The distribution of covert microbial natural enemies of a globally invasive crop pest, fall armyworm, in Africa: Enemy release and spillover events.

Invasive species pose a significant threat to biodiversity and agriculture world-wide. Natural enemies play an important part in controlling pest populations, yet we understand very little about the presence and prevalence of natural enemies during the early invasion stages. Microbial natural enemies of fall armyworm Spodoptera frugiperda are known in its native region, however, they have not yet been identified in Africa where fall armyworm has been an invasive crop pest since 2016. Larval samples were screened from Malawi, Rwanda, Kenya, Zambia, Sudan and Ghana for the presence of four different microbial natural enemies; two nucleopolyhedroviruses, Spodoptera frugiperda NPV (SfMNPV) and Spodoptera exempta NPV (SpexNPV); the fungal pathogen Metarhizium rileyi; and the bacterium Wolbachia. This study aimed to identify which microbial pathogens are present in invasive fall armyworm, and determine the geographical, meteorological and temporal variables that influence prevalence. Within 3 years of arrival, fall armyworm was exposed to all four microbial natural enemies. SfMNPV probably arrived with fall armyworm from the Americas, but this is the first putative evidence of host spillover from Spodoptera exempta (African armyworm) to fall armyworm for the endemic pathogen SpexNPV and for Wolbachia. It is also the first confirmed incidence of M. rileyi infecting fall armyworm in Africa. Natural enemies were localised, with variation being observed both nationally and temporally. The prevalence of SfMNPV (the most common natural enemy) was predominantly explained by variables associated with the weather; declining with increasing rainfall and increasing with temperature. However, virus prevalence also increased as the growing season progressed. The infection of an invasive species with a natural enemy from its native range and novel pathogens specific to its new range has important consequences for understanding the population ecology of invasive species and insect-pathogen interactions. Additionally, while it is widely known that temporal and geographic factors affect insect populations, this study reveals that these are important in understanding the distribution of microbial natural enemies associated with invasive pests during the early stages of invasion, and provide baseline data for future studies.

Trans-generational viral transmission and immune priming are dose-dependent.

It is becoming increasingly apparent that trans-generational immune priming (i.e. the transfer of the parental immunological experience to its progeny resulting in offspring protection from pathogens that persist across generations) is a common phenomenon not only in vertebrates, but also invertebrates. Likewise, it is known that covert pathogenic infections may become 'triggered' into an overt infection by various stimuli, including exposure to heterologous infections. Yet, rarely have both phenomena been explored in parallel. Using as a model system the African armyworm Spodoptera exempta, an eruptive agricultural pest and its endemic dsDNA virus (Spodoptera exempta nucleopolyhedrovirus, SpexNPV), the aim of this study was to explore the impact of parental inoculating-dose on trans-generational pathogen transmission and immune priming (in its broadest sense). Larvae were orally challenged with one of five doses of SpexNPV and survivors from these treatments were mated and their offspring monitored for viral mortality. Offspring from parents challenged with low viral doses showed evidence of 'immune priming' (i.e. enhanced survival following SpexNPV challenge); in contrast, offspring from parents challenged with higher viral doses exhibited greater susceptibility to viral challenge. Most offspring larvae died of the virus they were orally challenged with; in contrast, most offspring from parents that had been challenged with the highest doses were killed by the vertically transmitted virus (90%) and not the challenge virus. These results demonstrate that the outcome of a potentially lethal virus challenge is critically dependent on the level of exposure to virus in the parental generation-either increasing resistance at very low parental viral doses (consistent with trans-generational immune priming) or increasing susceptibility at higher parental doses (consistent with virus triggering). We discuss the implications of these findings for understanding both natural epizootics of baculoviruses and for using them as biological control agents.

The role of STAT3/p53 and PI3K-Akt-mTOR signaling pathway on DEHP-induced reproductive toxicity in pubertal male rat.

Di (2-ethylhexyl) phthalate (DEHP) is a known environmental endocrine disruptor that impairs development of testis and spermatogenesis. This study aims to explore the effects of STAT3/p53 and PI3K-Akt-mTOR signaling pathway on DEHP-induced reproductive toxicity in pubertal male rat. 24 6-week-old male Sprague-Dawley rats were randomly divided into 4 groups (Control, low-dose, middle-dose and high-dose group) and were treated with increasing concentration of DEHP (0, 250, 500, 1000 mg/kg/day) respectively for 28 consecutive days by intragastric administration. Our results showed that DEHP exposure induced obvious morphological changes of testis, decreased organ coefficient of testis and sperm count, and increased testicular cell apoptosis in the 500 and 1000 mg/kg/day DEHP groups (p < .05). The serum testosterone decreased in a dose-dependent manner after treatment with DEHP. Furthermore, the exposure of DEHP elevated the levels of oxidative stress accompanied by upregulated expression of p53 and reduced expression of STAT3. In addition, compared with the control group, the expression of PI3K, p-Akt and p-mTOR proteins significantly decreased, whereas the downstream autophagy-related proteins phosphorylated ULK1, Beclin-1, Atg7, LC3-II obviously increased in the 250 mg/kg/day DEHP group (p < .05). The expression of p62 was reduced in DEHP-treated groups. Our data indicated that autophagy could be activated to protect testes from DEHP-induced reproductive damage by inhibiting PI3K-Akt-mTOR signaling pathway in the 250 mg/kg/day DEHP group. STAT3/p53-mediated mitochondrial apoptosis pathway might play a major role to cause testis injury and reproductive dysfunction in the 500 and 1000 mg/kg/day DEHP groups.

Duplication and expression of horizontally transferred polygalacturonase genes is associated with host range expansion of mirid bugs.

BACKGROUD: Horizontal gene transfer and gene duplication are two major mechanisms contributing to the evolutionary adaptation of organisms. Previously, polygalacturonase genes (PGs) were independently horizontally transferred and underwent multiple duplications in insects (e.g., mirid bugs and beetles). Here, we chose three phytozoophagous mirid bugs (Adelphocoris suturalis, A. fasciaticollis, A. lineolatus) and one zoophytophagous mirid bug (Nesidiocoris tenuis) to detect whether the duplication, molecular evolution, and expression levels of PGs were related to host range expansion in mirid bugs. RESULTS: By RNA-seq, we reported 30, 20, 19 and 8 PGs in A. suturalis, A. fasciaticollis, A. lineolatus and N. tenuis, respectively. Interestingly, the number of PGs was significantly positive correlation to the number of host plants (P = 0.0339) in mirid bugs. Most PGs (> 17) were highly expressed in the three phytozoophagous mirid bugs, while only one PG was relatively highly expressed in the zoophytophagous mirid bug. Natural selection analysis clearly showed that a significant relaxation of selection pressure acted on the PGs in zoophytophagous mirid bugs (K = 0.546, P = 0.0158) rather than in phytozoophagous mirid bugs (K = 1, P = 0.92), suggesting a function constraint of PGs in phytozoophagous mirid bugs. CONCLUSION: Taken together with gene duplication, molecular evolution, and expression levels, our results suggest that PGs are more strictly required by phytozoophagous than by zoophytophagous mirid bugs and that the duplication of PGs is associated with the expansion of host plant ranges in mirid bugs.

Preference of the aphid Myzus persicae (Hemiptera: Aphididae) for tobacco plants at specific stages of potato virus Y infection.

Potato virus Y (PVY) is a common pathogen affecting agricultural production worldwide and is mainly transmitted by Myzus persicae in a non-persistent manner. Insect-borne plant viruses can modify the abundance, performance, and behavior of their vectors by altering host plant features; however, most studies have overlooked the fact that the dynamic progression of virus infection in plants can have variable effects on their vectors. We addressed this point in the present study by dividing the PVY infection process in tobacco into three stages (early state, steady state and late state); delineated by viral copy number. We then compared the differential effects of PVY-infected tobacco (Nicotiana tabacum) plants on the host selection and feeding behavior of M. persicae. We used Y-shaped olfactory apparatus and electrical penetration graph (EPG) methods to evaluate host selection and feeding behavior, respectively. Interestingly, we found that PVY-infected plants at the steady state attracted more aphids than healthy plants, whereas no differences were observed for those at the early and late states. In terms of feeding behavior, intracellular punctures (closely related to PVY acquisition and transmission) were more abundant on PVY-infected tobacco plants at the early and steady states of infection than in uninfected plants. These results indicate that PVY-infected host plants can alter the host selection and feeding behavior of aphids in a stage-dependent manner, which is an important consideration when studying the interactions among host plants, viruses, and insect vectors.

Nutritional composition of honey bee food stores vary with floral composition.

Sufficiently diverse and abundant resources are essential for generalist consumers, and form an important part of a suite of conservation strategies for pollinators. Honey bees are generalist foragers and are dependent on diverse forage to adequately meet their nutritional needs. Through analysis of stored pollen (bee bread) samples obtained from 26 honey bee (Apis mellifera L.) hives across NW-England, we quantified bee bread nutritional content and the plant species that produced these stores from pollen. Protein was the most abundant nutrient by mass (63%), followed by carbohydrates (26%). Protein and lipid content (but not carbohydrate) contributed significantly to ordinations of floral diversity, linking dietary quality with forage composition. DNA sequencing of the ITS2 region of the nuclear ribosomal DNA gene identified pollen from 89 distinct plant genera, with each bee bread sample containing between 6 and 35 pollen types. Dominant genera included dandelion (Taraxacum), which was positively correlated with bee bread protein content, and cherry (Prunus), which was negatively correlated with the amount of protein. In addition, proportions of amino acids (e.g. histidine and valine) varied as a function of floral species composition. These results also quantify the effects of individual plant genera on the nutrition of honey bees. We conclude that pollens of different plants act synergistically to influence host nutrition; the pollen diversity of bee bread is linked to its nutrient content. Diverse environments compensate for the loss of individual forage plants, and diversity loss may, therefore, destabilize consumer communities due to restricted access to alternative resources.

Gut microbial community supplementation and reduction modulates African armyworm susceptibility to a baculovirus.

Gut microbiota stimulates the immune system and inhibits pathogens, and thus, it is critical for disease prevention. Probiotics represent an effective alternative to antibiotics used for the therapy and prevention of bacterial diseases. Probiotic bacteria are commonly used in vertebrates, although their use in invertebrates is still rare. We manipulated the gut microbiome of the African Armyworm (Spodoptera exempta Walker) using antibiotics and field-collected frass, in an attempt to understand the interactions of the gut microbiome with the nucleopolyhedrovirus, SpexNPV. We found that S. exempta individuals with supplemented gut microbiome were significantly more resistant to SpexNPV, relative to those with a typical laboratory gut microbiome. Illumina MiSeq sequencing revealed the bacterial phyla in the S. exempta gut belonged to 28 different classes. Individuals with an increased abundance of Lactobacillales had a higher probability of surviving viral infection. In contrast, there was an increased abundance of Enterobacteriales and Pseudomonadales in individuals dying from viral infection, corresponding with decreased abundance of these two Orders in surviving caterpillars, suggesting a potential role for them in modulating the interaction between the host and its pathogen. These results have important implications for laboratory studies testing biopesticides.

Global insect decline is the result of wilful political failure: A battle plan for entomology.

The Millennium Ecosystem Assessment assessed ecosystem change, human wellbeing and scientific evidence for sustainable use of biological systems. Despite intergovernmental acknowledgement of the problem, global ecological decline has continued, including declines in insect biodiversity, which has received much media attention in recent years. Several roadmaps to averting biological declines have failed due to various economic and political factors, and so biodiversity loss continues, driven by several interacting human pressures. Humans are innately linked with nature but tend to take it for granted. The benefits we gain from the insect world are broad, yet aversion or phobias of invertebrates are common, and stand firmly in the path of their successful conservation. Providing an integrated synthesis for policy teams, conservation NGOs, academic researchers and those interested in public engagement, this article considers: (1) The lack of progress to preserve and protect insects. (2) Examples relating to insect decline and contributions insects make to people worldwide, and consequently what we stand to lose. (3) How to engage the public, governmental organizations and researchers through "insect contributions to people" to better address insect declines. International political will has consistently acknowledged the existence of biodiversity decline, but apart from a few narrow cases of charismatic megafauna, little meaningful change has been achieved. Public values are reflected in political willpower, the progress being made across the world, changing views on insects in the public should initiate a much-needed political sea-change. Taking both existing activity and required future actions, we outline an entomologist's "battle plan" to enormously expand our efforts and become the champions of insect conservation that the natural world needs.

Japanese Honeybees (Apis cerana japonica Radoszkowski, 1877) May Be Resilient to Land Use Change.

Pollinators are being threatened globally by urbanisation and agricultural intensification, driven by a growing human population. Understanding these impacts on landscapes and pollinators is critical to ensuring a robust pollination system. Remote sensing data on land use attributes have previously linked honeybee nutrition to land use in the Western Honeybee (Apis mellifera L.). Here, we instead focus on the less commonly studied Apis cerana japonica-the Japanese Honeybee. Our study presents preliminary data comparing forage (honey and pollen) with land use across a rural-urban gradient from 22 sites in Kyushu, southern Japan. Honey samples were collected from hives between June 2018 and August 2019. Pollen were collected and biotyped from hives in urban and rural locations (n = 4). Previous studies of honey show substantial variation in monosaccharide content. Our analysis of A. cerana japonica honey found very little variation in glucose and fructose (which accounted for 97% of monosaccharides), despite substantial differences in surrounding forage composition. As expected, we observed temporal variation in pollen foraged by A. cerana japonica, likely dependent on flowering phenology. These preliminary results suggest that the forage and nutrition of A. cerana japonica may not be negatively affected by urban land use. This highlights the need for further comparative studies between A. cerana japonica and A. mellifera as it could suggest a resilience in pollinators foraging in their native range.

A One-Health Model for Reversing Honeybee (Apis mellifera L.) Decline.

Global insect decline impacts ecosystem resilience; pollinators such as honeybees (Apis mellifera L.) have suffered extensive losses over the last decade, threatening food security. Research has focused discretely on in-hive threats (e.g., Nosema and Varroa destructor) and broader external causes of decline (e.g., agrochemicals, habitat loss). This has notably failed to translate into successful reversal of bee declines. Working at the interdisciplinary nexus of entomological, social and ecological research, we posit that veterinary research needs to adopt a "One-Health" approach to address the scope of crises facing pollinators. We demonstrate that reversing declines will require integration of hive-specific solutions, a reappraisal of engagement with the many stakeholders whose actions affect bee health, and recontextualising both of these within landscape scale efforts. Other publications within this special issue explore novel technologies, emergent diseases and management approaches; our aim is to place these within the "One-Health" context as a pathway to securing honeybee health. Governmental policy reform offers a particularly timely pathway to achieving this goal. Acknowledging that healthy honeybees need an interdisciplinary approach to their management will enhance the contributions of veterinary research in delivering systemic improvements in bee health.

Up-regulation of cryptochrome 1 gene expression in cotton bollworm (Helicoverpa armigera) during migration over the Bohai Sea.

Cryptochromes (CRYs) are flavoproteins and play a pivotal role in circadian clocks which mediate behavior of organisms such as feeding, mating and migrating navigation. Herein, we identified novel transcripts in Helicoverpa armigera of six isoforms of cry1 and seven isoforms of cry2 by Sanger sequencing. Phylogenetic analysis showed that the transcripts of cry1 and cry2 align closely with other insect crys, indicating within-species divergence of Hacry. A dn/ds analysis revealed that the encoding sequence of the cry1 was under purifying selection by a strong negative selection pressure whereas the cry2 was less constraint and showed a less strong purification selection than cry1. In general, Hacrys were more abundantly transcribed in wild migrating populations than that in laboratory maintained populations, and expression of the cry2 was lower than cry1 in all samples tested. Moreover, when compared with the migrating parental population, offspring reared in laboratory conditions showed a significant reduction on transcription of the cry1 but not cry2. These results strongly suggest that cry1 was more related to the migration behavior of H. armigera than cry2.

Bacterial communities associated with honeybee food stores are correlated with land use.

Microbial communities, associated with almost all metazoans, can be inherited from the environment. Although the honeybee (Apis mellifera L.) gut microbiome is well documented, studies of the gut focus on just a small component of the bee microbiome. Other key areas such as the comb, propolis, honey, and stored pollen (bee bread) are poorly understood. Furthermore, little is known about the relationship between the pollinator microbiome and its environment. Here we present a study of the bee bread microbiome and its relationship with land use. We estimated bacterial community composition using both Illumina MiSeq DNA sequencing and denaturing gradient gel electrophoresis (DGGE). Illumina was used to gain a deeper understanding of precise species diversity across samples. DGGE was used on a larger number of samples where the costs of MiSeq had become prohibitive and therefore allowed us to study a greater number of bee breads across broader geographical axes. The former demonstrates bee bread comprises, on average, 13 distinct bacterial phyla; Bacteroidetes, Firmicutes, Alpha-proteobacteria, Beta-proteobacteria, and Gamma-proteobacteria were the five most abundant. The most common genera were Pseudomonas, Arsenophonus, Lactobacillus, Erwinia, and Acinetobacter. DGGE data show bacterial community composition and diversity varied spatially and temporally both within and between hives. Land use data were obtained from the 2007 Countryside Survey. Certain habitats, such as improved grasslands, are associated with low diversity bee breads, meaning that these environments may be poor sources of bee-associated bacteria. Decreased bee bread bacterial diversity may result in reduced function within hives. Although the dispersal of microbes is ubiquitous, this study has demonstrated landscape-level effects on microbial community composition.

Witch's Broom Disease of Lime (Candidatus Phytoplasma aurantifolia): Identifying High-Risk Areas by Climatic Mapping.

Biological invasions of vectorborne diseases can be devastating. Bioclimatic modeling provides an opportunity to assess and predict areas at risk from complex multitrophic interactions of pathogens, highlighting areas in need of increased monitoring effort. Here, we model the distribution of an economically critical vectorborne plant pathogen 'Candidatus Phytoplasma aurantifolia', the etiological agent of Witches' Broom Disease of Lime. This disease is a significant limiting factor on acid lime production (Citrus aurantifolia, Swingle) in the Middle East and threatens its production globally. We found that temperature, humidity, and the vector populations significantly determine disease distribution. Following this, we used bioclimatic modeling to predict potential novel sites of infections. The model outputs identified potential novel sites of infection in the citrus producing regions of Brazil and China. We also used our model to explore sites in Oman where the pathogen may not be infectious, and suggest nurseries be established there. Recent major turbulence in the citrus agricultural economy has highlighted the importance of this work and the need for appropriate and targeted monitoring programs to safeguard lime production.

Invasive mutualisms between a plant pathogen and insect vectors in the Middle East and Brazil.

Complex multi-trophic interactions in vectorborne diseases limit our understanding and ability to predict outbreaks. Arthropod-vectored pathogens are especially problematic, with the potential for novel interspecific interactions during invasions. Variations and novelties in plant-arthropod-pathogen triumvirates present significant threats to global food security. We examined aspects of a phytoplasma pathogen of citrus across two continents. 'Candidatus Phytoplasma aurantifolia' causes Witches' Broom Disease of Lime (WBDL) and has devastated citrus production in the Middle East. A variant of this phytoplasma currently displays asymptomatic or 'silent' infections in Brazil. We first studied vector capacity and fitness impacts of the pathogen on its vectors. The potential for co-occurring weed species to act as pathogen reservoirs was analysed and key transmission periods in the year were also studied. We demonstrate that two invasive hemipteran insects-Diaphorina citri and Hishimonus phycitis-can vector the phytoplasma. Feeding on phytoplasma-infected hosts greatly increased reproduction of its invasive vector D. citri both in Oman and Brazil; suggesting that increased fitness of invasive insect vectors thereby further increases the pathogen's capacity to spread. Based on our findings, this is a robust system for studying the effects of invasions on vectorborne diseases and highlights concerns about its spread to warmer, drier regions of Brazil.

Honeybee nutrition is linked to landscape composition.

Declines in insect pollinators in Europe have been linked to changes in land use. Pollinator nutrition is dependent on floral resources (i.e., nectar and pollen), which are linked to landscape composition. Here, we present a stratified analysis of the nutritional composition of beebread in managed honeybee hives with a view to examining potential sources of variation in its nutritional composition. Specifically, we tested the hypothesis that beebread composition correlates with local land use and therefore available floral resources. The results demonstrated that the starch, lipid, and moisture contents of beebread are all highly conserved across hives, whereas levels of protein and nonreducing sugar increased as the year progressed, reducing sugars, however, decreased during the first half of the year and then increased toward the end. Local land use around hives was quantified using data from the Countryside Survey 2007 Land Cover Map. Bee-bread protein content was negatively correlated with increasing levels of arable and horticultural farmland surrounding hives and positively correlated with the cover of natural grasslands and broadleaf woodlands. Reducing sugar content was also positively correlated with the amount of broad-leaved woodland in a 3 Km² radius from the hives. Previous studies on a range of invertebrates, including honeybees, indicate that dietary protein intake may have a major impact on correlates of fitness, including longevity and immune function. The finding that beebread protein content correlates with land use suggests that landscape composition may impact on insect pollinator well-being and provides a link between landscape and the nutritional ecology of socially foraging insects in a way not previously considered.