspring dependence on parental care and the role of parental transfer of oral fluids in burying beetles.

BACKGROUND: Immature stages of many animals can forage and feed on their own, whereas others depend on their parents' assistance to obtain or process food. But how does such dependency evolve, and which offspring and parental traits are involved? Burying beetles (Nicrophorus) provide extensive biparental care, including food provisioning to their offspring. Interestingly, there is substantial variation in the reliance of offspring on post-hatching care among species. Here, we examine the proximate mechanisms underlying offspring dependence, focusing on the larvae of N. orbicollis, which are not able to survive in the absence of parents. We specifically asked whether the high offspring dependence is caused by (1) a low starvation tolerance, (2) a low ability to self-feed or (3) the need to obtain parental oral fluids. Finally, we determined how much care (i.e. duration of care) they require to be able to survive. RESULTS: We demonstrate that N. orbicollis larvae are not characterized by a lower starvation tolerance than larvae of the more independent species. Hatchlings of N. orbicollis are generally able to self-feed, but the efficiency depends on the kind of food presented and differs from the more independent species. Further, we show that even when providing highly dependent N. orbicollis larvae with easy ingestible liquefied mice carrion, only few of them survived to pupation. However, adding parental oral fluids significantly increased their survival rate. Finally, we demonstrate that survival and growth of dependent N. orbicollis larvae is increased greatly by only a few hours of parental care. CONCLUSIONS: Considering the fact that larvae of other burying beetle species are able to survive in the absence of care, the high dependence of N. orbicollis larvae is puzzling. Even though they have not lost the ability to self-feed, an easily digestible, liquefied carrion meal is not sufficient to ensure their survival. However, our results indicate that the transfer of parental oral fluids is an essential component of care. In the majority of mammals, offspring rely on the exchange of fluids (i.e. milk) to survive, and our findings suggest that even in subsocial insects, such as burying beetles, parental fluids can significantly affect offspring survival.

Increased lipid accumulation but not reduced metabolism explains improved starvation tolerance in cold-acclimated arthropod predators.

Predatory arthropods are used for biological control in greenhouses, but there is increasing interest to extend their use to the outdoor environment where temperatures are typically lower. Acclimation at low temperature increases the ability of ectotherms to cope with subsequent more extreme cold, but may involve costs or benefits to other performance traits. A recent study in mesostigmatid mites (Gaeolaelaps aculeifer) showed that starvation tolerance was improved following a period of cold exposure. However, the physiological mechanisms that underlie improved starvation tolerance following cold exposure were not investigated. To examine whether cold acclimation would also improve starvation tolerance in an insect, we repeated the starvation study in another arthropod predator, the pirate bug Orius majusculus, as well as in G. aculeifer. Before tests, the two species were acclimated at 10, 15, or 20 °C for 7 (G. aculeifer) or 16 (O. majusculus) days. We then analyzed the effects of thermal exposure on body composition, consumption, and basal metabolic rate in both species. Our results confirmed that exposure to low temperature improves starvation tolerance in these arthropod predators. Body composition analyses revealed that both species had accumulated larger lipid stores during exposure to colder temperature, which at least in part can explain the larger starvation tolerance following cold exposure. In contrast, consumption and basal metabolic rate were not changed by thermal acclimation. Our study indicates that predatory arthropods exposed to cold increase their physiological robustness and ability to endure environmental challenges, including low temperature and low prey availability.

Long-term balancing selection at the Phosphorus Starvation Tolerance 1 (PSTOL1) locus in wild, domesticated and weedy rice (Oryza).

BACKGROUND: The ability to grow in phosphorus-depleted soils is an important trait for rice cultivation in many world regions, especially in the tropics. The Phosphorus Starvation Tolerance 1 (PSTOL1) gene has been identified as underlying the ability of some cultivated rice varieties to grow under low-phosphorus conditions; however, the gene is absent from other varieties. We assessed PSTOL1 presence/absence in a geographically diverse sample of wild, domesticated and weedy rice and sequenced the gene in samples where it is present. RESULTS: We find that the presence/absence polymorphism spans cultivated, weedy and wild Asian rice groups. For the subset of samples that carry PSTOL1, haplotype sequences suggest long-term selective maintenance of functional alleles, but with repeated evolution of loss-of-function alleles through premature stops and frameshift mutations. The loss-of-function alleles have evolved convergently in multiple rice species and cultivated rice varieties. Greenhouse assessments of plant growth under low- and high-phosphorus conditions did not reveal significant associations with PSTOL1 genotype variation; however, the striking signature of balancing selection at this locus suggests that further phenotypic characterizations of PSTOL1 allelic variants is warranted and may be useful for crop improvement. CONCLUSIONS: These findings suggest balancing selection for both functional and non-functional PSTOL1 alleles that predates and transcends Asian rice domestication, a pattern that may reflect fitness tradeoffs associated with geographical variation in soil phosphorus content.

SUMOylation of OsPSTOL1 is essential for regulating phosphate starvation responses in rice and Arabidopsis.

Although rice is one of the main sources of calories for most of the world, nearly 60% of rice is grown in soils that are low in phosphorus especially in Asia and Africa. Given the limitations of bioavailable inorganic phosphate (Pi) in soils, it is important to develop crops tolerant to low phosphate in order to boost food security. Due to the immobile nature of Pi, plants have developed complex molecular signalling pathways that allow them to discern changes in Pi concentrations in the environment and adapt their growth and development. Recently, in rice, it was shown that a specific serine-threonine kinase known as Phosphorus-starvation tolerance 1 (PSTOL1) is important for conferring low phosphate tolerance in rice. Nonetheless, knowledge about the mechanism underpinning PSTOL1 activity in conferring low Pi tolerance is very limited in rice. Post-translation modifications (PTMs) play an important role in plants in providing a conduit to detect changes in the environment and influence molecular signalling pathways to adapt growth and development. In recent years, the PTM SUMOylation has been shown to be critical for plant growth and development. It is known that plants experience hyperSUMOylation of target proteins during phosphate starvation. Here, we demonstrate that PSTOL1 is SUMOylated in planta, and this affects its phosphorylation activity. Furthermore, we also provide new evidence for the role of SUMOylation in regulating PSTOL1 activity in plant responses to Pi starvation in rice and Arabidopsis. Our data indicated that overexpression of the non-SUMOylatable version of OsPSTOL1 negatively impacts total root length and total root surface area of rice grown under low Pi. Interestingly, our data also showed that overexpression of OsPSTOL1 in a non-cereal species, Arabidopsis, also positively impacts overall plant growth under low Pi by modulating root development. Taken together our data provide new evidence for the role of PSTOL1 SUMOylation in mediating enhanced root development for tolerating phosphate-limiting conditions.

Corrigendum: SUMOylation of OsPSTOL1 is essential for regulating phosphate starvation responses in rice and Arabidopsis.

[This corrects the article DOI: 10.3389/fpls.2024.1274610.].

No evidence for direct thermal carryover effects on starvation tolerance in the obligate blood-feeder, Glossina morsitans morsitans.

Thermal stress during development can prime animals to cope better with similar conditions in later life. Alternatively, negative effects of thermal stress can persist across life stages and result in poorer quality adults (negative carryover effects). As mean temperatures increase due to climate change, evidence for such effects across diverse taxa is required. Using Glossina morsitans morsitans, a species of tsetse fly and vector of trypanosomiasis, we asked whether (i) adaptive developmental plasticity allows flies to survive for longer under food deprivation when pupal and adult temperatures are matched; or (ii) temperature stress during development persists into adulthood, resulting in a greater risk of death. We did not find any advantage of matched pupal and adult temperature in terms of improved starvation tolerance, and no direct negative carryover effects were observed. There was some evidence for indirect carryover effects-high pupal temperature produced flies of lower body mass, which, in turn, resulted in greater starvation risk. However, adult temperature had the largest impact on starvation tolerance by far: flies died 60% faster at 31°C than those experiencing 25°C, consequently reducing survival time from a median of 8 (interquartile range (IQR) 7-9) to 5 (IQR 5-5.25) days. This highlights differences in temperature sensitivity between life stages, as there was no direct effect of pupal temperature on starvation tolerance. Therefore, for some regions of sub-Saharan Africa, climate change may result in a higher mortality rate in emerging tsetse while they search for their first blood meal. This study reinforces existing evidence that responses to temperature are life stage specific and that plasticity may have limited capacity to buffer the effects of climate change.

Prey species and abundance affect growth and photosynthetic performance of the polyphagous sea slug Elysia crispata.

Some sacoglossan sea slugs steal functional macroalgal chloroplasts (kleptoplasts). In this study, we investigated the effects of algal prey species and abundance on the growth and photosynthetic capacity of the tropical polyphagous sea slug Elysia crispata. Recently hatched sea slugs fed and acquired chloroplasts from the macroalga Bryopsis plumosa, but not from Acetabularia acetabulum. However, adult sea slugs were able to switch diet to A. acetabulum, rapidly replacing the great majority of the original kleptoplasts. When fed with B. plumosa, higher feeding frequency resulted in significantly higher growth and kleptoplast photosynthetic yield, as well as a slower relative decrease in these parameters upon starvation. Longevity of A. acetabulum-derived chloroplasts in E. crispata was over twofold that of B. plumosa. Furthermore, significantly lower relative weight loss under starvation was observed in sea slugs previously fed on A. acetabulum than on B. plumosa. This study shows that functionality and longevity of kleptoplasts in photosynthetic sea slugs depend on the origin of the plastids. Furthermore, we have identified A. acetabulum as a donor of photosynthetically efficient chloroplasts common to highly specialized monophagous and polyphagous sea slugs capable of long-term retention, which opens new experimental routes to unravel the unsolved mysteries of kleptoplasty.

Identification and functional characterization of insulin-like peptides in a pine beetle.

Insulin - including insulin-like peptides (ILPs), relaxins and insulin-like growth factors (IGFs) - is an evolutionarily conserved hormone in all metazoans. It is involved in various physiological processes, such as metabolism, growth, reproduction, lifespan and stress resistance. However, there are no reports on the functional role of ILPs in the Chinese white pine beetle, Dendroctonus armandi. In this study, we have cloned and identified two ILP cDNAs in D. armandi. The expression levels of DaILP1 and DaILP2 were significantly changed in different developmental stages. Both ILPs were expressed mostly in the head and fat body. Moreover, starvation induces the reduction of ILP1 mRNA level in adults and larvae, while ILP2 only in larvae of D. armandi, respectively. Additionally, RNA-interference (RNAi) using double stranded RNA to knock down ILP1 and ILP2 reduced the mRNA levels of the target genes, and caused a significant reduction in body weight of D. armandi. Moreover, silencing ILP1 led to an increase of trehalose and glycogen and significantly enhanced starvation resistance in both adults and larvae. The results show that the ILP signaling pathway plays a significant role in growth and carbohydrate metabolism of D. armandi and may provide a potential molecular target for pest control.

An analysis of direct and indirect effects in Drosophila melanogaster undergoing a few cycles of experimental evolution for stress-related traits.

The physiological mechanisms underpinning adaptations to starvation and cold stresses have been extensively studied in Drosophila, yet the understanding of correlated changes in stress-related and life-history traits, as well as the energetics of stress tolerance, still remains elusive. To answer the questions empirically in this context, we allowed D. melanogaster to evolve for either increased starvation or cold tolerance (24-generations / regime) in an experimental evolution system, and examined whether selection of either trait affects un-selected stress trait, as well as the impacts potential changes in life-history and mating success-related traits. Our results revealed remarkable changes in starvation/cold tolerance (up to 1.5-fold) as a direct effect of selection, while cold tolerance had been dramatically reduced (1.26-fold) in the starvation tolerant (ST) lines compared to control counterparts, although no such changes were evident in cold-tolerant (CT) lines. ST lines exhibited a higher level of body lipids and a reduced level of trehalose content, while CT lines accumulated a greater levels of body lipid and trehalose contents. Noticeably, we found that selection for starvation or cold tolerance positively correlates with larval development time, longevity, and copulation duration, indicating that these traits are among the most common targets of selection trajectories shaping stress tolerance. Altogether, this study highlights the complexity of mechanisms evolved in ST lines that contribute to enhanced starvation tolerance, but also negatively impact cold tolerance. Nevertheless, mechanisms foraging enhanced cold tolerance in CT lines appear not to target starvation tolerance. Moreover, the parallel changes in life history/mating success traits across stress regimes could indicate some generic pathways evolved in stressful environments, targeting life-history and mating success characteristics to optimize fitness.

Cross-Generational Effects of Heat Stress on Fitness and Wolbachia Density in Aedes aegypti Mosquitoes.

Aedes aegypti mosquitoes infected with Wolbachia symbionts are now being released into the field to control the spread of pathogenic human arboviruses. Wolbachia can spread throughout vector populations by inducing cytoplasmic incompatibility and can reduce disease transmission by interfering with virus replication. The success of this strategy depends on the effects of Wolbachia on mosquito fitness and the stability of Wolbachia infections across generations. Wolbachia infections are vulnerable to heat stress, and sustained periods of hot weather in the field may influence their utility as disease control agents, particularly if temperature effects persist across generations. To investigate the cross-generational effects of heat stress on Wolbachia density and mosquito fitness, we subjected Ae. aegypti with two different Wolbachia infection types (wMel, wAlbB) and uninfected controls to cyclical heat stress during larval development over two generations. We then tested adult starvation tolerance and wing length as measures of fitness and measured the density of wMel in adults. Both heat stress and Wolbachia infection reduced adult starvation tolerance. wMel Wolbachia density in female offspring was lower when mothers experienced heat stress, but male Wolbachia density did not depend on the rearing temperature of the previous generation. We also found cross-generational effects of heat stress on female starvation tolerance, but there was no cross-generational effect on wing length. Fitness costs of Wolbachia infections and cross-generational effects of heat stress on Wolbachia density may reduce the ability of Wolbachia to invade populations and control arbovirus transmission under specific environmental conditions.

Standard metabolic rate predicts growth trajectory of juvenile Chinese crucian carp (Carassius auratus) under changing food availability.

Phenotypic traits vary greatly within populations and can have a significant influence on aspects of performance. The present study aimed to investigate the effects of individual variation in standard metabolic rate (SMR) on growth rate and tolerance to food deprivation in juvenile Chinese crucian carp (Carassius auratus) under varying levels of food availability. To address this issue, 19 high and 16 low SMR individuals were randomly assigned to a satiation diet for 3 weeks, whereas another 20 high and 16 low SMR individuals were assigned to a restricted diet (approximately 50% of satiation) for the same period. Then, all fish were completely food-deprived for another 3 weeks. High SMR individuals showed a higher growth rate when fed to satiation, but this advantage of SMR did not exist in food-restricted fish. This result was related to improved feeding efficiency with decreased food intake in low SMR individuals, due to their low food processing capacity and maintenance costs. High SMR individuals experienced more mass loss during food deprivation as compared to low SMR individuals. Our results here illustrate context-dependent costs and benefits of intraspecific variation in SMR whereby high SMR individuals show increased growth performance under high food availability but had a cost under stressful environments (i.e. food shortage).

Novel Alleles of Phosphorus-Starvation Tolerance 1 Gene (PSTOL1) from Oryza rufipogon Confers High Phosphorus Uptake Efficiency.

Limited phosphorus availability in the soil is one of the major constraints to the growth and productivity of rice across Asian, African and South American countries, where 50% of the rice is grown under rain-fed systems on poor and problematic soils. With an aim to determine novel alleles for enhanced phosphorus uptake efficiency in wild species germplasm of rice Oryza rufipogon, we investigated phosphorus uptake1 (Pup1) locus with 11 previously reported SSR markers and sequence characterized the phosphorus-starvation tolerance 1 (PSTOL1) gene. In the present study, we screened 182 accessions of O. rufipogon along with Vandana as a positive control with SSR markers. From the analysis, it was inferred that all of the O. rufipogon accessions undertaken in this study had an insertion of 90 kb region, including Pup1-K46, a diagnostic marker for PSTOL1, however, it was absent among O. sativa cv. PR114, PR121, and PR122. The complete PSTOL1 gene was also sequenced in 67 representative accessions of O. rufipogon and Vandana as a positive control. From comparative sequence analysis, 53 mutations (52 SNPs and 1 nonsense mutation) were found in the PSTOL1 coding region, of which 28 were missense mutations and 10 corresponded to changes in the amino acid polarity. These 53 mutations correspond to 17 haplotypes, of these 6 were shared and 11 were scored only once. A major shared haplotype was observed among 44 accessions of O. rufipogon along with Vandana and Kasalath. Out of 17 haplotypes, accessions representing 8 haplotypes were grown under the phosphorus-deficient conditions in hydroponics for 60 days. Significant differences were observed in the root length and weight among all the genotypes when grown under phosphorus deficiency conditions as compared to the phosphorus sufficient conditions. The O. rufipogon accession IRGC 106506 from Laos performed significantly better, with 2.5 times higher root weight and phosphorus content as compared to the positive control Vandana. In terms of phosphorus uptake efficiency, the O. rufipogon accessions IRGC 104639, 104712, and 105569 also showed nearly two times higher phosphorus content than Vandana. Thus, these O. rufipogon accessions could be used as the potential donor for improving phosphorus uptake efficiency of elite rice cultivars.

The Drosophila melanogaster Muc68E Mucin Gene Influences Adult Size, Starvation Tolerance, and Cold Recovery.

Mucins have been implicated in many different biological processes, such as protection from mechanical damage, microorganisms, and toxic molecules, as well as providing a luminal scaffold during development. Nevertheless, it is conceivable that mucins have the potential to modulate food absorption as well, and thus contribute to the definition of several important phenotypic traits. Here we show that the Drosophila melanogaster Muc68E gene is 40- to 60-million-yr old, and is present in Drosophila species of the subgenus Sophophora only. The central repeat region of this gene is fast evolving, and shows evidence for repeated expansions/contractions. This and/or frequent gene conversion events lead to the homogenization of its repeats. The amino acid pattern P[ED][ED][ST][ST][ST] is found in the repeat region of Muc68E proteins from all Drosophila species studied, and can occur multiple times within a single conserved repeat block, and thus may have functional significance. Muc68E is a nonessential gene under laboratory conditions, but Muc68E mutant flies are smaller and lighter than controls at birth. However, at 4 d of age, Muc68E mutants are heavier, recover faster from chill-coma, and are more resistant to starvation than control flies, although they have the same percentage of lipids as controls. Mutant flies have enlarged abdominal size 1 d after chill-coma recovery, which is associated with higher lipid content. These results suggest that Muc68E has a role in metabolism modulation, food absorption, and/or feeding patterns in larvae and adults, and under normal and stress conditions. Such biological function is novel for mucin genes.

Effects of Starvation on Deltamethrin Tolerance in Bed Bugs, Cimex lectularius L. (Hemiptera: Cimicidae).

Bed bugs, Cimex lectularius L., are a major pest in the urban environment. Their presence often results in physical, psychological, and financial distress of homeowners and apartment dwellers. Although many insecticide bioassays have been performed on this pest, little attention has been paid to bed bug feeding status, which is closely linked to metabolism, molting, and mass. Therefore, we evaluated the toxicity of topically applied deltamethrin on insecticide susceptible adult male bed bugs fed 2 d, 9 d, and 21 d prior to testing. When toxicity was evaluated on a "per-bug" basis, there was no difference between 2 d [LD50 = 0.498 (0.316 - 0.692) ng·bug(-1)] and 9 d [LD50 = 0.572 (0.436 - 0.724) ng·bug(-1)] starved bugs, while 21 d starved bugs had a significantly lower LD50 [0.221 (0.075 - 0.386) ng·bug(-1)]. When toxicity was evaluated in terms of body mass, 9 d starved bugs had the highest LD50 values [0.138 (0.102 - 0.176) ng·mg(-1)], followed by 2 d starved bugs [0.095 (0.060 - 0.134) ng·mg(-1)], and then 21 d starved bugs [0.058 (0.019-0.102) ng·mg(-)¹]; the LD50 values of 2 d and 9 d starved bugs were significantly different from 21 d starved bugs. These results indicate that feeding status plays an important role in the toxicity of deltamethrin. In addition, the lack of differences between 2 d and 9 d starved bugs indicate that the blood meal itself has little impact on tolerance, but rather it is some physiological change following feeding that confers increased tolerance to bed bugs.