Predicting the dispersal and invasion dynamics of ambrosia beetles through demographic reconstruction and process-explicit modeling.

Evaluating potential routes of invasion of pathogens and vectors of sanitary importance is essential for planning and decision-making at multiple scales. An effective tool are process-explicit models that allow coupling environmental, demographic and dispersal information to evaluate population growth and range dynamics as a function of the abiotic conditions in a region. In this work we simulate multiple dispersal/invasion routes in Mexico that could be taken by ambrosia beetles and a specific symbiont, Harringtonia lauricola, responsible for a severe epiphytic of Lauraceae in North America. We used Xyleborus bispinatus Eichhoff 1868 as a study subject and estimated its demography in the laboratory in a temperature gradient (17, 20, 26, 29, 35 °C), which we then used to parameterize a process-based model to estimate its metapopulation dynamics. The maximum intrinsic growth rate of X. bispinatus is 0.13 with a thermal optimum of 26.2 °C. The models suggest important regions for the establishment and dispersal the states of Veracruz, Chiapas and Oaxaca (high host and secondary vectors diversity), the Isthmus of Tehuantepec (connectivity region), and Michoacán and Jalisco (important avocado plantations). The use of hybrid process-based models is a promising tool to refine the predictions applied to the study of biological invasions and species distributions.

The influence of humidity and temperature on the vertical richness and abundance of blood-sucking flies (Culicidae and Ceratopogonidae) in a montane cloud forest in Mexico.

Because the vertical distribution and diversity of blood-sucking flies are poorly known, we determined the diversity, structure, and composition of culicids between vertical vegetation strata. We evaluated the influence of microclimatic variables during different times of the day over a year. We used eight CDC traps baited with CO2 at a height of 1.5 m and 12-15 m. We conducted rank-abundance curves, similarity analysis (ANOSIM and SIMPER), and multivariate clustering with incidence and abundance data. We used GAM models to analyze the influence of strata (understory vs canopy), humidity, and temperature on insect richness and abundance. During the day, the difference between strata was mainly due to higher abundance of Wyeomyia arthrostigma and Wyeomyia ca. adelpha in the understory. During the night, the differences were mainly due to higher abundance of Culex stigmatosoma, Culex salinarius, and Aedes allotecnon in the canopy, and Wyeomyia arthrostigma in the understory. Seasonality played a role in the similarity between the strata. Diversity during the day was positively related to humidity and temperature, and nocturnal diversity increased with temperature but decreased with higher humidity. The effects of environmental factors on the spatiotemporal distribution of fly species are essential for epidemiological surveillance.

Antifungal and Insecticidal Activities of Selected Plant Species from Cloud Forest of Veracruz, Mexico: A Contribution to the Search of Novel Control Agents against Ambrosia Pest Complexes.

The antifungal and insecticidal activities of 34 extracts from 27 plant species were evaluated against fungal phytopathogens of the genus Fusarium and Xyleborus Scolytine ambrosia beetles involved in Fusarium dieback (FD) and laurel wilt (LW) diseases. Sixteen extracts caused mycelial growth inhibition (MGI) above 23 % at 2 mg mL-1 against F. solani, those from S. nudum and M. argyrophylla exhibited the highest MGI (57 % and 49 %, respectively). Thirteen extracts displayed significant antifungal activity against F. kuroshium, those from C. nocturnum and M. argyrophylla exhibited the highest MGI (100 % and 54.9 %, respectively). Additionally, ten plants extracts caused mortality in at least one of the beetle species tested, mainly from Solanaceae species. In the most active species, 39 phenolics were identified that may have contributed to their biological effects. This study is one of the first to report the potential of plant-derived natural products against the causative agents of FD and LW.

Microorganisms Associated with the Ambrosial Beetle Xyleborus affinis with Plant Growth-Promotion Activity in Arabidopsis Seedlings and Antifungal Activity Against Phytopathogenic Fungus Fusarium sp. INECOL_BM-06.

Plants interact with a great diversity of microorganisms or insects throughout their life cycle in the environment. Plant and insect interactions are common; besides, a great variety of microorganisms associated with insects can induce pathogenic damage in the host, as mutualist phytopathogenic fungus. However, there are other microorganisms present in the insect-fungal association, whose biological/ecological activities and functions during plant interaction are unknown. In the present work evaluated, the role of microorganisms associated with Xyleborus affinis, an important beetle species within the Xyleborini tribe, is characterized by attacking many plant species, some of which are of agricultural and forestry importance. We isolated six strains of microorganisms associated with X. affinis shown as plant growth-promoting activity and altered the root system architecture independent of auxin-signaling pathway in Arabidopsis seedlings and antifungal activity against the phytopathogenic fungus Fusarium sp. INECOL_BM-06. In addition, evaluating the tripartite interaction plant-microorganism-fungus, interestingly, we found that microorganisms can induce protection against the phytopathogenic fungus Fusarium sp. INECOL_BM-06 involving the jasmonic acid-signaling pathway and independent of salicylic acid-signaling pathway. Our results showed the important role of this microorganisms during the plant- and insect-microorganism interactions, and the biological potential use of these microorganisms as novel agents of biological control in the crops of agricultural and forestry is important.

Electroantennographic Responses of Wild and Laboratory-Reared Females of Xyleborus affinis Eichhoff and Xyleborus ferrugineus (Fabricius) (Coleoptera: Curculionidae: Scolytinae) to Ethanol and Bark Volatiles of Three Host-Plant Species.

Chemical ecology studies on ambrosia beetles are typically conducted with either wild or laboratory-reared specimens. Unlike laboratory-reared insects, important aspects that potentially influence behavioral responses, such as age, physiological state, and prior experience are unknown in wild specimens. In this study, we compared the electroantennographic (EAG) responses of laboratory-reared and wild X. affinis and X. ferrugineus to 70% ethanol and bark odors (host kairomones) of Bursera simaruba, Mangifera indica, and Persea schiedeana aged for 2, 24, and 48 h. Chemical analyses of each odor treatment (bark species x length of aging) were performed to determine their volatilome composition. EAG responses were different between laboratory-reared and wild X. ferrugineus when exposed to ethanol, whereas wild X. affinis exhibited similar EAG responses to the laboratory-reared insects. Ethanol elicited the strongest olfactory responses in both species. Among the bark-odors, the highest responses were triggered by B. simaruba at 48 h in X. affinis, and P. schiedeana at 24 and 48 h in X. ferrugineus. Volatile profiles varied among aged bark samples; 3-carene and limonene were predominant in B. simaruba, whereas α-copaene and α-cubebene were abundant in P. schiedeana. Further studies are needed to determine the biological function of B. simaruba and P. schiedeana terpenes on X. affinis and X. ferrugineus, and their potential application for the development of effective lures.

Antifungal Effect of Copper Nanoparticles against Fusarium kuroshium, an Obligate Symbiont of Euwallacea kuroshio Ambrosia Beetle.

Copper nanoparticles (Cu-NPs) have shown great antifungal activity against phytopathogenic fungi, making them a promising and affordable alternative to conventional fungicides. In this study, we evaluated the antifungal activity of Cu-NPs against Fusarium kuroshium, the causal agent of Fusarium dieback, and this might be the first study to do so. The Cu-NPs (at different concentrations) inhibited more than 80% of F. kuroshium growth and were even more efficient than a commercial fungicide used as a positive control (cupric hydroxide). Electron microscopy studies revealed dramatic damage caused by Cu-NPs, mainly in the hyphae surface and in the characteristic form of macroconidia. This damage was visible only 3 days post inoculation with used treatments. At a molecular level, the RNA-seq study suggested that this growth inhibition and colony morphology changes are a result of a reduced ergosterol biosynthesis caused by free cytosolic copper ions. Furthermore, transcriptional responses also revealed that the low- and high-affinity copper transporter modulation and the endosomal sorting complex required for transport (ESCRT) are only a few of the distinct detoxification mechanisms that, in its conjunction, F. kuroshium uses to counteract the toxicity caused by the reduced copper ion.

Characterization of Two Fusarium solani Species Complex Isolates from the Ambrosia Beetle Xylosandrus morigerus.

Ambrosia beetles are insect vectors of important plant diseases and have been considered as a threat to forest ecosystems, agriculture, and the timber industry. Several factors have been suggested as promoters of the pathogenic behavior of ambrosia beetles; one of them is the nature of the fungal mutualist and its ability to establish an infectious process. In Mexico, Xylosandrus morigerus is an invasive ambrosia beetle that damages many agroecosystems. Herein, two different isolates from the X. morigerus ambrosia beetle belonging to the Fusarium genus are reported. Both isolates belong to the Fusarium solani species complex (FSSC) but not to the Ambrosia Fusarium clade (AFC). The two closely related Fusarium isolates are pathogenic to different forest and agronomic species, and the morphological differences between them and the extracellular protease profile suggest intraspecific variability. This study shows the importance of considering these beetles as vectors of different species of fungal plant pathogens, with some of them even being phylogenetically closely related and having different pathogenic abilities, highlighting the relevance of the fungal mutualist as a factor for the ambrosia complex becoming a pest.

Application of Deep Learning to Community-Science-Based Mosquito Monitoring and Detection of Novel Species.

Mosquito-borne diseases account for human morbidity and mortality worldwide, caused by the parasites (e.g., malaria) or viruses (e.g., dengue, Zika) transmitted through bites of infected female mosquitoes. Globally, billions of people are at risk of infection, imposing significant economic and public health burdens. As such, efficient methods to monitor mosquito populations and prevent the spread of these diseases are at a premium. One proposed technique is to apply acoustic monitoring to the challenge of identifying wingbeats of individual mosquitoes. Although researchers have successfully used wingbeats to survey mosquito populations, implementation of these techniques in areas most affected by mosquito-borne diseases remains challenging. Here, methods utilizing easily accessible equipment and encouraging community-scientist participation are more likely to provide sufficient monitoring. We present a practical, community-science-based method of monitoring mosquito populations using smartphones. We applied deep-learning algorithms (TensorFlow Inception v3) to spectrogram images generated from smartphone recordings associated with six mosquito species to develop a multiclass mosquito identification system, and flag potential invasive vectors not present in our sound reference library. Though TensorFlow did not flag potential invasive species with high accuracy, it was able to identify species present in the reference library at an 85% correct identification rate, an identification rate markedly higher than similar studies employing expensive recording devices. Given that we used smartphone recordings with limited sample sizes, these results are promising. With further optimization, we propose this novel technique as a way to accurately and efficiently monitor mosquito populations in areas where doing so is most critical.

Modeling the impact of temperature on the population abundance of the ambrosia beetle Xyleborus affinis (Curculionidae: Scolytinae) under laboratory-reared conditions.

Modeling the impact of temperature on each life stage of a beetle population represents a continuing challenge. This study evaluates the effects of five temperature treatments (20, 23, 26, 29 and 32 °C) on population abundance and timing of a colony of ambrosia beetles Xyleborus affinis reared under laboratory conditions and use this data to develop demographic and phenological models. Abundances at each life stage (eggs, larvae, pupae and adult) were examined through periodic destructive sampling; given that it was not possible to track individuals. To assess the effects of temperature on oviposition, development and survival rates we developed a novel estimation strategy based on cohorts, which does not require individual developmental data. Since oviposition was entirely unwitnessed, we assessed competing empirical ovipositional models. Rates of development were computed using a modal rate curve for each life stage, and rates were projected to cohorts in life stages assuming log-normal developmental variance. Temperature-driven survival rates were assumed to be logistic with a quadratic exponent to capture modal temperature dependence. Parameters were estimated simultaneously using minimum negative log posterior likelihood, assuming Poisson distribution of observations and using priors to inform unobserved developmental rates and enforce mechanistic constraints on oviposition models. A parabolic function best described oviposition rate. Optimal developmental temperatures were 30.5 °C, 29 °C and 27.5 °C, with maximum developmental rates of 0.26/day, 0.12/day and 0.23/day for eggs, larvae and pupae, respectively. The survival rates in the range 20-29 °C were equal to 1 in the eggs-to-larvae transition, from 0.72 to 0.35 in larvae-to-pupae transition, and from 0.2 to 0.89 in pupae-to-adults transition. This procedure effectively characterized the direct thermal effects on development and survival of each life stage in the X. affinis under laboratory conditions and would be suitable for estimating temperature dependence for other species in which individual observations are not possible.

Synthesis and Insecticidal Evaluation of Chiral Neonicotinoids Analogs: The Laurel Wilt Case.

Xyleborus sp beetles are types of ambrosia beetles invasive to the United States and recently also to Mexico. The beetle can carry a fungus responsible for the Laurel Wilt, a vascular lethal disease that can host over 300 tree species, including redbay and avocado. This problem has a great economic and environmental impact. Indeed, synthetic chemists have recently attempted to develop new neonicotinoids. This is also due to severe drug resistance to "classic" insecticides. In this research, a series of neonicotinoids analogs were synthesized, characterized, and evaluated against Xyleborus sp. Most of the target compounds showed good to excellent insecticidal activity. Generally, the cyclic compounds also showed better activity in comparison with open-chain compounds. Compounds R-13, 23, S-29, and 43 showed a mortality percent of up to 73% after 12 h of exposure. These results highlight the enantioenriched compounds with absolute R configuration. The docking results correlated with experimental data which showed both cation-π interactions in relation to the aromatic ring and hydrogen bonds between the search cavity 3C79 and the novel molecules. The results suggest that these sorts of interactions are responsible for high insecticidal activity.

Growth temperature effect on mandibles' ontogeny and sexual dimorphism in the ambrosia beetle Xyleborus affinis (Curculionidae: Scolytinae).

Ambrosia beetles from the genus Xyleborus are important vectors of fungal pathogens in forest and agricultural systems, yet the influence of temperature on their morphological development has been poorly studied. Because host colonization and ambrosial fungi cultivation is mostly restricted to females, it is possible to speculate on strong sexual dimorphism expression in secondary sexual characters and ecological segregation between sexes. Here, we determined the effect of different growing temperatures (17, 23, 26 and 29 °C) on mandible ontogeny of larvae and adult individuals of X. affinis, and sexual dimorphism in adults, in shape and size variation using geometric morphometrics. Mandible shape change showed significant differences in magnitude and direction through larval ontogeny among temperature treatments. Sexual shape and size dimorphism were found in adult mandibles, and the degree of sexual dimorphism was dependent on growth temperature, with a significant effect of the interaction between temperature and sex on mandible shape and size variation. Higher morphological differences were observed at the base of mandibles among temperature treatments in adults and a gradual narrowing trend with temperature increments. These findings could have consequences on feeding performance and fungus cultivation inside colonies, potentially influencing their ability to establish populations in new geographical areas.

Xyleborus volvulus (Coleoptera: Curculionidae): Biology and Fungal Associates.

The ambrosia beetle Xyleborus volvulus Fabricius has been reported as a potential vector of the plant pathogen Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva that is affecting avocado orchards in South Florida. In this study, we examined its life cycle, process of gallery formation, gallery structure, and fungal associates by rearing one generation on avocado sawdust medium under control conditions. The adult foundress excavated a vertical tunnel that constituted the main gallery with a length of 2.5 cm, followed by the construction of up to six secondary galleries with a total length of 4.4 cm. The time period for one generation (egg to adult) was 28 days. Teneral males emerged 3 days after the emergence of the first females. The F1 generation did not significantly contribute to gallery expansion. Four species of Raffaelea and nine yeast species were recovered from galleries and beetles. Raffaelea arxii and Candida berthetii were the most frequent symbionts recovered from new adults and galleries. Candida berthetii dominated during the early stages of the gallery development, whereas R. arxii was most frequent in later stages. Other Raffaelea species were inconsistently isolated from galleries, which suggests a strong association between Xyleborus volvulus and both R. arxii and C. berthetii These results suggest that R. arxii is the primary nutritional symbiont of X. volvulus and that yeast species may be pioneer colonizers that assist with the growth of fungal symbionts.IMPORTANCE Ambrosia beetles cultivate fungi in tunnels bored into weakened host trees. This obligate interaction is required for their survival as beetles feed on these symbiotic fungi, and the fungi benefit from transportation by the beetles. Xyleborus volvulus carries many nonpathogenic symbionts; however, recently the acquisition of Raffaelea lauricola (the causal agent of a lethal vascular disease of lauraceous trees) by this beetle has altered its status from wood degrader to potential pest in avocado. We conducted a study to understand the relationship of this beetle and its fungal associates. Our results show that X. volvulus has a multipartite flexible association with different Raffaelea species. The lack of fidelity in the mutualistic association may explain the acquisition of R. lauricola Knowing the beetle biology and its mutualistic interactions furthers an understanding of the beetle's role as a potential vector and in disease transmission.

Genomic Signals of Adaptation towards Mutualism and Sociality in Two Ambrosia Beetle Complexes.

Mutualistic symbiosis and eusociality have developed through gradual evolutionary processes at different times in specific lineages. Like some species of termites and ants, ambrosia beetles have independently evolved a mutualistic nutritional symbiosis with fungi, which has been associated with the evolution of complex social behaviors in some members of this group. We sequenced the transcriptomes of two ambrosia complexes (Euwallacea sp. near fornicatus⁻Fusarium euwallaceae and Xyleborus glabratus⁻Raffaelea lauricola) to find evolutionary signatures associated with mutualism and behavior evolution. We identified signatures of positive selection in genes related to nutrient homeostasis; regulation of gene expression; development and function of the nervous system, which may be involved in diet specialization; behavioral changes; and social evolution in this lineage. Finally, we found convergent changes in evolutionary rates of proteins across lineages with phylogenetically independent origins of sociality and mutualism, suggesting a constrained evolution of conserved genes in social species, and an evolutionary rate acceleration related to changes in selective pressures in mutualistic lineages.

Impact of Rearing Conditions on the Ambrosia Beetle's Microbiome.

Ambrosia beetles, along with termites and leafcutter ants, are the only fungus-farming lineages within the tree of life. Bacteria harbored by ambrosia beetles may play an essential role in the nutritional symbiotic interactions with their associated fungi; however, little is known about the impact of rearing conditions on the microbiota of ambrosia beetles. We have used culture-independent methods to explore the effect of rearing conditions on the microbiome associated with Xyleborus affinis, Xyleborus bispinatus, and Xyleborus volvulus, evaluating different media in laboratory-controlled conditions and comparing wild and laboratory conditions. Our results revealed that rearing conditions affected the fungal and bacterial microbiome structure and had a strong influence on bacterial metabolic capacities. We propose that the rearing conditions influence the ambrosia-associated fungal and bacterial communities. Furthermore, bacterial microbiome flexibility may help beetles adapt to different substrates.

Universal primers for the amplification and sequence analysis of actin-1 from diverse mosquito species.

We report the development of universal primers for the reverse-transcription polymerase chain reaction (RT-PCR) amplification and nucleotide sequence analysis of actin cDNAs from taxonomically diverse mosquito species. Primers specific to conserved regions of the invertebrate actin-1 gene were designed after actin cDNA sequences of Anopheles gambiae, Bombyx mori, Drosophila melanogaster, and Caenorhabditis elegans. The efficacy of these primers was determined by RT-PCR with the use of total RNA from mosquitoes belonging to 30 species and 8 genera (Aedes, Anopheles, Culex, Deinocerites, Mansonia, Psorophora, Toxorhynchites, and Wyeomyia). The RT-PCR products were sequenced, and sequence data were used to design additional primers. One primer pair, denoted as Act-2F (5'-ATGGTCGGYATGGGNCAGAAGGACTC-3') and Act-8R (5'-GATTCCATACCCAGGAAGGADGG-3'), successfully amplified an RT-PCR product of the expected size (683-nt) in all mosquito spp. tested. We propose that this primer pair can be used as an internal control to test the quality of RNA from mosquitoes collected in vector surveillance studies. These primers can also be used in molecular experiments in which the detection, amplification or silencing of a ubiquitously expressed mosquito housekeeping gene is necessary. Sequence and phylogenetic data are also presented in this report.

Indicators for elevated risk of human exposure to host-seeking adults of the Rocky Mountain wood tick (Dermacentor andersoni) in Colorado.

The human-biting adult stage of the Rocky Mountain wood tick (Dermacentor andersoni) can cause tick paralysis in humans and domestic animals and is the primary tick vector in the intermountain west of the pathogens causing Colorado tick fever, Rocky Mountain spotted fever, and tularemia. We conducted drag sampling studies in Poudre Canyon and Rocky Mountain National Park of Larimer County, CO, to determine microhabitat use patterns by host-seeking D. andersoni adults and find environmental factors signaling elevated risk of tick exposure. Big sagebrush (Artemisia tridentata) was found to serve as a general indicator of areas with elevated risk of exposure to host-seeking D. andersoni adults; this likely results from a shared climate tolerance of big sagebrush and D. andersoni. Grass was the favored substrate for host-seeking ticks. Drag sampling of open grass or grass bordering rock or shrub produced abundances of D. andersoni adults significantly higher than sampling of brush. Sampling sites in Rocky Mountain National Park, relative to Poudre Canyon, were characterized by more intense usage by elk (Cervus elaphus) but decreased brush coverage, smaller brush size, and lower abundances of host-seeking D. andersoni adults. There has been a tremendous increase in the population of elk in Rocky Mountain National Park over the last decades and we speculate that this has resulted in an ecological cascade where overgrazing of vegetation by elk is followed by suppression of rodent populations, decreased tick abundance, and, ultimately, reduced risk of human exposure to D. andersoni and its associated pathogens.

Respuesta conductual de Aedes aegypti (Linnaeus, 1762) frente a adulticidas piretroides de uso frecuente en salúd pública / Behavioural response of Aedes aegypti (Linnaeus, 1762) exposed to pyretrhoids insecticides of frequently use in public health

Objetivos. Evaluar la respuesta conductual de la generación F1 de Aedes aegyti (L) colectados en el área metropolitana de Monterrey (Nuevo León, México) frente a tres adulticidas piretroides de uso frecuente en salud pública. Materiales y métodos. Se utilizó un sistema modular novedoso denominado HITSS (High-Throughput Screening System), para evaluar dos respuestas de comportamiento (irritación de contacto y repelencia espacial), así como la toxicidad de tres insecticidas DDT, permetrina y bifentrina a diferentes concentraciones (0,025, 0,25, 25 y 250 nmol/cm2). Resultados. En la concentración 2,5 nmol/cm2, el DDT (4,3 +/- 2,4) y la permetrina (8,0 +/- 1,4) son los insecticidas que tienen mayor efecto irritante (p < 0,05); la bifentrina requiere dosis 20 veces más alta para lograr efectos similares. En repelencia espacial, los tres insecticidas evaluados producen respuestas similares en todas las concentraciones; para DDT de 7 a 14%; permetrina de 9 a 15% y bifentrina de 19 a 27%. La permetrina y bifentrina a concentraciones 0,025 nmol/cm2, producen efectos knockdown superiores a 34%, con una mortalidad 19%, el DDT requiere concentraciones diez veces más altapara lograr efectos similares. Conclusiones. El sistema HITTS puede ser usado para evaluar la respuesta conductual frente a insecticidas.

Objectives. To assess the behavioural response of the F1 generation of Aedes aegyti (L) collected in the Metropolitanarea of Monterrey (Nuevo Leon, Mexico) vs. three adulticides pyrethroids frequently used in public health. Materials and methods. We used a novel modular system called HITSS (High-Throughput Screening System) to evaluate two behavioral responses (contact irritation and spatial repellency), as well as the toxicity of three insecticides DDT, permeathrin and bifenthrin at different concentrations (0.025, 0.25, 25 and 250 nmol/cm2). Results. In the concentration 2.5 nmol/cm2, DDT (4.3 +/- 2.4) and permethrin (8.0 +/- 1.4) were the insecticides that are more irritating effect (p < 0.05); the bifenthrin requires 20 times higher doses to achieve similar effects. In spatial repellency, the three insecticides tested have had a similar responses at all concentrations; DDT: 7 to 14%, permethrin: 9 to 15% and bifenthrin: 19% to 27%. Permethrin and bifenthrin in 0.025 nmol/cm2 concentrations, effects knockdown above 34% with a mortality rate 19%, DDT requires 10 times higher concentrations to achieve similar effects. Conclusions. HITTS system can be used to assess the behavioural response to insecticides.