An association between Dnmt1 and Wnt in the production of oocytes in the whitefly Bemisia tabaci.

The function of DNA methylation in insects and the DNA methyltransferase (Dnmt) genes that influence methylation remains uncertain. We used RNA interference to reduce the gene expression of Dnmt1 within the whitefly Bemisia tabaci (Hemiptera:Aleyrodidae; Gennadius), a hemipteran species that relies on Dnmt1 for proper gametogenesis. We then used RNA-seq to test an a priori hypothesis that meiosis-related genetic pathways would be perturbed. We generally did not find an overall effect on meiosis-related pathways. However, we found that genes in the Wnt pathway, genes associated with the entry into meiosis in vertebrates, were differentially expressed. Our results are consistent with Dnmt1 knockdown influencing specific pathways and not causing general transcriptional response. This is a finding that is also seen with other insect species. We also characterised the methylome of B. tabaci and assessed the influence of Dnmt1 knockdown on cytosine methylation. This species has methylome characteristics comparable to other hemipterans regarding overall level, enrichment within gene bodies, and a bimodal distribution of methylated/non-methylated genes. Very little differential methylation was observed, and difference in methylation were not associated with differences in gene expression. The effect on Wnt presents an interesting new candidate pathway for future studies.

takeout gene expression is associated with temporal kin recognition.

A key component of parental care is avoiding killing and eating one's own offspring. Many organisms commit infanticide but switch to parental care when their own offspring are expected, known as temporal kin recognition. It is unclear why such types of indirect kin recognition are so common across taxa. One possibility is that temporal kin recognition may evolve through alteration of simple mechanisms, such as co-opting mechanisms that influence the regulation of timing and feeding in other contexts. Here, we determine whether takeout, a gene implicated in coordinating feeding, influences temporal kin recognition in Nicrophorus orbicollis. We found that takeout expression was not associated with non-parental feeding changes resulting from hunger, or a general transition to the full parental care repertoire. However, beetles that accepted and provided care to their offspring had a higher takeout expression than beetles that committed infanticide. Together, these data support the idea that the evolution of temporal kin recognition may be enabled by co-option of mechanisms that integrate feeding behaviour in other contexts.

The role of Dmnt1 during spermatogenesis of the insect Oncopeltus fasciatus.

BACKGROUND: The function of DNA methyltransferase genes of insects is a puzzle, because an association between gene expression and methylation is not universal for insects. If the genes normally involved in cytosine methylation are not influencing gene expression, what might be their role? We previously demonstrated that gametogenesis of Oncopeltus fasciatus is interrupted at meiosis following knockdown of DNA methyltransferase 1 (Dnmt1) and this is unrelated to changes in levels of cytosine methylation. Here, using transcriptomics, we tested the hypothesis that Dmnt1 is a part of the meiotic gene pathway. Testes, which almost exclusively contain gametes at varying stages of development, were sampled at 7 days and 14 days following knockdown of Dmnt1 using RNAi. RESULTS: Using microscopy, we found actively dividing spermatocysts were reduced at both timepoints. However, as with other studies, we saw Dnmt1 knockdown resulted in condensed nuclei after mitosis-meiosis transition, and then cellular arrest. We found limited support for a functional role for Dnmt1 in our predicted cell cycle and meiotic pathways. An examination of a priori Gene Ontology terms showed no enrichment for meiosis. We then used the full data set to reveal further candidate pathways influenced by Dnmt1 for further hypotheses. Very few genes were differentially expressed at 7 days, but nearly half of all transcribed genes were differentially expressed at 14 days. We found no strong candidate pathways for how Dnmt1 knockdown was achieving its effect through Gene Ontology term overrepresentation analysis. CONCLUSIONS: We, therefore, suggest that Dmnt1 plays a role in chromosome dynamics based on our observations of condensed nuclei and cellular arrest with no specific molecular pathways disrupted.

Insect homolog of oxytocin/vasopressin associated with parenting of males but not females in a subsocial beetle.

Parental care is thought to evolve through modification of behavioral precursors, which predicts that mechanistic changes occur in the genes underlying those traits. The duplicated gene system of oxytocin/vasopressin has been broadly co-opted across vertebrates to influence parenting, from a preduplication ancestral role in water balance. It remains unclear whether co-option of these genes for parenting is limited to vertebrates. Here, we experimentally tested for associations between inotocin gene expression and water balance, parental acceptance of offspring, and active parenting in the subsocial beetle Nicrophorus orbicollis, to test whether this single-copy homolog of the oxytocin/vasopressin system has similarly been co-opted for parental care in a species with elaborate parenting. As expected, inotocin was associated with water balance in both sexes. Inotocin expression increased around sexual maturation in both males and females, although more clearly in males. Finally, inotocin expression was not associated with acceptance of larvae, but was associated with a transition to male but not female parenting. Moreover, level of offspring provisioning behavior and gene expression were positively correlated in males but uncorrelated in females. Our results suggest a broad co-option of this system for parenting that may have existed prior to gene duplication.

The role of Dnmt1 in oocyte development.

The whitefly Bemisia tabaci is a globally important crop pest that is difficult to manage through current commercially available methods. While RNA interference (RNAi) is a promising strategy for managing this pest, effective target genes remain unclear. We suggest DNA methyltransferase 1 (Dnmt1) as a potential target gene due to its effect on fecundity in females in other taxa of insects. We investigated the role of Dnmt1 in B. tabaci using RNAi and immunohistochemistry to confirm its potential conserved function in insect reproduction, which will define its usefulness as a target gene. Using RNAi to downregulate Dnmt1 in female B. tabaci, we show that Dnmt1 indeed has a conserved role in reproduction, as knockdown interfered with oocyte development. Females in which Dnmt1 was knocked down had greatly reduced fecundity and fertility; this supports Dnmt1 as a suitable target gene for RNAi-mediated pest management of B. tabaci.

Ovarian apoptosis is regulated by carbohydrate intake but not by protein intake in speckled cockroaches.

When the likelihood of reproducing successfully is low, any prior investment in developing oocytes may be wasted. One means of recouping this investment is oosorption - where ova are absorbed and resources salvaged so they can be re-allocated to other traits. Food-limited female speckled cockroaches (Nauphoeta cinerea) appear to use this strategy. However, it is unclear if total food intake or the availability of specific nutrients induces this process. Here, we used the geometric framework of nutrition to determine how protein, carbohydrate and energy intake affect levels of ovarian apoptosis and necrosis (controlled versus uncontrolled cell death) in the terminal oocytes of female N. cinerea. We then compare the effects of nutrient intake on apoptosis (a key step towards oosorption) and offspring production to better understand the relationship between diet, apoptosis and female fitness. We found that even when food was abundant, females experienced high levels of apoptosis if their diet lacked carbohydrate. Necrosis was reduced when energy intake was high, but largely irrespective of nutrient ratio. Offspring production peaked on a low protein, high carbohydrate nutrient ratio (1P:7.96C), similar to that which minimized apoptosis (1P:7.34C) but not in the region of nutrient space that minimized necrosis. Thus, females consuming an ideal nutrient blend for reproduction can invest heavily in their current brood without needing to salvage nutrients from developing ova. However, offspring production was more dependent on carbohydrate consumption than apoptosis was, suggesting that the importance of carbohydrate in reproduction goes beyond regulating oosorption. This reliance on carbohydrate for female reproduction may reflect the unusual reproductive and nutritional physiology of speckled cockroaches; attributes that make this species an exciting model for understanding how diet regulates reproduction.

High-quality host plant diets partially rescue female fecundity from a poor early start.

Nutrition is a dynamic environmental factor and compensatory growth may help animals handle seasonal fluctuations in their diets. Yet, how the dynamic changes in nutrition affect female reproduction is understudied. We took advantage of a specialist insect herbivore, Narnia femorata Stål (Hemiptera: Coreidae), that feeds and reproduces on cactus across three seasons. We first examined how cactus quality can affect female reproductive success. Then, we investigated the extent to which reproductive success can be improved by a switch in diet quality at adulthood. We placed N. femorata juveniles onto prickly pear cactus pads with early-season (low-quality) or late-season (high-quality) fruit and tracked survivorship and development time. A subset of the females raised on low-quality diets were provided with an improved adult diet to simulate a seasonal change in diet. Adult female survival and egg production were tracked over time. All fitness-related traits were lower for females fed low-quality diets compared with females fed high-quality diets. However, when females had access to an improved adult diet, egg production was partially rescued. These findings show that a seasonal improvement in diet can enhance reproduction, but juvenile nutrition still has lasting effects that females cannot overcome.

The trade-off between investment in weapons and fertility is mediated through spermatogenesis in the leaf-footed cactus bug Narnia femorata.

Males have the ability to compete for fertilizations through both precopulatory and postcopulatory intrasexual competition. Precopulatory competition has selected for large weapons and other adaptations to maximize access to females and mating opportunities, while postcopulatory competition has resulted in ejaculate adaptations to maximize fertilization success. Negative associations between these strategies support the hypothesis that there is a trade-off between success at pre- and postcopulatory mating success. Recently, this trade-off has been demonstrated with experimental manipulation. Males of the leaf-footed cactus bug Narnia femorata use hind limbs as the primary weapon in male-male competition. However, males can drop a hind limb to avoid entrapment. When this autotomy occurs during development, they invest instead in large testes. While evolutionary outcomes of the trade-offs between pre- and postcopulatory strategies have been identified, less work has been done to identify proximate mechanisms by which the trade-off might occur, perhaps because the systems in which the trade-offs have been investigated are not ones that have the molecular tools required for exploring mechanism. Here, we applied knowledge from a related model species for which we have developmental knowledge and molecular tools, the milkweed bug Oncopeltus fasciatus, to investigate the proximate mechanism by which autotomized N. femorata males developed larger testes. Autotomized males had evidence of a higher rate of transit amplification divisions in the spermatogonia, which would result more spermatocytes and thus in greater sperm numbers. Identification of mechanisms underlying a trade-off can help our understanding of the direction and constraints on evolutionary trajectories and thus the evolutionary potential under multiple forms of selection.

The essential role of Dnmt1 in gametogenesis in the large milkweed bug Oncopeltus fasciatus.

Given the importance of DNA methylation in protection of the genome against transposable elements and transcriptional regulation in other taxonomic groups, the diversity in both levels and patterns of DNA methylation in the insects raises questions about its function and evolution. We show that the maintenance DNA methyltransferase, DNMT1, affects meiosis and is essential to fertility in milkweed bugs, Oncopeltus fasciatus, while DNA methylation is not required in somatic cells. Our results support the hypothesis that Dnmt1 is required for the transition of germ cells to gametes in O. fasciatus and that this function is conserved in male and female gametogenesis. They further suggest that DNMT1 has a function independent of DNA methylation in germ cells. Our results raise thequestion as to how a gene that is so critical to fitness across multiple insect species is able to diverge widely across the insect tree of life.

Whitefly Endosymbionts: Biology, Evolution, and Plant Virus Interactions.

Whiteflies (Hemiptera: Aleyrodidae) are sap-feeding global agricultural pests. These piercing-sucking insects have coevolved with intracellular endosymbiotic bacteria that help to supplement their nutrient-poor plant sap diets with essential amino acids and carotenoids. These obligate, primary endosymbionts have been incorporated into specialized organs called bacteriomes where they sometimes coexist with facultative, secondary endosymbionts. All whitefly species harbor the primary endosymbiont Candidatus Portiera aleyrodidarum and have a variable number of secondary endosymbionts. The secondary endosymbiont complement harbored by the cryptic whitefly species Bemisia tabaci is particularly complex with various assemblages of seven different genera identified to date. In this review, we discuss whitefly associated primary and secondary endosymbionts. We focus on those associated with the notorious B. tabaci species complex with emphasis on their biological characteristics and diversity. We also discuss their interactions with phytopathogenic begomoviruses (family Geminiviridae), which are transmitted exclusively by B. tabaci in a persistent-circulative manner. Unraveling the complex interactions of these endosymbionts with their insect hosts and plant viruses could lead to advancements in whitefly and whitefly transmitted virus management.

Debugging: Strategies and Considerations for Efficient RNAi-Mediated Control of the Whitefly Bemisia tabaci.

The whitefly Bemisia tabaci is a globally important pest that is difficult to control through insecticides, transgenic crops, and natural enemies. Post-transcriptional gene silencing through RNA interference (RNAi) has shown potential as a pest management strategy against B. tabaci. While genomic data and other resources are available to create highly effective customizable pest management strategies with RNAi, current applications do not capitalize on species-specific biology. This lack of specificity has the potential to have substantial ecological impacts. Here, we discuss both short- and long-term considerations for sustainable RNAi pest management strategies for B. tabaci, focusing on the need for species specificity incorporating both life history and population genetic considerations. We provide a conceptual framework for selecting sublethal target genes based on their involvement in physiological pathways, which has the greatest potential to ameliorate unintended negative consequences. We suggest that these considerations allow an integrated pest management approach, with fewer negative ecological impacts and reduced likelihood of the evolution of resistant populations.

Species Composition, Temporal Abundance and Distribution of Insect Captures Inside and Outside Commercial Peanut Shelling Facilities.

Insect populations were studied within two commercial peanut shelling facilities located in the southeastern United States. Commercially available pheromone/kairomone-baited dome traps and pheromone-baited flight traps were deployed throughout processing and shipping portions of the shelling plants and serviced weekly over one year. Lasioderma serricorne, Tribolium castaneum, Typhaea stercorea, Carpophilus spp., Plodia interpunctella and Cadra cautella were the most common captures across locations. Lasioderma serricorne made up 87% and 88% of all captures in dome traps in plants one and two, respectively. While L. serricorne was not captured during the winter months in flight traps, it was captured with near 100% frequency in dome traps, suggesting that populations persisted throughout the year inside the facilities. Tribolium castaneum populations were active year round. Across insect species and trap type, temperature was a significant covariate for explaining variation in insect counts. After accounting for the effect of temperature, there were always more insects captured in the processing portions of the facilities compared to the shipping areas. A negative linear relationship was observed between captures of L. serricorne and T. castaneum and trap distance from in-shell peanuts entering the shelling facilities. Conversely, fungivores were more evenly distributed throughout all parts of the shelling plants. These data suggest that management efforts should be focused where in-shell peanuts enter to reduce breeding and harborage sites for grain feeding insects.

Impact of heat stress on development and fertility of Drosophila suzukii Matsumura (Diptera: Drosophilidae).

Drosophila suzukii is a globally invasive fruit pest that costs millions in yield losses and increased pest management costs. Management practices for D. suzukii currently rely heavily on calendar-based applications of broad-spectrum insecticides, but decision-based applications are theoretically possible with refined population modeling and monitoring. Temperature conditions are strongly deterministic of insect growth rates, fecundity, fertility, and resulting population densities. Therefore, information about the effects of temperature can be incorporated into population modeling to accurately predict D. suzukii population densities in the field which is crucial to maximize pesticide application efficiency and improve sustainability. Here, we investigated the effects of chronic heat stress during all of juvenile development on egg-to-adult viability and fertility. We also investigated egg-to-adult viability under heat stress after heat shock of the maternal parent. We found that heat stress during development results in lower egg-to-adult viability, and reduced lifespan and fertility for surviving adults. However, heat-shock treatment of females prior to egg laying increased the egg-to-adult viability of their eggs under heat stress. Female flies that developed at 30 °C had smaller ovaries than the untreated group and male flies had less sperm in their testes, and no sperm in their seminal vesicles. We conclude that heat stress during development is likely to have negative effect on D. suzukii population dynamics in the field. However, the intensity of such negative impact will depend on the phenotypic state of their maternal parents.

Dnmt1 is essential for egg production and embryo viability in the large milkweed bug, Oncopeltus fasciatus.

BACKGROUND: The function of cytosine (DNA) methylation in insects remains inconclusive due to a lack of mutant and/or genetic studies. RESULTS: Here, we provide evidence for the functional role of the maintenance DNA methyltransferase 1 (Dnmt1) in an insect using experimental manipulation. Through RNA interference (RNAi), we successfully posttranscriptionally knocked down Dnmt1 in ovarian tissue of the hemipteran Oncopeltus fasciatus (the large milkweed bug). Individuals depleted for Dnmt1, and subsequently DNA methylation, failed to reproduce. Eggs were inviable and declined in number, and nuclei structure of follicular epithelium was aberrant. Erasure of DNA methylation from gene or transposon element bodies did not reveal a direct causal link to steady-state mRNA levels in somatic cells. These results reveal an important function of Dnmt1 seemingly not contingent on directly controlling gene expression. CONCLUSIONS: This study provides direct experimental evidence for a functional role of Dnmt1 in egg production and embryo viability and uncovers a trivial role, if any, for DNA methylation in control of gene expression in O. fasciatus.

A study of the transit amplification divisions during spermatogenesis in Oncopetus fasciatus to assess plasticity in sperm numbers or sperm viability under different diets.

Oncopeltus fasciatus males fed the ancestral diet of milkweed seeds prioritize reproduction over lifespan as evidenced by higher rates of fertility and shorter lifespans than males from the same population fed the adapted diet of sunflower seeds. We examined the proximate mechanisms by which milkweed-fed males maintained late-life fertility. We tested the hypothesis that older milkweed-fed males maintained fertility by producing more, higher quality sperm. Our results, that older males have more sperm, but their sperm do not have higher viability, are in general agreement with other recent studies on how nutrition affects male fertility in insects. We further examined the mechanisms by which sperm are produced by examining the progression of spermatogonial cells through the cell cycle during the transit amplification divisions. We demonstrated that diet affects the likelihood of a spermatocyst being in the S-phase or M-phase of the cell cycle. Given work in model systems, these results have implications for subtle effects on sperm quality either through replication stress or epigenetic markers. Thus, viability may not be the best marker for sperm quality and more work is called for on the mechanisms by which the germline and the production of sperm mediate the cost of reproduction.

Variation in mandible development and its relationship to dependence on parents across burying beetles.

BACKGROUND: In species with parental care, there is striking variation in offspring dependence at birth, ranging from feeding independence to complete dependency on parents for nutrition. Frequently, highly dependent offspring further evolve reductions or alterations of morphological traits that would otherwise promote self-sufficiency. Here, we examine evidence for morphological evolution associated with dependence in burying beetles (Nicrophorus spp.), in which dependence upon parents appears to have several independent origins. In many species, precocial first instar larvae can survive without parenting, but several altricial species die at this stage on their own. We focused specifically on the mandibles, which are expected to be related to feeding ability and therefore independence from parents. RESULTS: We find no evidence that the size of the mandible is related to dependence on parents. However, we do find a developmental and phylogenetic correlation between independence and the presence of serrations on the inner edge of the mandible. Mandibles of independent species bear serrations at hatching, whereas dependent species hatch with smooth mandibles, only developing serrations in the second instar when these larvae gain the ability to survive on their own. Phylogenetic evidence suggests that serrations coincide with independence repeatedly. We note a single exception to this trend, a beetle with a serrated mandible that cannot survive without parents. However, this exception occurs in a species that has recently evolved the loss of independence. CONCLUSIONS: We argue that the absence of mandible serrations occurs due to alternative selection pressures incurred in larvae dependent upon parents to survive. We suggest that this may have led to a variable function for mandibles, perhaps related to increased competitive ability among siblings or increased efficiency in receiving nutrition from parents. Furthermore, we propose that the phylogenetic pattern we see is consistent with the long-held evolutionary hypothesis that evolutionary change in behavior and physiology precede morphological change.

A Simple Flight Mill for the Study of Tethered Flight in Insects.

Flight in insects can be long-range migratory flights, intermediate-range dispersal flights, or short-range host-seeking flights. Previous studies have shown that flight mills are valuable tools for the experimental study of insect flight behavior, allowing researchers to examine how factors such as age, host plants, or population source can influence an insects' propensity to disperse. Flight mills allow researchers to measure components of flight such as speed and distance flown. Lack of detailed information about how to build such a device can make their construction appear to be prohibitively complex. We present a simple and relatively inexpensive flight mill for the study of tethered flight in insects. Experimental insects can be tethered with non-toxic adhesives and revolve around an axis by means of a very low friction magnetic bearing. The mill is designed for the study of flight in controlled conditions as it can be used inside an incubator or environmental chamber. The strongest points are the very simple electronic circuitry, the design that allows sixteen insects to fly simultaneously allowing the collection and analysis of a large number of samples in a short time and the potential to use the device in a very limited workspace. This design is extremely flexible, and we have adjusted the mill to accommodate different species of insects of various sizes.

Oosorption in response to poor food: complexity in the trade-off between reproduction and survival.

Plasticity in reproductive physiology is one avenue by which environmental signals, such as poor quality food, can be coordinated with adaptive responses. Insects have the ability to resorb oocytes that are not oviposited. Oosorption is proposed to be an adaptive mechanism to optimize fitness in hostile environments, recouping resources that might otherwise be lost, and reinvesting them into future reproductive potential. We tested the hypothesis that oosorption is an evolved mechanism by which females can reallocate resources from current reproductive effort to survival and future reproduction, when conditions for reproduction are poor, by examining the reproductive physiology and life-history outcome under poor quality food in populations of the milkweed bug (Oncopeltus fasciatus) that have adapted to live on sunflower seed. Females fed a diet of pumpkin seeds, known to be a poor host food, had higher levels of ovarian apoptosis (oosorption), lower reproductive output, but no reduction in life span under poor nutrition, as predicted under the oosorption hypothesis. However, the schedule of reproduction was surprising given the "wait to reproduce" assumption of oosorption as early fecundity was unaffected.

Separate and combined effects of nutrition during juvenile and sexual development on female life-history trajectories: the thrifty phenotype in a cockroach.

We have yet to understand fully how conditions during different periods of development interact to influence life-history structure. Can the negative effects of poor juvenile nutrition be overcome by a good adult diet, or are life-history strategies set by early experience? Here, we tested the influence and interaction of different nutritional quality during juvenile and sexual development on female resource allocation physiology, life history and courtship behaviour in the cockroach, Nauphoeta cinerea. Nymphs were raised on either a good-quality or poor-quality diet. After adult eclosion, females were either switched to the opposite diet or remained on their original diet. We assessed mating behaviour and lifetime reproductive success for half of the females from each treatment. We evaluated reproductive investment, somatic investment and resource reallocation from reproduction to the soma via oocyte apoptosis in the remaining females. We found that poor juvenile conditions resulted in a fat phenotype with slow juvenile growth and short reproductive lifespan that could not be retrieved with a change in diet. Good juvenile conditions resulted in the converse, but again fixed, phenotype in adulthood. Thus, juvenile nutrition sets adult patterns of resource allocation.

Effects of mating delay and nutritional signals on resource recycling in a cyclically breeding cockroach.

Ovarian apoptosis has been found to occur in the female cockroach Nauphoeta cinerea in response to lack of mates. It has been proposed that ovarian apoptosis in continuously breeding insects is an adaptive mechanism for recouping resources in poor conditions (oosorption). However, N. cinerea is a cyclically breeding insect and ovarian apoptosis may represent ageing and clearance of old unused oocytes. To test the hypothesis that oocyte resorption via apoptosis reflects the reclamation of resources, we delayed mating in combination with positive and negative nutritional signals. Females without access to food during sexual maturation invested less in reproduction and had elevated rates of ovarian apoptosis in the terminal oocyte. Starvation also induced apoptosis in non-vitellogenic oocytes of the vitellarium and germinarium, which would be used for future reproductive events. This is paradoxical as theory states that oosorption is an adaptive means of rerouting resources into investment in future reproduction, yet these oocytes do not represent a cache of resources and their loss could limit future reproduction.