Temporal dynamics of apoptosis-induced proliferation in pupal wing development: implications for regenerative ability.

BACKGROUND: The ability of animals to regenerate damaged tissue is a complex process that involves various cellular mechanisms. As animals age, they lose their regenerative abilities, making it essential to understand the underlying mechanisms that limit regenerative ability during aging. Drosophila melanogaster wing imaginal discs are epithelial structures that can regenerate after tissue injury. While significant research has focused on investigating regenerative responses during larval stages our comprehension of the regenerative potential of pupal wings and the underlying mechanisms contributing to the decline of regenerative responses remains limited. RESULTS: Here, we explore the temporal dynamics during pupal development of the proliferative response triggered by the induction of cell death, a typical regenerative response. Our results indicate that the apoptosis-induced proliferative response can continue until 34 h after puparium formation (APF), beyond this point cell death alone is not sufficient to induce a regenerative response. Under normal circumstances, cell proliferation ceases around 24 h APF. Interestingly, the failure of reinitiating the cell cycle beyond this time point is not attributed to an incapacity to activate the JNK pathway. Instead, our results suggest that the function of the ecdysone-responsive transcription factor E93 is involved in limiting the apoptosis-induced proliferative response during pupal development. CONCLUSIONS: Our study shows that apoptosis can prolong the proliferative period of cells in the wing during pupal development as late as 34 h APF, at least 10 h longer than during normal development. After this time point, the regenerative response is diminished, a process mediated in part by the ecdysone-responsive transcription factor E93.

Effects of temperature on metabolic rate during metamorphosis in the alfalfa leafcutting bee.

Spring conditions, especially in temperate regions, may fluctuate abruptly and drastically. Environmental variability can expose organisms to temperatures outside of their optimal thermal ranges. For ectotherms, sudden changes in temperature may cause short- and long-term physiological effects, including changes in respiration, morphology, and reproduction. Exposure to variable temperatures during active development, which is likely to occur for insects developing in spring, can cause detrimental effects. Using the alfalfa leafcutting bee, Megachile rotundata, we aimed to determine if oxygen consumption could be measured using a new system and to test the hypothesis that female and male M. rotundata have a thermal performance curve with a wide optimal range. Oxygen consumption of M. rotundata pupae was measured across a large range of temperatures (6-48°C) using an optical oxygen sensor in a closed respirometry system. Absolute and mass-specific metabolic rates were calculated and compared between bees that were extracted from their brood cells and those remaining in the brood cell to determine whether pupae could be accurately measured inside their brood cells. The metabolic response to temperature was non-linear, which is an assumption of a thermal performance curve; however, the predicted negative slope at higher temperatures was not observed. Despite sexual dimorphism in body mass, sex differences only occurred in mass-specific metabolic rates. Higher metabolic rates in males may be attributed to faster development times, which could explain why there were no differences in absolute metabolic rate measurements. Understanding the physiological and ecological effects of thermal environmental variability on M. rotundata will help to better predict their response to climate change.

Cadmium exposure through the food chain reduces the parasitic fitness of Chouioia cunea to Hyphantria cunea pupae: An ecotoxicological risk to pest control.

The toxicity of heavy metals on various trophic levels along the food chain has been extensively investigated, but no studies have focused on parasitic natural enemy insects. Herein, we constructed a food chain consisting of soil-Fraxinus mandshurica seedlings-Hyphantria cunea pupae-Chouioia cunea to analyze the effects of Cd exposure through food chain on the fitness of parasitic natural enemy insects and its corresponding mechanism. The results showed that the transfer of Cd between F. mandshurica leaves and H. cunea pupae and between H. cunea pupae and C. cunea was a bio-minimization effect. After parasitizing Cd-accumulated pupa, the number of offspring larvae, and the number, individual size (body weight, body length, abdomen length) and life span of offspring adults decreased significantly, while the duration of embryo development extended significantly. The contents of malondialdehyde and H2O2 in Cd-exposed offspring wasps increased significantly, accompanied by a significantly decrease in antioxidant capacity. The cellular immunity parameters significantly decreased in Cd-accumulated pupae, including the number of hemocytes, melanization activity and the expression level of cellular immunity genes (e.g. Hemolin-1 and PPO1). The humoral immunity disorder was found in the Cd-accumulated pupae, as evidenced by that the expression level of immune recognition gene (PGRP-SA), signal transduction genes (IMD, Dorsal, and Tube), as well as all antimicrobial peptide genes (e.g. Lysozym and Attacin) decreased significantly. Cd exposure decreased the content of glucose, trehalose, amino acid, and free fatty acid in H. cunea pupae. The expression of Hk2 in glycolysis pathway and the expression of Idh2, Idh3, Cs, and OGDH in TCA cycle pathway were significantly down-regulated in Cd-accumulated pupae. Taken together, exposure to Cd through the food chain causes oxidative damage on the offspring wasps and disrupts energy metabolism of the host insect, ultimately reducing the parasitic fitness of C. cunea to H. cunea pupae.

Comparative transcriptome analysis of Liriomyza trifolii (Burgess) and Liriomyza sativae (Blanchard) (Diptera: Agromyzidae) in response to rapid cold hardening.

The ability of insets to react efficiently to fluctuation in temperature is crucial for them to survive in variable surroundings. Rapid cold hardening (RCH) is a process that increase cold tolerance in most insect species. The molecular mechanisms of RCH remain largely unknown, and whether it is associated with transcriptional changes is unclear. In this study, we compared the transcriptomes of Liriomyza trifolii and L. sativae exposed to RCH to investigate the transcript abundance due to RCH in both species. RNA-seq revealed 93,166 assembled unigenes, and 34,303 of these were annotated in the L. trifolii and L. sativae transcriptome libraries. After a 4-h treatment at 1°C (RCH) compared with control, 268 and 606 unigenes were differentially expressed in L. trifolii and L. sativae, respectively. When comparing pupae exposed to 2h cold shock directly with pupae went through 4h acclimation prior to 2h cold shock, 60 and 399 unigenes were differentially expressed in L trifolii and L sativae, respectively. Genes that were commonly expressed in both L. trifolii and L. sativae, included cytochrome P450, cuticular protein, glucose dehydrogenase, solute carrier family 22 and cationic amino acid transporter. Additionally, several pathways including galactose metabolism and peroxisome were significantly enriched during RCH. Our results show that the transcriptional response is correlated with RCH in the pupal stage of the two Liriomyza species, but more transcriptional changes were identified in L sativae than in L. trifolii.

The pupal moulting fluid has evolved social functions in ants.

Insect societies are tightly integrated, complex biological systems in which group-level properties arise from the interactions between individuals1-4. However, these interactions have not been studied systematically and therefore remain incompletely known. Here, using a reverse engineering approach, we reveal that unlike solitary insects, ant pupae extrude a secretion derived from the moulting fluid that is rich in nutrients, hormones and neuroactive substances. This secretion elicits parental care behaviour and is rapidly removed and consumed by the adults. This behaviour is crucial for pupal survival; if the secretion is not removed, pupae develop fungal infections and die. Analogous to mammalian milk, the secretion is also an important source of early larval nutrition, and young larvae exhibit stunted growth and decreased survival without access to the fluid. We show that this derived social function of the moulting fluid generalizes across the ants. This secretion thus forms the basis of a central and hitherto overlooked interaction network in ant societies, and constitutes a rare example of how a conserved developmental process can be co-opted to provide the mechanistic basis of social interactions. These results implicate moulting fluids in having a major role in the evolution of ant eusociality.

Metamorphosis and denucleation of the brain in the miniature wasp Megaphragma viggianii (Hymenoptera: Trichogrammatidae).

Holometabolan brains undergo structural and allometric changes and complex reorganizations during metamorphosis. In minute egg parasitoids, brain formation is shifted to the late larva and young pupa, due to extreme de-embryonization. The brains of Megaphragma wasps undergo denucleation, the details of which remained unknown. We describe the morphological and volumetric changes in the brain of Megaphragma viggianii (Trichogrammatidae) during pupal development with emphasis on the lysis of nuclei and show that the absolute and relative volume of the brain decrease by a factor of 5 from prepupa to adult at the expense of the cell body rind. The first foci of lysis appear during early pupal development, but most nuclei (up to 97%) are lost between pharate adult and adult. The first signs of lysis (destruction of the nuclear envelopes) occur in pupae with red eyes. The number of lysis foci (organelle destruction and increasing number of lysosomes and degree of chromatin compaction) strongly increases in pupae with black eyes. The cell body rind volume strongly decreases during pupal development (in larger insects it increases slightly or remains unchanged). Elucidation of the lysis of nuclei in neurons and of the functioning of an anucleate brain is an important objective for neuroscience.

The possibility of using xenogeneic phagocytes in wound treatment.

Metamorphosis in the insect larva is associated with disintegration, engulf and digestion of larval tissues. These processes are accompanied by a significant shift in physiological parameters like high activity of hydrolytic enzymes and decrease of pH. In the way, the metamorphosing larva resembles the processes occurring in the wound at the stage of inflammation. Based on this thesis, we put forward the idea of the possibility of using insect phagocytes in the wound treatment. The search for a suitable insect cell line and the study of its properties were the purpose of the work. The abilities of insect phagocytes to retain viability and functional activity under conditions physiological for humans were also investigated. We found that blue blowfly Calliphora vicina larvae had histolysocytes, a specialized population of professional phagocytes involved in the histolysis. In vitro, histolysocytes possess high phagocytic activity to fragments of vertebrate soft tissues and debris. These cells retain viability and functional activity for a long time under conditions that are physiological for vertebrate cells. Moreover histolysocytes can realize the humoral control over the bacteria through the synthesis of antimicrobial peptides. So histolysocytes have the potential to be used as xenogeneic phagocytes in the wound treatment. The data obtained allow proceeding to experiments on laboratory animals for studying the effect of such therapy on the wound healing process.

Postmortem Interval Estimation and Validation Through a Comparative Study of South American Flies Reared in the Field Versus Laboratory Conditions.

Studies under constant temperatures are the most common to estimate the Postmortem Interval (PMI). It is imperative that forensic sciences have data from studies carried out in the field. Therefore, this work aims to: (1) evaluate the parameters (weight, length, development time) associated with the life cycles of Lucilia ochricornis (Wiedemann) (Diptera: Calliphoridae) and Lucilia purpurascens (Walker) under experimental conditions in the field considering fluctuating temperatures, and (2) compare these results with those known and published by the same authors for cultures realized in the laboratory under constant temperatures; which will permit us to contrast the most widely used existing methodologies for forensic application in estimating the minimum postmortem interval (PMImin). For each season of the year, cultures of both species were made in the field, collecting information on temperature, humidity, and photoperiod to perform laboratory cultures, later comparing: development time, length, weight, and Accumulated Degree-Hours (ADH) in both types of cultures. Methods for estimating the PMI were obtained and validated with the information of the cultures grown in the field. The two types of cultures showed differences between each other for both species. The forensic use methods to estimate PMI were enhanced and their precision increased when maximum larval length data were used, and it was also concluded that feeding larval stages are the most accurate to be used in making estimates because the larva is growing. The estimation of the PMI through the use of necrophagous flies development remains reliable for obtaining the PMImin.

Ovicidal, larvicidal and pupicidal efficacy of silver nanoparticles synthesized by Bacillus marisflavi against the chosen mosquito species.

Microbial synthesis of silver nanoparticles is more advantageous and is eco-friendly to combat the various vectors that cause diseases in humans. Hence, in the present study a Bacillus strain is isolated from marine habitat and is evaluated for its ability to synthesize silver nanoparticles (AgNPs) and its efficacy evaluated against the immature stages of selected mosquito species. The effective candidate was confirmed to be Bacillus marisflavi after 16S rRNA sequencing. The synthesis of AgNPs was confirmed by UV-Vis spectrophotometer. Atomic Force Microscopic (AFM) analysis showed spherical nanoparticles. Size analysis using Scanning Electron Microscope (SEM) showed particles of nano size averaging 78.77 nm. The diameter of the particles analyzed by Dynamic Light Scattering (DLS) showed 101.6 nm with a poly-dispersive index of 0.3. Finally the elemental nature of the nanoparticles was identified by Fourier-transform infrared spectroscopy (FTIR). LC50 and LC90 values for the ovicidal, larvicidal and pupicidal efficacy of the AgNPs against the egg, larvae and pupae of Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi respectively were evaluated. The present study revealed that the nanoparticles have an excellent toxic effect against the disease transmitting vector mosquitoes. Hence, the rapid synthesis of AgNPs would be an appropriate eco-friendly tool for biocontrol of vector mosquitoes.

Demography and perturbation analyses of the coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae): Implications for management.

The coffee berry borer (CBB) Hypothenemus hampei Ferrari is the most serious pest of coffee worldwide. Management of the CBB is extremely difficult because its entire life cycle occurs inside the fruit, where it is well protected. Knowing which life stages contribute most to population growth, would shed light on the population dynamics of this pest and help to improve CBB management programs. Two staged-classified matrices were constructed for CBB populations reared in the lab on artificial diets and CBB populations from artificial infestations in the field. Matrices were used to determine demographic parameters, to conduct elasticity analyses, and to perform prospective perturbation analysis. Higher values of the intrinsic rate of natural increase (rm) and population growth rate (λ): were observed for CBB populations growing in the lab than in the field (rm: 0.058, λ: 1.74 lab; rm: 0.053, λ: 1.32 field). Sensitivity values for both CBB populations were highest for the transitions from larva to pupa (G2: 0.316 lab, 0.352 field), transition from pupa to juvenile (G3: 0.345 lab, 0.515 field) and survival of adult females (P5: 0.324 lab, 0.389 field); these three vital rates can be important targets for CBB management. Prospective perturbation analyses indicated that an effective management for the CBB should consider multiple developmental stages; perturbations of >90% for each transition are necessary to reduce λ to <1. However, when the three vital rates with highest sensitivity are impacted at the same time, the percentage of perturbation is reduced to 25% for each transition; with these reductions in survival of larvae, pupae and adult females the value of λ was reduced from 1.32 to 0.96. Management programs for CBB should be focused on the use of biological and cultural measures that are known to affect these three important targets.

Diet impacts on the biological aspects of pink bollworm, Pectinophora gossypiella (Lepidoptera: Gelechiidae) under controlled laboratory conditions.

BACKGROUND: Pink bollworm, Pectinophora gossypiella (Lepidoptera: Gelechiidae) is a native pest of Asia and preferably invasion on cotton (Gossypium hirsutum L.) crop as a commendatory host plant. Commercially, G. hirsutum is known as white gold and is an important cash crop all over the globe. Limited studies were published to focus on certain dietary compositions against different cotton pests. Therefore, the present study was undertaken in the laboratory under controlled conditions (temperature: 27 ± 2°C and relative humidity: 60 ± 10%) to determine the impact of three different treatment diets (wheat germ meal, okra, and chickpea) on the biological aspects (lifetime, developmental period) of P. gossypiella. RESULTS: Results revealed that the shortest larval time of P. gossypiella was observed on the okra feed diet while the longest period was recorded on the wheat germ diet. Meanwhile, the pupation delay was noted on the wheat germ diet. The dietary influence was also observed on adult stages of female and male P. gossypiella (43.00 and 37.50 days respectively) and compared with a standard diet (56.50 and 52.50 days respectively). Furthermore, larval weighed more on the okra and chickpea diet followed by the wheat germ diet, whereas highest pupal weight was observed on the standard diet followed by the chickpea diet and okra diet. CONCLUSION: Developmental parameters were significantly variant across all treatment diets, whereas the higher significant difference was reported on the okra diet. Therefore, the existing data of this study offers fruitful interventions for the future as a modified diet for large-scale and rapid mass production of P. gossypiella larvae.

Characterization and Developmental Expression Patterns of Four Hexamerin Genes in the Bumble Bee, Bombus terrestris (Hymenoptera: Apidae).

Hexamerins are members of the hemocyanin superfamily and play essential roles in providing amino acids and energy for the nonfeeding stages of insects. In this study, we cloned and analyzed the expression patterns of four hexamerin genes (hex 70a, hex 70b, hex 70c, and hex 110) at different worker development stages and queen diapause statuses in the bumble bee, Bombus terrestris. The results of this study showed that hex 110 has the longest open reading frame (ORF; 3,297 bp) compared to the ORFs of hex 70a (2,034 bp), hex 70b (2,067 bp), and hex 70c (2,055 bp). The putative translation product of Hex 70a, Hex 70b, Hex70c, and Hex 110 has 677, 688, 684, and 1,098aa with predicted molecular mass of 81.13, 79.69, 81.58, and 119 kDa. In the development stages of workers, the expression levels of hex 70a, hex 70b, and hex 70c increased gradually from the larval stage and exhibited high expression levels at the pink eyed and brown eyed pupae stage, whereas hex 110 exhibited the highest expression level at the larval period. Four hexamerin genes were highly expressed at the prediapause status of queen (P < 0.05), and compared to the eclosion queen, the lowest upregulation was 3.7-fold, and the highest upregulation was 1,742-fold. The expression levels of hex 70b, hex 70c, and hex 110 at diapause were significantly higher than those at postdiapause (P < 0.05). In conclusion, hexamerins may play important roles in queen diapause and metamorphosis of larval and pupal stages.

60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression.

Radiation is considered as a promising insect pest control strategy for minimizing postharvest yield losses. Among various techniques, irradiation is a method of choice as it induces lethal biochemical or molecular changes that cause a downstream cascade of abrupt physiological abnormalities at the cellular level. In this study, we evaluated the effect of 60Co-γ radiation on various developmental stages of Zeugodacus cucurbitae Coquillett and subsequent carry-over effects on the progeny. For this purpose, we treated eggs with 30- and 50-Gy radiation doses of 60Co-γ. We found that radiation significantly affected cellular antioxidants, insect morphology, and gene expression profiles. Our results indicate that in response to various doses of irradiation reactive oxygen species, catalase, peroxidase, and superoxide dismutase activities were increased along with a significant increase in the malondialdehyde (MDA) content. We observed higher mortality rates during the pupal stage of the insects that hatched from irradiated eggs (50 Gy). Furthermore, the life span of the adults was reduced in response to 50 Gy radiation. The negative effects carried over to the next generation were marked by significantly lower fecundity in the F1 generation of the irradiation groups as compared to control. The radiation induced morphological abnormalities at the pupal, as well as the adult, stages. Furthermore, variations in the gene expression following irradiation are discussed. Taken together, our results signify the utility of 60Co-γ radiation for fruit fly postharvest management.

Household-level risk factors for Aedes aegypti pupal density in Guayaquil, Ecuador.

BACKGROUND: Vector-borne diseases are a major cause of disease burden in Guayaquil, Ecuador, especially arboviruses spread by Aedes aegypti mosquitoes. Understanding which household characteristics and risk factors lead to higher Ae. aegypti densities and consequent disease risk can help inform and optimize vector control programs. METHODS: Cross-sectional entomological surveys were conducted in Guayaquil between 2013 and 2016, covering household demographics, municipal services, potential breeding containers, presence of Ae. aegypti larvae and pupae, and history of using mosquito control methods. A zero-truncated negative binomial regression model was fitted to data for estimating the household pupal index. An additional model assessed the factors of the most productive breeding sites across all of the households. RESULTS: Of surveyed households, 610 satisfied inclusion criteria. The final household-level model found that collection of large solid items (e.g., furniture and tires) and rainfall the week of and 2 weeks before collection were negatively correlated with average pupae per container, while bed canopy use, unemployment, container water volume, and the interaction between large solid collection and rainfall 2 weeks before the sampling event were positively correlated. Selection of these variables across other top candidate models with ∆AICc < 1 was robust, with the strongest effects from large solid collection and bed canopy use. The final container-level model explaining the characteristics of breeding sites found that contaminated water is positively correlated with Ae. aegypti pupae counts while breeding sites composed of car parts, furniture, sewerage parts, vases, were all negatively correlated. CONCLUSIONS: Having access to municipal services like bulky item pickup was effective at reducing mosquito proliferation in households. Association of bed canopy use with higher mosquito densities is unexpected, and may be a consequence of large local mosquito populations or due to limited use or effectiveness of other vector control methods. The impact of rainfall on mosquito density is multifaceted, as it may both create new habitat and "wash out" existing habitat. Providing services and social/technical interventions focused on monitoring and eliminating productive breeding sites is important for reducing aquatic-stage mosquito densities in households at risk for Ae. aegypti-transmitted diseases.

Interference Efficiency and Effects of Bacterium-mediated RNAi in the Fall Armyworm (Lepidoptera: Noctuidae).

RNAi is an effective tool for gene function analysis and a promising strategy to provide environmentally friendly control approaches for pathogens and pests. Recent studies support the utility of bacterium-mediated RNAi as a cost-effective method for gene function study and a suitable externally applied delivery mechanism for pest control. Here, we developed a bacterium-mediated RNAi system in Spodoptera frugiperda based on four target genes, specifically, Chitinase (Sf-CHI), Chitin synthase B (Sf-CHSB), Sugar transporter SWEET1 (Sf-ST), and Hemolin (Sf-HEM). RNAi conducted by feeding larvae with bacteria expressing dsRNAs of target genes or injecting pupae and adults with bacterially synthesized dsRNA induced silencing of target genes and resulted in significant negative effects on growth and survival of S. frugiperda. However, RNAi efficiency and effects were variable among different target genes and dsRNA delivery methods. Injection of pupae with dsCHI and dsCHSB induced a significant increase in wing malformation in adults, suggesting that precise regulation of chitin digestion and synthesis is crucial during wing formation. Injection of female moths with dsHEM resulted in lower mating, fecundity, and egg hatching, signifying a critical role of Sf-HEM in the process of egg production and/or embryo development. Our collective results demonstrate that bacterium-mediated RNAi presents an alternative technique for gene function study in S. frugiperda and a potentially effective strategy for control of this pest, and that Sf-CHI, Sf-CHSB, Sf-ST, and Sf-HEM encoding genes can be potent targets.

Neonicotinoids can cause arrested pupal ecdysis in Lepidoptera.

Recently, we reported a novel mode of action in monarch butterfly (Danaus plexippus) larvae exposed to neonicotinoid insecticides: arrest in pupal ecdysis following successful larval ecdysis. In this paper, we explore arrested pupal ecdysis in greater detail and propose adverse outcome pathways to explain how neonicotinoids cause this effect. Using imidacloprid as a model compound, we determined that final-instar monarchs, corn earworms (Helicoverpa zea), and wax moths (Galleria mellonella) showed high susceptibility to arrested pupal ecdysis while painted ladies (Vanessa cardui) and red admirals (Vanessa atalanta) showed low susceptibility. Fall armyworms (Spodoptera frugiperda) and European corn borers (Ostrinia nubilalis) were recalcitrant. All larvae with arrested ecdysis developed pupal cuticle, but with incomplete shedding of larval cuticle and unexpanded pupal appendages; corn earworm larvae successfully developed into adults with unexpanded appendages. Delayed initiation of pupal ecdysis was also observed with treated larvae. Imidacloprid exposure was required at least 26 h prior to pupal ecdysis to disrupt the molt. These observations suggest neonicotinoids may disrupt the function of crustacean cardioactive peptide (CCAP) neurons, either by directly acting on their nicotinic acetylcholine receptors or by acting on receptors of inhibitory neurons that regulate CCAP activity.

Pupal behavior emerges from unstructured muscle activity in response to neuromodulation in Drosophila.

Identifying neural substrates of behavior requires defining actions in terms that map onto brain activity. Brain and muscle activity naturally correlate via the output of motor neurons, but apart from simple movements it has been difficult to define behavior in terms of muscle contractions. By mapping the musculature of the pupal fruit fly and comprehensively imaging muscle activation at single-cell resolution, we here describe a multiphasic behavioral sequence in Drosophila. Our characterization identifies a previously undescribed behavioral phase and permits extraction of major movements by a convolutional neural network. We deconstruct movements into a syllabary of co-active muscles and identify specific syllables that are sensitive to neuromodulatory manipulations. We find that muscle activity shows considerable variability, with sequential increases in stereotypy dependent upon neuromodulation. Our work provides a platform for studying whole-animal behavior, quantifying its variability across multiple spatiotemporal scales, and analyzing its neuromodulatory regulation at cellular resolution.

How do we find out how the brain works? One way is to use imaging techniques to visualise an animal's brain in action as it performs simple behaviours: as the animal moves, parts of its brain light up under the microscope. For laboratory animals like fruit flies, which have relatively small brains, this lets us observe their brain activity right down to the level of individual brain cells. The brain directs movements via collective activity of the body's muscles. Our ability to track the activity of individual muscles is, however, more limited than our ability to observe single brain cells: even modern imaging technology still cannot monitor the activity of all the muscle cells in an animal's body as it moves about. Yet this is precisely the information that scientists need to fully understand how the brain generates behaviour. Fruit flies perform specific behaviours at certain stages of their life cycle. When the fly pupa begins to metamorphose into an adult insect, it performs a fixed sequence of movements involving a set number of muscles, which is called the pupal ecdysis sequence. This initial movement sequence and the rest of metamorphosis both occur within the confines of the pupal case, which is a small, hardened shell surrounding the whole animal. Elliott et al. set out to determine if the fruit fly pupa's ecdysis sequence could be used as a kind of model, to describe a simple behaviour at the level of individual muscles. Imaging experiments used fly pupae that were genetically engineered to produce an activity-dependent fluorescent protein in their muscle cells. Pupal cases were treated with a chemical to make them transparent, allowing easy observation of their visually 'labelled' muscles. This yielded a near-complete record of muscle activity during metamorphosis. Initially, individual muscles became active in small groups. The groups then synchronised with each other over the different regions of the pupa's body to form distinct movements, much as syllables join to form words. This synchronisation was key to progression through metamorphosis and was co-ordinated at each step by specialised nerve cells that produce or respond to specific hormones. These results reveal how the brain might direct muscle activity to produce movement patterns. In the future, Elliott et al. hope to compare data on muscle activity with comprehensive records of brain cell activity, to shed new light on how the brain, muscles, and other factors work together to control behaviour.

First Detections of Culiseta longiareolata (Diptera: Culicidae) in Belgium and the Netherlands.

Culiseta (Allotheobaldia) longiareolata (Macquart) (Diptera: Culicidae) is an ornithophilic mosquito species that occurs in the southern Palaearctic Region from the Azores to Central Asia, the Ethiopian Region, India, and Pakistan. Although it has a widespread distribution range, the species was only recently reported in Western and Central Europe. Between 2017 and 2020, larvae, pupae, and adults of Cs. longiareolata (n = 161) were found at 13 distinct locations in Belgium (n = 4) and The Netherlands (n = 9). Collected mosquitoes were morphologically identified and the identification was then validated by COI DNA barcoding. These are the first records of the species in the above-mentioned countries. The present results suggest that Cs. longiareolata could be increasing its distribution range in temperate regions, indicating a warming climate. As the species might be a potential vector of bird pathogens (e.g., West Nile virus), its spread in Western Europe is noteworthy.

The Pupal Parasitoid Trichopria drosophilae Is Attracted to the Same Yeast Volatiles as Its Adult Host.

There is increasing evidence that microorganisms, particularly fungi and bacteria, emit volatile compounds that mediate the foraging behaviour of insects and therefore have the potential to affect key ecological relationships. However, to what extent microbial volatiles affect the olfactory response of insects across different trophic levels remains unclear. Adult parasitoids use a variety of chemical stimuli to locate potential hosts, including those emitted by the host's habitat, the host itself, and microorganisms associated with the host. Given the great capacity of parasitoids to utilize and learn odours to increase foraging success, parasitoids of eggs, larvae, or pupae may respond to the same volatiles the adult stage of their hosts use when locating their resources, but compelling evidence is still scarce. In this study, using Saccharomyces cerevisiae we show that Trichopria drosophilae, a pupal parasitoid of Drosophila species, is attracted to the same yeast volatiles as their hosts in the adult stage, i.e. acetate esters. Parasitoids significantly preferred the odour of S. cerevisiae over the blank medium in a Y-tube olfactometer. Deletion of the yeast ATF1 gene, encoding a key acetate ester synthase, decreased attraction of T. drosophilae, while the addition of synthetic acetate esters to the fermentation medium restored parasitoid attraction. Bioassays with individual compounds revealed that the esters alone were not as attractive as the volatile blend of S. cerevisiae, suggesting that other volatile compounds also contribute to the attraction of T. drosophilae. Altogether, our results indicate that pupal parasitoids respond to the same volatiles as the adult stage of their hosts, which may aid them in locating oviposition sites.

Effect of Drosophila suzukii on Blueberry VOCs: Chemical Cues for a Pupal Parasitoid, Trichopria anastrephae.

Biocontrol agents such as parasitic wasps use long-range volatiles and host-associated cues from lower trophic levels to find their hosts. However, this chemical landscape may be altered by the invasion of exotic insect species. The spotted-wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), is a highly polyphagous fruit pest native to eastern Asia and recently arrived in South America. Our study aimed to characterize the effect of SWD attack on the volatile organic compounds (VOCs) of blueberries, a common host fruit, and to correlate these odor changes with the olfactory-mediated behavioral response of resident populations of Trichopria anastrephae parasitoids, here reported for the first time in Uruguay. Using fruit VOC chemical characterization followed by multivariate analyses of the odor blends of blueberries attacked by SWD, we showed that the development of SWD immature stages inside the fruit generates a different odor profile to that from control fruits (physically damaged and free of damage). These differences can be explained by the diversity, frequency, and amounts of fruit VOCs. The behavioral response of T. anastrephae in Y-tube bioassays showed that female wasps were significantly attracted to volatiles from SWD-attacked blueberries when tested against both clean air and undamaged blueberries. Therefore, T. anastrephae females can use chemical cues from SWD-infested fruits, which may lead to a successful location of their insect host. Since resident parasitoids are able to locate this novel potential host, biological control programs using local populations may be plausible as a strategy for control of SWD.