Comparative metatranscriptomics reveals effect of host plant on microbiota gene expression of Anastrepha obliqua (Diptera: Tephritidae) larvae.

The microbiota associated with phytophagous insects perform several functions that help insects exploit plant resources. Thus, microorganisms contribute to the dispersal of phytophagous species to new host plants, thereby promoting diversification. In this study, metatranscriptomic analysis was used to compare the gene expression of the microbiome of Anastrepha obliqua Macquart larvae feeding on 3 of its host plants: Spondias purpurea L (red mombin), Mangifera indica L (mango), and Averrhoa carambola L (starfruit). To identify differential gene expression in relation to the host plant, transcript abundance was compared. The results of the taxonomic and functional beta-diversity analysis showed that there were significant differences in the structures and activities of the microbial communities depending on the infested plant. Among the microorganisms, bacteria and fungi were active components of the microbiota. Differential expression analyses showed that the different active genes in each of the plants analyzed were mainly grouped into categories related to carbohydrate and amino acid metabolism, with some of these genes coding for cytochrome o ubiquinol oxidase, cytochrome c, and the enzyme isocitrate dehydrogenase. The microbiota of A. carambola larvae differed more at the level of community structure and gene function, possibly due to the different nutritional composition of the A. carambola and the presence of a set of secondary metabolites specific to the family Oxalidaceae. In conclusion, the transcriptional activity of the microbiota of A. obliqua larvae is influenced by diet, which is important because it could influence the performance of the insect on each of its different host plants.

Genomic Traces of the Fruit Fly Anastrepha obliqua Associated with Its Polyphagous Nature.

Anastrepha obliqua (Macquart) (Diptera: Tephritidae) is an important pest in the neotropical region. It is considered a polyphagous insect, meaning it infests plants of different taxonomic families and readily colonizes new host plants. The change to new hosts can lead to diversification and the formation of host races. Previous studies investigating the effect of host plants on population structure and selection in Anastrepha obliqua have focused on the use of data from the mitochondrial DNA sequence and microsatellite markers of nuclear DNA, and there are no analyses at the genomic level. To better understand this issue, we used a pooled restriction site-associated DNA sequencing (pooled RAD-seq) approach to assess genomic differentiation and population structure across sympatric populations of Anastrepha obliqua that infest three host plants-Spondias purpurea (red mombin), Mangifera indica (mango) of the family Anacardiaceae and Averrhoa carambola (carambola) of the family Oxalidaceae-in sympatric populations of the species Anastrepha obliqua of Inter-Andean Valley of the Cauca River in southwestern Colombia. Our results show genomic differentiation of populations from carambola compared to mango and red mombin populations, but the genetic structure was mainly established by geography rather than by the host plant. On the other hand, we identified 54 SNPs in 23 sequences significantly associated with the use of the host plant. Of these 23 sequences, we identified 17 candidate genes and nine protein families, of which four protein families are involved in the nutrition of these flies. Future studies should investigate the adaptive processes undergone by phytophagous insects in the Neotropics, using fruit flies as a model and state-of-the-art molecular tools.

Mutualistic interaction of aphids and ants in pepper, Capsicum annuum and Capsicum frutenscens (Solanaceae) / Interacción mutualista de pulgones y hormigas en pimiento, Capsicum annuum y Capsicum frutenscens (Solanaceae)

Abstract Introduction: Adequate biological identification is fundamental for establishing integrated pest management programs and identifying the trophic and mutualist relationships that can affect pest population dynamics. Aphids are the main pest of pepper Capsicum spp. (Solanaceae) crops in Southwestern Colombia, due to their role as vectors of viruses. However, the identification of aphid species is complex, limiting the investigations performed to address their interactions with other organisms. Ants and aphids present a facultative mutualistic relationship, that promotes the growth of hemipteran colonies, for this reason, the study of the ecological mutualistic association between aphids and ants is important. Objective: The main objective was to discriminate the aphid species present in commercial crops of Capsicum spp., and to identify the ant community that attends the aphid colonies and its effects on the size of the aphid colonies. Methods: Aphid species, and their ant mutualist, were collected from Capsicum annuum and Capsicum frutescens, in the Cauca valley, Southwestern Colombia. We used the DNA barcoding approach to identify aphid species, and the ants were identified by morphology-based taxonomy. To evaluate the effect of ant care on the size and structure of aphid colonies, generalized linear models were calculated using as the response variables the total number of aphids for each colony and the proportion of nymphs. Results: The aphid species that attack pepper crops, are: Aphis gossypii and Myzus persicae (Hemiptera: Aphididae), with A. gossypii being the species that interacts with ants (19 ant species). A. gossypii colonies attended by ants had larger sizes and more nymphs per colony, than those not attended. Conclusions: Although the aphid-ant interaction is not species-specific, it is necessary to consider its role in the propagation of viral diseases in peppers and to determine how this interaction may affect regional biological control strategies.

Resumen Introducción: La adecuada identificación biológica es fundamental para establecer programas de manejo integrado de plagas e identificar las relaciones tróficas y mutualistas que pueden afectar la dinámica poblacional de insectos plaga. Los áfidos son las principales plagas del ají Capsicum spp. (Solanaceae) en el suroccidente colombiano, debido a su rol como vectores de virus. Sin embargo, su identificación es compleja, y limita las investigaciones que intentan revelar sus interacciones con otros organismos. Las hormigas y los áfidos presentan una relación mutualista facultativa, que promueve el crecimiento de las colonias de los hemípteros, por esta razón, el estudio de la asociación ecológica y mutualista entre áfidos y hormigas es importante. Objetivo: El principal objetivo de esta investigación fue discriminar las especies de áfidos presentes en cultivos comerciales de Capsicum spp., e identificar la comunidad de hormigas que atiende las colonias de áfidos y su efecto en el tamaño de las colonias de áfidos. Métodos: Los áfidos, y las hormigas mutualistas de estos áfidos, se recolectaron de Capsicum annuum y Capsicum frutescens, en el valle del rio Cauca, en el suroccidente colombiano. Se empleó el Código de barras del ADN para identificar las especies de áfidos, y las hormigas se identificaron empleando taxonomía basada en morfología. Para evaluar el efecto que tiene el cuidado de las hormigas sobre el tamaño de las colonias de áfidos, se empleó un modelo lineal generalizado, utilizando como variables de respuesta el número total de áfidos por cada colonia y la proporción de ninfas por colonia. Resultados: Las especies de áfidos que atacan los cultivos de ají, son: Aphis gossypii y Myzus persicae (Hemiptera: Aphididae), siendo A. gossypii la especie que interactúa con hormigas (19 especies). Las colonias de A. gossypii atendidas por hormigas presentan mayor tamaño y número de ninfas, que aquellas desatendidas. Conclusiones: Aunque la interacción áfido-hormiga no es especie específica, es necesario considerar su rol en la propagación de enfermedades virales en plantas cultivadas y determinar cómo esta interacción puede afectar la implementación de estrategias de control biológico.

Variations in the Bacterial Communities in Anastrepha obliqua (Diptera: Tephritidae) According to the Insect Life Stage and Host Plant.

Insects have established close relationships with a wide variety of microorganisms, which play a key role in insect ecology and evolution. Fruit flies in the Tephritidae family have economic importance at the global level, including species such as Anastrepha obliqua, which is an important pest in the neotropical region. Although several studies have been performed on the microbiota associated with fruit flies, there are still large gaps in our knowledge about the bacterial communities on the genus Anastrepha. During this study, we used high-throughput sequencing to characterize the bacterial communities of the polyphagous fly A. obliqua, and we evaluated the effect of the life stage (larvae and adults) and host plant (three plant species) on the structure of these communities. Our results show that the bacterial communities in A. obliqua appears to be structured according to the insect life stage and the host plant. The predominant genera belonging to the phylum Proteobacteria were Wolbachia and Enterobacter in both larvae and adults, and they displayed differences in abundance between them, with Wolbachia sp. being more abundant in larvae and Enterobacter sp. being more abundant in adults. Differences in the structures of the bacterial communities were also observed according to the host plant with higher abundance of Enterobacter and Acetobacter bacteria in mango and plum fruits. Based on our results, it can be hypothesized that the bacterial communities on A. obliqua reorganize according to the needs of these insects during their different life stages and could also play an important role in the establishment of this fly species on different host plants. This study represents the first approach to understanding microorganism-insect interactions in fruit flies in Colombia.

Bacterial communities of Aphis gossypii and Myzus persicae (Hemiptera: Aphididae) from pepper crops (Capsicum sp.).

Insects harbor a wide variety of microorganisms that form complex and changing communities and play an important role in the biology and evolution of their hosts. Aphids have been used as model organisms to study microorganism-insect interactions. Almost all aphids are infected with the obligate endosymbiont Buchnera aphidicola and can host different bacteria that allow them to acquire traits of agronomic importance, such as resistance to high temperatures and/or defense against natural enemies. However, the bacterial communities of most aphid species remain poorly characterized. In this study, we used high-throughput DNA sequencing to characterize the bacterial communities of Aphis gossypii and Myzus persicae from two cultivable pepper species, Capsicum frutescens (Tabasco variety) and C. annuum (Cayenne variety), in four localities of southwestern Colombia. In addition, we evaluated the dynamics of A. gossypii-associated microorganisms on a seasonal basis. Our results show that the bacterial communities of A. gossypii and M. persicae are dominated by the primary endosymbiont B. aphidicola, while the presence of the facultative symbiont Arsenophonus sp. was only detected in one A. gossypii population from cayenne pepper. In addition to these two known symbionts, eight bacterial OTUs were identified that presented a frequency of 1% or more in at least one of the analyzed populations. The results show that the bacterial communities of aphids associated with pepper crops appears to be structured according to the host aphid species and the geographical location, while no differences were observed in the diversity of bacteria between host plants. Finally, the diversity and abundance of the A. gossypii bacterial community was variable among the four sampling points evaluated over the year and showed a relation with the aphid's population dynamics. This study represents the first approach to the knowledge of the bacterial community present in chili pepper aphids from Colombia. Nevertheless, more in-depth studies, including replicates, are required to confirm the patterns observed in the microbial communities of aphids from pepper crops.

Saving DNA from museum specimens: The success of DNA mini-barcodes in haplotype reconstruction in the genus Anastrepha (Diptera: Tephritidae).

The fragmentation of DNA in historical specimens is very common, so obtaining sequences that allow molecular identification and the study of diversity is quite challenging. In this study, we used preserved and fresh specimens of the fruit fly genus Anastrepha, a genus of economic impact of fruit crops of the Neotropic. From these specimens, we evaluated: (1) the success PCR amplification rates of mini-barcodes fragments of the cytochrome c oxidase subunit I (COI) gene, and (2) the usefulness of mini-barcodes in the reconstruction of haplotypes for the identification of species and the diversity analysis. We used 93 specimens from 12 species, which had been preserved in 70% ethanol for more than 20 years. Internal primers were designed in the COI region and primers available in the literature were also evaluated. We obtained amplifications for 62.36% of the samples processed, and reconstructed haplotypes between 171 bp and 632 bp. Variable amplification rates between combinations of primers and between species were obtained, and molecular identification of some museum specimens was achieved. It was also possible to compare the haplotypes obtained in four species from which both fresh and museum samples were available. Our results also show the importance of the adjustment of the primers for the amplification, allowing to amplify fragments of up to 400 bp. The use available resources in biological collections is key to increasing knowledge of species of interest, and by means of the amplification of mini-barcodes, short sequences can be obtained that allow the molecular identification of specimens and the reconstruction of haplotypes with multiple purposes.

Molecular analysis of the expression of a crtB transgene and the endogenous psy2-y 1 and psy2-y 2 genes of cassava and their effect on root carotenoid content.

A conventional breeding program was established to transfer the bacterial phytoene synthase transgene-crtB-from a transgenic, white-rooted cassava to yellow-rooted cassava plants carrying the endogenous phytoene synthase alleles named psy2-y 1 and/or psy2-y 2. Combining endogenous phytoene synthase enzymes (PSYs) with CRTB in a single cassava plant would allow the molecular dissection of individual allele contributions to carotenoid synthesis and/or accumulation in cassava roots. The simultaneous expression of the crtB transgene and psy2-y 2 in individuals planted in the field coincided with higher total, HPLC-quantified carotenoid content in roots, although the variability among replications (plants) precluded the detection of statistically significant differences. Nevertheless, the highest total carotenoid content in roots within a family coincided with one individual of the F1 progeny carrying both psy2-y 2 and crtB genes. The results also indicated the presence of at least one more key gene-different from psy or crtB-which too is necessary for the synthesis and/or accumulation of Pro-Vitamin A carotenoids in cassava roots.

Genetic diversity and population structure of Anastrepha striata (Diptera: Tephritidae) in three natural regions of southwestern Colombia using mitochondrial sequences.

Anastrepha striata is widely distributed across the Americas and is a pest of economically important crops, especially crops of the Myrtaceae family. Insect population structures can be influenced by the presence of physical barriers or characteristics associated with habitat differences. This study evaluated the effect of the Western Andes on the population structure of A. striata. Individuals were collected from Psidium guajava fruits from three natural regions of southwestern Colombia (Pacific Coast, mountainous region and the inter-Andean valley of the Cauca River). Based on a 1318 bp concatenated of the genes Cytochrome Oxidase subunit I (COI) and NADH dehydrogenase subunit 6 (ND6), 14 haplotypes with few changes among them (between 1 and 3) were found. There was only one dominant haplotype in all three regions. No genetic structure associated with the three eco-geographical regions of the study was found. Moreover, the Western Andes are not an effective barrier for the genetic isolation of the populations from the Pacific Coast compared with the inter-Andean valley populations. This genetic homogeneity could be partially due to anthropogenic intervention, which acts as a dispersal agent of infested fruits. Another hypothesis to explain the lack of structure would be the relatively recent arrival of A. striata to the region, as indicated by an analysis of the demographic history, which reveals a process of population expansion. This study represents the first attempt to understand the population genetics of A. striata in Colombia and could contribute to the integral management of this pest.

Contrasting demographic history and gene flow patterns of two mangrove species on either side of the Central American Isthmus.

Comparative phylogeography offers a unique opportunity to understand the interplay between past environmental events and life-history traits on diversification of unrelated but co-distributed species. Here, we examined the effects of the quaternary climate fluctuations and palaeomarine currents and present-day marine currents on the extant patterns of genetic diversity in the two most conspicuous mangrove species of the Neotropics. The black (Avicennia germinans, Avicenniaceae) and the red (Rhizophora mangle, Rhizophoraceae) mangroves have similar geographic ranges but are very distantly related and show striking differences on their life-history traits. We sampled 18 Atlantic and 26 Pacific locations for A. germinans (N = 292) and R. mangle (N = 422). We performed coalescence simulations using microsatellite diversity to test for evidence of population change associated with quaternary climate fluctuations. In addition, we examined whether patterns of genetic variation were consistent with the directions of major marine (historical and present day) currents in the region. Our demographic analysis was grounded within a phylogeographic framework provided by the sequence analysis of two chloroplasts and one flanking microsatellite region in a subsample of individuals. The two mangrove species shared similar biogeographic histories including: (1) strong genetic breaks between Atlantic and Pacific ocean basins associated with the final closure of the Central American Isthmus (CAI), (2) evidence for simultaneous population declines between the mid-Pleistocene and early Holocene, (3) asymmetric historical migration with higher gene flow from the Atlantic to the Pacific oceans following the direction of the palaeomarine current, and (4) contemporary gene flow between West Africa and South America following the major Atlantic Ocean currents. Despite the remarkable differences in life-history traits of mangrove species, which should have had a strong influence on seed dispersal capability and, thus, population connectivity, we found that vicariant events, climate fluctuations and marine currents have shaped the distribution of genetic diversity in strikingly similar ways.

Differential expression patterns among heat-shock protein genes and thermal responses in the whitefly Bemisia tabaci (MEAM 1).

There is convincing evidence that heat-shock proteins (HSP) are upregulated by stress conditions in insects; however, the relative contribution of each HSP gene to the heat-shock response remains unclear. Here we considered the whitefly Bemisia tabaci (MEAM 1), a phloem feeder and invasive species whose molecular stress response is an important mechanism for overcoming heat stress. We assessed the expression of the hsp23, 40, 70 and 90 genes at the mRNA level when submitted to heat shocks of 40 and 44°C/1h (control at 25°C). For this, we evaluated a set of available and suitable reference genes in order to perform data normalization using the real-time polymerase chain reaction (qRT-PCR) technique, and then confirmed the production of HSP70 protein based on Western blot. Results were compared with the hardening capacity of B. tabaci, measured by fitness components as a response to heat shocks, using 40°C as the induction temperature. Three of the four genes (hsp23, 70 and 90) were upregulated by heat stress at mRNA, showing differential expression patterns. Hsp70 expression was confirmed at the protein level. Hardening significantly increased fitness following heat stress, suggesting that HSPs may contribute to hardening capacity in B. tabaci. Potential role of each gene in the heat-shock response for whiteflies is discussed.