It is imperative to determine the expression patterns and functions of genes essential for reproduction in mosquito vectors, such as , to optimize vector control strategies, such as the sterile insect technique (SIT). The reproductive organs of mosquitoes are responsible for various processes, including spermatogenesis, seminal fluid secretion, and female insemination during mating. Using Illumina RNA-seq and qRT-PCR validation, this study aimed to identify and categorize genes associated with the male accessory glands, as well as potentially other reproductive structures, of . Analysis of young and old male accessory gland tissues revealed 345 and 26 upregulated transcripts, respectively. Further qRT-PCR analysis showed that six genes were highly upregulated in old male accessory gland (MAG) tissue, including , , and . Additionally, 17 genes were found to be highly upregulated in young MAG tissue with some of them speculated to have reproductive functions; among the notably upregulated genes were those encoding , , and two . The function of (CPIJ003539) has been validated in male through in-cage insemination and fertility assays, revealing that females mated with knockdown males had impaired sperm storage and egg production abilities. These findings provide promising prospects for the development of targeted vector control strategies for mosquitoes to achieve more successful outcomes concerning efficient reduction of the pest populations.

Caloric intake can greatly affect many aspects of an organism's life. A deficiency of calories can lead to stress resulting in decreased fecundity, insufficient calories to maintain tissues and increased lifespan. Conversely, increasing caloric density increases fecundity and decreases lifespan. Despite decades of work exploring food quality and quantity on physiology in the model species Meigan 1830 (Diptera: Drosophilidae) and the melanogaster group in general, relatively little work explores the physiological responses to diet manipulation in other species, like the obscura species group. Here, we looked at the effects of five different caloric densities (0.5×, 0.75×, 1.0×, 1.5× and 3.0×) on food intake, body weight, body fat, fecundity and longevity in Frolova & Astaurov, 1929 (Diptera: Drosophilidae). Comparing longevity and fecundity across diets, we found that heavy caloric concentration (3.0×) decreases lifespan and that calorie restriction (0.5× and 0.75×) led to significant decreases in fecundity and body weight. However, calorie concentration did not significantly increase body fat. By expanding our understanding of the physiological responses to diet stress to , we establish the framework for comparative work across species. With this information, we can then identify which physiological responses to diet manipulation might be most conserved and comparable across species.

Organisms subjected to periodic nutrient limitation early in life exhibit improvements in aspects of survival, including resistance to some environmental stressors. Recent findings indicate that forms of periodic fasting such as intermittent fasting and time restricted feeding can improve starvation resistance. However, it remains unclear to what extent this survival improvement persists across different genetic backgrounds. In this study, we examine fasting-induced starvation resistance across a broad survey of wild-derived lineages and document genetic variation within this trait. We adopt a standard dietary intervention and show improvement to starvation resistance within a common laboratory lineage, replicating previous results. Next, we examine fasting-induced starvation resistance across isofemale lines collected across latitudes and in different seasons, and among inbred lines derived from flies collected on different continents. We discover genetic variation of fasting-induced starvation resistance, and show that fasting improved starvation resistance as often as it worsened starvation resistance. Fasted flies generally showed reduced fat concentration, and their starvation survival varied with sex, season of collection, and geographic origin. While specific lineages common to the laboratory can show a specific fasting-induced phenotype, we show that this result is not consistent across genetic backgrounds, reinforcing the idea that phenotypes observed in historic laboratory strains may not be conserved across a species.

Lethal time (LTime) and lethal temp (LTemp) are commonly used laboratory indices of arthropod cold tolerance, with the former often being employed to predict winter survival in the field. In the present study, we compare the cold tolerance of different life-history stages (nondiapausing and diapausing females, as well as males and juveniles) of a major agricultural pest: the two-spot spider mite Koch (Acarina: Tetranychidae). Diapausing females from European populations of this species are shown to be freeze avoiding, supercooling to -23.6 ± 0.37  °C and with an LTemp of -23.2 °C. However, nondiapausing females [supercooling point (SCP) -19.1 ± 0.49 °C, LTemp -14.32 °C], males (SCP -21.27 ± 0.52  °C, LTemp -16 °C) and juveniles (SCP -25.34 ± 0.29 °C, LTemp -18.3 °C) are subclassified as strongly chill tolerant juveniles. LTime is 148.3 days for non-acclimated diapausing females, whereas nondiapausing females, males and juveniles reach 50% mortality by 21.7 days. When individuals are acclimated at 10 °C for a period of 7 days, no effect is found. Cold tolerance is suggested to be a major contributor to the successful spread of T. urticae across temperate countries, although it is dependent on a diapause trait, suggesting a potential target for control. Winter field trial data from diapausing females indicate that LTime is a reliable indicator of winter survival even within diapause, supporting the use of these indices as a valuable component within environmental niche models for the prediction of future pest invasions.

Diapause, a strategy to endure unfavourable conditions (e.g. cold winters) is commonly found in ectothermic organisms and is characterized by an arrest of development and reproduction, a reduction of metabolic rate, and an increased resistance to adversity. Diapause, in addition to adaptations for surviving low winter temperatures, significantly influences phenology, voltinism and ultimately population growth. We review the literature on diapause and overwintering behaviour of two bark beetle species that affect spruce-dominated forests in the northern hemisphere, and describe and compare how these strategies can influence population dynamics. The European spruce bark beetle Ips typographus (L.) (Coleoptera, Curculionidae) is the most important forest pest of Norway spruce in Europe. It enters an adult reproductive diapause that might be either facultative or obligate. Obligate diapausing beetles are considered strictly univoltine, entering this dormancy type regardless of environmental cues. Facultative diapausing individuals enter diapause induced by photoperiod, modified by temperature, thus being potentially multivoltine. The spruce beetle Dendroctonus rufipennis (Kirby) (Coleoptera: Curculionidae) infests all spruce species in its natural range in North America. A facultative prepupal diapause is averted by relatively warm temperatures, resulting in a univoltine life cycle, whereas cool temperatures induce prepupal diapause leading to a semivoltine cycle. An adult obligate diapause in D. rufipennis could limit bi- or multivoltinism. We discuss and compare the influence of diapause and overwinter survival on voltinism and population dynamics of these two species in a changing climate and provide an outlook on future research.

Many of the characteristics associated with mammalian sleep are also observed in Drosophila melanogaster Meigen, making the fruit fly a powerful model organism for studying the genetics of this important process. Included among the similarities is the presence of sexual dimorphic sleep patterns, which, in flies, are manifested as increased mid-day sleep ('siesta') in males compared with females. In the present study, targeted mis-expression of the genes transformer (tra) and tra2 is used to either feminize or masculinize specific neural and non-neural tissues in the fly. Feminization of male D. melanogaster using three different GAL4 drivers that are expressed in the mushroom bodies induces a female-like reduced siesta, whereas the masculinization of females using these drivers triggers the male-like increased siesta. A similar reversal of sex-specific sleep is also observed by mis-expressing tra in the fat body, which is a key tissue in energy metabolism and hormone secretion. In addition, the daily expression levels of takeout, an important circadian clock output gene, are sexually dimorphic. Taken together, these experiments suggest that sleep sexual dimorphism in D. melanogaster is driven by multiple neural and non-neural circuits, within and outside the brain.

Insects inhabiting the temperate zones measure seasonal changes in day or night length to enter the overwintering diapause. Diapause induction occurs after the duration of the night exceeds a critical night length (CNL). Our understanding of the time measurement mechanisms is continuously evolving subsequent to Bünning's proposal that circadian systems play the clock role in photoperiodic time measurement (Bünning, 1936). Initially, the photoperiodic clocks were considered to be either based on circadian oscillators or on simple hour-glasses, depending on 'positive' or 'negative' responses in Nanda-Hamner and Bünsow experiments (Nanda & Hammer, 1958; Bünsow, 1960). However, there are also species whose responses can be regarded as neither 'positive', nor as 'negative', such as the Northern Drosophila species Drosophila ezoana, which is investigated in the present study. In addition, modelling efforts show that the 'positive' and 'negative' Nanda-Hamner responses can also be provoked by circadian oscillators that are damped to different degrees: animals with highly sustained circadian clocks will respond 'positive' and those with heavily damped circadian clocks will respond 'negative'. In the present study, an experimental assay is proposed that characterizes the photoperiodic oscillators by determining the effects of non-24-h light/dark cycles (T-cycles) on critical night length. It is predicted that there is (i) a change in the critical night length as a function of T-cycle period in sustained-oscillator-based clocks and (ii) a fixed night-length measurement (i.e. no change in critical night length) in damped-oscillator-based clocks. Drosophila ezoana flies show a critical night length of approximately 7 h irrespective of T-cycle period, suggesting a damped-oscillator-based photoperiodic clock. The conclusion is strengthened by activity recordings revealing that the activity rhythm of D. ezoana flies also dampens in constant darkness.

Circadian coordination of metabolism, physiology, and behaviour is found in all living kingdoms. Clock genes are transcriptional regulators, and their rhythmic activities generate daily rhythms in clock-controlled genes which result in cellular and organismal rhythms. Insects provide numerous examples of rhythms in behaviour and reproduction, but less is known about control of metabolic processes by circadian clocks in insects. Recent data suggest that several pathways involved in protecting cells from oxidative stress may be modulated by the circadian system, including genes involved in glutathione (GSH) biosynthesis. Specifically, rhythmic expression of the gene encoding the catalytic subunit () of the rate-limiting GSH biosynthetic enzyme was detected in heads. The aim of this study was to determine which clocks in the fly multi-oscillatory circadian system are responsible for rhythms. Genetic disruption of tissue-specific clocks in revealed that transcriptional rhythms in mRNA levels occur independently of the central pacemaker neurons, because these rhythms persisted in heads of behaviourally arrhythmic flies with a disabled central clock but intact peripheral clocks. Disrupting the clock specifically in glial cells abolished rhythmic expression of , suggesting that glia play an important role in transcriptional regulation, which may contribute to maintaining homeostasis in the fly nervous system.

Hypotheses exploring the influence of dietary conditions on the life history trade-off between survival and reproductive success are extensively tested in female insects, but are rarely explored in males. Here, the impact of dietary quality and female access on age-specific reproduction and survival of male Mexican fruit flies, Loew (Diptera: Tephritidae), are examined. There is a clear cost of female access for males with access to dietary protein, measurable as a decrease in life expectancy, which is further influenced by the age when females are introduced. A protein deficient diet reduces the lifespan benefit of virginity and masks the detrimental effect of female access on male life expectancy. Dietary protein is not necessary for reproductive success, but access to protein at eclosion improves the lifetime reproductive success of males compared to when it is delayed. Overall, reproductive success diminishes as the male flies age, regardless of the dietary conditions, providing evidence for reproductive senescence in males. Delaying the males' access to a protein source fails to influence the negative effect of age on reproductive ability. Because age specific reproductive rates decline with age, regardless of diet, male fitness does not benefit from lifespan extension. Therefore, males can be expected to allocate available resources towards reproductive effort in favour of extended lifespan, regardless of mate and protein availability.

A large-arena bioassay is used to examine sex differences in spatiotemporal patterns of bed bug L. behavioural responses to either a human host or CO gas. After release in the centre of the arena, 90% of newly-fed bed bugs move to hiding places in the corners within 24 h. They require 3 days to settle down completely in the arena, with generally low activity levels and the absence of responses to human stimuli for 5 days. After 8-9 days, persistent responses can be recorded. Sex differences are observed, in which females are more active during establishment, respond faster after feeding, expose themselves more than males during the daytime, and respond more strongly to the host signal. The number of bed bugs that rest in harbourages is found to vary significantly according to light setting and sex. Both sexes stay inside harbourages more in daylight compared with night, and males hide more than females during the daytime but not during the night. The spatial distribution of the bed bugs is also found to change with the presence of CO, and peak aggregation around the odour source is observed after 24 min. Both male and female bed bugs move from hiding places or the border of the arena toward the centre where CO is released. Peak responses are always highest during the night. Bed bug behaviour and behaviour-regulating features are discussed in the context of control methods.

The lower and upper thermal activity thresholds of adult and larval von Heyden (Acari: Erythraeidae) are compared with those of its prey Koch (Acari: Tetranychidae). Adult female retain ambulatory function (CT) and movement of appendages (chill coma) at significantly lower temperatures (5.9 and -2.1 °C, respectively) than those of larval (8.1 and -1.7 °C) and (10.6 and 10.3 °C). There is no significant difference between the temperature at which adult and larval and cease walking as the temperature is raised (CT) (46.7, 46.3 and 47.3 °C, respectively). However, both life stages of cease movement (heat coma) below the upper locomotory limits of (46.8, 46.7 and 48.7 °C, respectively). Adult have significantly faster walking speeds than larvae and across a range of temperatures. The lower thermal activity threshold data indicate that would make an effective biological control agent in temperate climates; however, the extent of the low temperature tolerances of the species suggests the potential to establish in a northern European climate.

(Diptera: Culicidae) has adapted to urban environments; the urbanisation process provides suitable habitats for this disease vector subsequently increasing the probability of the transmission of pathogens in high-density environments. Urban environments provide metal stressed larval habitats. However, little is known about the physiological cost of metal stress or how this might affect the performance of this mosquito species. This study aims to characterise the sub-lethal physiological consequences of metal stress in . Various parameters of mosquito physiology under larval metal stress are assessed including larval metallothionein expression and the effects of larval metal stress on adult performance and their progeny. Results show that environmentally relevant larval metal stress compromises larval and adult development and performance, and results in larval metal tolerance along with an increase in lipid consumption. These performance costs are coupled to a dramatic increase in metallothionein expression in the midgut. Metal stress results in lowered adult body mass and neutral storage lipids at emergence, starvation tolerance, fecundity and starvation tolerance of offspring compared to non-metal stressed individuals. Ironically, larval metal stress results in increased adult longevity. Together, these findings indicate that even low levels of environmentally relevant larval metal stress have considerable physiological consequences for this important disease vector.

In a wind-tunnel study, the upwind flight and source location of female to plumes of carbon dioxide (CO) gas and odour from human feet is tested. Both odour sources are presented singly and in combination. Flight upwind along the plumes is evident for both CO and odour from human feet when the odours are presented alone. Likewise, both odour sources are located by more than 70% of mosquitoes in less than 3 min. When both CO and odour from human feet are presented simultaneously in two different choice tests (with plumes superimposed or with plumes separated), there is no evidence that females orientate along the plume of CO and only a few mosquitoes locate its source. Rather, the foot odour plume is navigated and the source of foot odour is located by over 80% of female . When a female is presented a plume of CO within a broad plume of human foot odour of relatively low concentration, the source of CO is not located; instead, flight is upwind in the diffuse plume of foot odour. Although upwind flight by at long range is presumably induced by CO and the threshold of response to skin odours is lowered, our findings suggest that once females have arrived near a prospective human host, upwind orientation and landing are largely governed by the suite of odours from a human foot, while orientation is no longer influenced by CO.

Diapause has long been recognized as a crucial ecological adaptation to spatio-temporal environmental variation. More recently, rapid evolution of the diapause response has been implicated in response to contemporary global warming and during the range expansion of invasive species. Although the molecular regulation of diapause remains largely unresolved, rapidly emerging next-generation sequencing (NGS) technologies provide exciting opportunities to address this longstanding question. Herein, a new assembly from life-history stages relevant to diapause in the Asian tiger mosquito, (Skuse) is presented, along with unique methods for the analysis of NGS data and transcriptome assembly. A digital normalization procedure that significantly reduces computational resources required for transcriptome assembly is evaluated. Additionally, a method for protein reference-based and genomic reference-based merged assembly of 454 and Illumina reads is described. Finally, a gene ontology analysis is presented, which creates a platform to identify physiological processes associated with diapause. Taken together, these methods provide valuable tools for analyzing the transcriptional underpinnings of many complex phenotypes, including diapause, and provide a basis for determining the molecular regulation of diapause in .

The 'choice' of whether to enter diapause or to develop directly has profound effects on the life histories of insects, and may thus have cascading consequences such as seasonal morphs and other less obvious forms of seasonal plasticity. Present knowledge of the control of diapause and seasonal morphs at the physiological and molecular levels is briefly reviewed. Examples, mainly derived from personal research (primarily on butterflies), are given as a starting point with the aim of outlining areas of research that are still poorly understood. These include: the role of the direction of change in photoperiod; the role of factors such as temperature and diet in modifying the photoperiodic responses; and the role of sex, parental effects and sex linkage on photoperiodic control. More generally, there is still a limited understanding of how external cues and physiological pathways regulating various traits are interconnected via gene action to form a co-adapted complete phenotype that is adaptive in the wild despite environmental fluctuation and change.

The reproductive ability of female tephritids can be limited and prevented by denying access to host plants and restricting the dietary precursors of vitellogenesis. The mechanisms underlying the delayed egg production in each case are initiated by different physiological processes that are anticipated to have dissimilar effects on lifespan and reproductive ability later in life. The egg laying abilities of laboratory reared females of the Mediterranean fruit fly ( Wiedmann) and melon fly ( Coquillett) from Hawaii are delayed or suppressed by limiting access to host fruits and dietary protein. In each case, this is expected to prevent the loss of lifespan associated with reproduction until protein or hosts are introduced. Two trends are observed in each species: Firstly, access to protein at eclosion leads to a greater probability of survival and higher reproductive ability than if it is delayed, and secondly, that delayed host access reduces lifetime reproductive ability without improving life expectancy. When host access and protein availability are delayed, the rate of reproductive senescence is reduced in the medfly, whereas the rate of reproductive senescence is generally increased in the melon fly. Overall, delaying reproduction lowers the fitness of females by constraining their fecundity for the remainder of the lifespan without extending the lifespan.

The orientation to and landing on a source of human odour by female Say (Diptera: Culicidae) is observed in a wind tunnel without an airflow or with a laminar airflow of 0.2 m s. Odours from human feet are collected by 'wearing' clean glass beads inside a stocking and presenting beads in a Petri dish in a wind tunnel. Mosquitoes are activated by brief exposure to a 1 L min jet of 4% CO positioned 10 cm from the release cage. In moving air at 0.2 m s, a mean of 3.45 ± 0.49 landings are observed in 10 min trials (5 mosquitoes per trial), whereas 6.50 ± 0.96 landings are recorded in still air. Furthermore, 1.45 ± 0.31mosquitoes are recorded on beads at any one time in moving air (a measure of individuals landing versus one landing multiple times) compared to 3.10 ± 0.31 in still air. Upwind flight to beads in moving air is demonstrated by angular headings of flight immediately prior to landing, whereas approaches to beads in still air are oriented randomly. The mean latency until first landing is 226.7 ± 17.98 s in moving air compared to 122.5 ± 24.18 in still air. Strategies used to locate a prospective host at close range in still air are considered.

Once captured by the antenna, 11- vaccenyl acetate (cVA) binds to an extracellular binding protein called LUSH that undergoes a conformational shift upon cVA binding. The stable LUSH-cVA complex is the activating ligand for pheromone receptors present on the dendrites of the aT1 neurones, comprising the only neurones that detect cVA pheromone. This mechanism explains the single molecule sensitivity of insect pheromone detection systems. The receptor that recognizes activated LUSH consists of a complex of several proteins, including Or67d, a member of the tuning odourant receptor family, Orco, a co-receptor ion channel, and SNMP, a CD36 homologue that may be an inhibitory subunit. In addition, genetic screens and reconstitution experiments reveal additional factors that are important for pheromone detection. Identification and functional dissection of these factors in Meigen should permit the identification of homologous factors in pathogenic insects and agricultural pests, which, in turn, may be viable candidates for novel classes of compounds to control populations of target insect species without impacting beneficial species.

Despite many decades of multilateral global efforts, a significant portion of the world population continues to be plagued with one or more mosquito-vectored diseases. These include malaria and filariasis as well as numerous arboviral-associated illnesses including Dengue and Yellow fevers. The dynamics of disease transmission by mosquitoes is complex, and involves both vector competence and vectorial capacity. One area of intensive effort is the study of chemosensory-driven behaviours in the malaria vector mosquito Giles, the modulation of which are likely to provide opportunities for disease reduction. In this context recent studies have characterized a large divergent family of odorant receptors (AgORs) that play critical roles in olfactory signal transduction. This work has facilitated high-throughput, cell-based calcium mobilization screens of AgOR-expressing HEK cells that have identified a large number of conventional AgOR ligands, as well as the first non-conventional Orco (olfactory receptor co-receptor) family agonist. As such, ligand-mediated modulation serves as a proof-of-concept demonstration that AgORs represent viable targets for high-throughput screening and for the eventual development of behaviour-modifying olfactory compounds. Such attractants or repellents could foster malaria reduction programmes.

The pupal parasitoid Pimpla turionellae (L.) uses self-produced vibrations transmitted on the plant substrate, so-called vibrational sounding, to locate immobile concealed pupal hosts. The wasps are able to use vibrational sounding reliably over a broad range of ambient temperatures and even show an increased signal frequency and intensity at low temperatures. The present study investigates how control of body temperature in the wasps by endothermic mechanisms may facilitate host location under changing thermal environments. Insect body temperature is measured with real-time IR thermography on plant-stem models at temperature treatments of 10, 18, 26 and 30 °C, whereas behaviour is recorded with respect to vibrational host location. The results reveal a low-level endothermy that likely interferes with vibrational sound production because it occurs only in nonsearching females. At the lowest temperature of 10 °C, the thoracic temperature is 1.15 °C warmer than the ambient surface temperature whereas, at the high temperatures of 26 and 30 ° C, the wasps cool down their thorax by 0.29 and 0.47 °C, respectively, and their head by 0.45 and 0.61 °C below ambient surface temperature. By contrast, regardless of ambient temperature, searching females always have a slightly elevated body temperature of at most 0.30 °C above the ambient surface temperature. Behavioural observations indicate that searching females interrupt host location more frequently at suboptimal temperatures, presumably due to the requirements of thermoregulation. It is assumed that both mechanisms, producing vibrations for host location and low-level endothermy, are located in the thorax. Endothermy by thoracic muscle work probably disturbs signal structure of vibrational sounding, so the processes cannot be used at the same time.

Tsetse flies, Glossina pallidipes (Diptera: Glossinidae) are said to have strong dispersal tendencies. Gene flow among these populations is estimated to be the theoretical equivalent of no more than one or two reproducing flies per generation, thereby raising the hypothesis of local regimes of natural selection. Flies were sampled from four environmentally diverse locations in Kenya to determine whether populations are homogeneous in desiccation tolerance and cuticular lipids. Cuticular hydrocarbon fractions known to act as sex pheromones do not differ among populations, thereby eliminating sexual selection as an isolating mechanism. Cuticular lipid quantities vary among populations and are not correlated with prevailing temperatures, humidities, and normalized density vegetation indices. Females demonstrate a stronger correlation than males between cuticular lipid mass and body weight. Desiccation rates also vary among populations, but are not correlated with the amounts of cuticular lipid. Chemical analysis of cuticular hydrocarbons by gas chromatography-mass spectroscopy shows that one of the four populations has more 11,15- and 11,21-dimethyl-31 hydrocarbon on females. These results are discussed in the context of population differences and estimates of gene flow.