Lethal effect of Goniozus legneri on Cactoblastis cactorum: A potential biocontrol agent for inundative releases.

Cactoblastis cactorum (Berg) (Lepidoptera: Pyralidae), the cactus moth, is native to South America with a widespread distribution in Argentina. The larvae consume the interior of Opuntia spp. (Cactaceae) plants. The moth was used as a biocontrol agent against invasive non-native Opuntia spp. in many countries around the world. The cactus moth arrived unintentionally in Florida, USA, expanded its range and threatened Opuntia-based agriculture and natural ecosystems in southern North America. The insect is also a pest of cultivated O. ficus-indica L. in Argentina. An endemic South American parasitoid, Goniozus legneri Gordth (Hymenoptera: Bethylidae), is used in inundative biological control programmes against lepidopteran pests. The goal of this work was to evaluate G. legneri as a biocontrol agent to be used in inundative releases against C. cactorum. Mortality of C. cactorum by G. legneri was assessed at different spatial scales, as well as the interactions with Apanteles opuntiarum Martínez & Berta (Hymenoptera: Braconidae), a common Argentine natural enemy of C. cactorum. The ability of G. legneri to paralyse, parasitise and kill C. cactorum was confirmed. The paralysis inflicted on C. cactorum larvae reduced larval damage to the plants by 85%. Using two parasitoid species increased the mortality of C. cactorum larvae, but it was highly dependent on the order of their arrival. The combined mortality caused by both parasitoids was higher than a single one, in particular when G. legneri arrived first (56 ± 1%), suggesting asymmetric competition due to the preference of G. legneri attacking previously parasitised larvae. Goniozus legneri has potential as an inundative biocontrol agent of C. cactorum, but its interaction with the classical biocontrol agent A. opuntiarum needs to be considered.

Best practices, errors, and perspectives of half a century of plant translocation in Italy.

Conservation translocations are becoming common conservation practice, so there is an increasing need to understand the drivers of plant translocation performance through reviews of cases at global and regional levels. The establishment of the Italian Database of Plant Translocation (IDPlanT) provides the opportunity to review the techniques used in 186 plant translocation cases performed in the last 50 years in the heart of the Mediterranean Biodiversity Hotspot. We described techniques and information available in IDPlanT and used these data to identify drivers of translocation outcomes. We tested the effect of 15 variables on survival of translocated propagules as of the last monitoring date with binomial logistic mixed-effect models. Eleven variables significantly affected survival of transplants: life form, site protection, material source, number of source populations, propagation methods, propagule life stage, planting methods, habitat suitability assessment, site preparation, aftercare, and costs. The integration of vegetation studies in the selection of suitable planting sites significantly increased the success of translocation efforts. Although posttranslocation watering had a generally positive effect on translocation outcome, other aftercare techniques did not always increase transplant survival. Finally, we found that how funds were spent appeared to be more important than the actual amount spent. Plant translocations in Italy and in the Mediterranean area should account for the complexity of speciation, gene flow, and plant migrations that has led to local adaptations and has important implications for the choice and constitution of source material.

Mejores prácticas, errores y perspectivas tras medio siglo de reubicaciones botánicas en Italia Resumen Las reubicaciones son una práctica cada vez más común en la conservación, por lo que hay una necesidad creciente por entender los factores del desempeño de las reubicaciones botánicas por medio de la revisión de casos regionales y globales. La creación de la Italian Database of Plant Translocation (IDPlanT) proporciona una oportunidad para revisar las técnicas usadas para los casos de reubicación de 186 plantas realizados durante los últimos cincuenta años en el centro del punto caliente de biodiversidad mediterránea. Describimos las técnicas y la información disponible en IDPlanT y usamos estos datos para identificar los factores involucrados en los resultados de las reubicaciones. Usamos modelos logísticos binomiales de efectos mixtos para analizar el efecto de 15 variables sobre la supervivencia de los propágulos reubicados a partir de la última fecha de monitoreo. Once de las variables afectaron de manera significativa la supervivencia de las plantas: forma de vida, protección del sitio, fuente de materiales, cantidad de poblaciones originarias, método de propagación, etapa de vida del propágulo, método de siembra, evaluación de idoneidad del hábitat, preparación del sitio, cuidados posteriores y costos. La integración de los estudios botánicos a la selección de los sitios idóneos para plantar aumentó el éxito de los esfuerzos de reubicación. Aunque el riego posterior a la reubicación tuvo un efecto positivo general sobre el resultado, las otras técnicas de cuidado posterior no siempre incrementaron la supervivencia de la planta reubicada. Por último, descubrimos que parece ser más importante cómo se utilizan los fondos que la cantidad actual empleada. Las reubicaciones botánicas en Italia y en el área del Mediterráneo deben considerar lo complejo de la especiación, el flujo génico y la migración botánica que han derivado en adaptaciones locales y que han tenido implicaciones importantes para la elección y constitución del material de origen.

Tackling local ecological homogeneity: Finding intraspecific trait variability in local populations of Mediterranean plants.

Local homogeneity, in ecology, is the often undisclosed assumption that variability within populations is negligible or mostly distributed evenly. In large areas, this can lead to the aggregation of different populations without regard for their unique needs and characteristics, such as drought sensitivity and functional trait distributions. Here, we discuss whether this assumption can be justified, and we hypothesize that discerning the source of variation between plasticity and adaptation could be a feasible approach to formulate an informed decision. We test this hypothesis on plants, resorting to a common garden experiment to determine the source of variation of several plant functional traits at a local scale (~60 km) of three wild species: Quercus ilex, Pistacia lentiscus, and Cistus salviifolius. Individuals of each species were sourced from three key sites chosen along a local aridity gradient. Our approach led to the rejection of the local homogeneity assumption for Q. ilex and C. salviifolius at this scale due to the adaptive divergence observed among neighboring populations. This case study provides evidence that addressing local homogeneity can highlight diverging populations in a relatively simple way. We conclude that gathering empirical evidence on intraspecific variability is a feasible approach that can provide researchers with solid bases to decide whether to adopt the local homogeneity assumption or not.

Phenotypic dissimilarity index: Correcting for intra- and interindividual variability when quantifying phenotypic variation.

In trait-based ecology, phenotypic variation (PVar) is often quantified with measures expressing average differences between populations standardized in the range 0-1. However, these measures disregard the within-population trait variability. In addition, some of them cannot be partitioned between populations. These aspects can either alter their interpretation or limit their applicability. To overcome these problems, we propose a new measure, the phenotypic dissimilarity (PhD) index, to quantify PVar between populations in scenarios of varying within-population interindividual trait variability. PhD can also quantify within-population PVar while accounting for intraindividual trait variability. Using simulated and real data, we show that using the PhD index becomes important when the within-population trait variability is not negligible, as in all ecological studies. By accounting for within-population trait variability, the PhD index does not overestimate PVar across an environmental gradient compared to other estimators. Traits inherently vary within species. Accounting for such variability is essential to understanding species' phenotypic responses to environmental cues. The proposed PhD index will provide ecologists with a tool for quantifying PVar within species and compare it between species at different levels of biological organization. We provide an R function to calculate the PhD index.

Effect of Water Stress on Physiological and Morphological Leaf Traits: A Comparison among the Three Widely-Spread Invasive Alien Species Ailanthus altissima, Phytolacca americana, and Robinia pseudoacacia.

Invasive alien species (IAS) are a problem, especially in drought-prone environments such as the Mediterranean Basin where the exacerbation of the already severe conditions could constrain the native species acclimatation degree, creating new opportunities for IAS. Climate change may drive IAS expansions, even if different IAS can vary in their acclimatation response. Thus, it is important to obtain a broader insight of how the different IAS face abiotic stress. This research aimed to compare the effect of the imposed water stress on physiological and morphological leaf traits of Ailanthus altissima (AA), Robinia pseudoacacia (RP), and Phytolacca americana (PA), which are widely spread IAS in the Mediterranean Basin. Our results showed a species-dependent effect of the water stress at a physiological and morphological level, as well as an interaction between species and stress duration. Despite a common strategy characterized by low stomatal control of the photosynthesis, AA, PA, and RP differ in their sensitivity to water stress. In particular, even if AA was characterized by a more water-spending strategy, it was more resistant to water stress than PA and RP. In this view, the key factor was its plasticity to increase leaf mass per area (LMA) in response to water stress.

Daily Temperature Effect on Seedling Growth Dynamic of Three Invasive Alien Species.

A greater relative growth rate (RGR) is positively correlated with a species' ability to deploy a larger leaf area either due to a greater total number of leaves (LN) in the canopy or due to an average size of individual leaves (LA). This study aimed to analyze and compare, (1) the temporal (i.e., daily) RGR, leaf production rate (LPR), and leaf area production rate (LAPR) changes during the early growth stages of three among the most invasive species in the world, namely, Ailanthus altissima, Phytolacca americana, and Robinia pseudoacacia. (2) the interspecific differences in the relationship between RGR, LPR, LAPR, and mean daily air temperature. Our results show that growth dynamics as a function of temperature differ between invasive alien species (IAS). While these differences are partly explained by differences due to the growth form of the investigated species, the three IAS have a different behavior to adjust RGR, LPR, and LAPR with air temperature changes even within the same growth form, and in agreement with species habitat requirements in their native range. In conclusion, the results help disentangle the relative role of RGR, LPR, and LAPR in defining non-native species growth responses to mean daily air temperature also in relation to a species' growth form.

Spatial and host related genomic variation in partially sympatric cactophagous moth species.

Surveys of patterns of genetic variation in natural sympatric and allopatric populations of recently diverged species are necessary to understand the processes driving intra- and interspecific diversification. The South American moths Cactoblastis cactorum, Cactoblastis doddi and Cactoblastis bucyrus are specialized in the use of cacti as host plants. These species have partially different geographic ranges and differ in patterns of host plant use. However, there are areas that overlap, particularly, in northwestern Argentina, where they are sympatric. Using a combination of genome-wide SNPs and mitochondrial data we assessed intra and interspecific genetic variation and investigated the relative roles of geography and host plants on genetic divergence. We also searched for genetic footprints of hybridization between species. We identified three well delimited species and detected signs of hybridization in the area of sympatry. Our results supported a hypothetical scenario of allopatric speciation in the generalist C. cactorum and genetic interchange during secondary geographic contact with the pair of specialists C. bucyrus and C. doddi that probably speciated sympatrically. In both cases, adaptation to new host plants probably played an important role in speciation. The results also suggested the interplay of geography and host plant use as drivers of divergence and limiting gene flow at intra and interspecific levels.

Ecological Niche Modeling to Calculate Ideal Sites to Introduce a Natural Enemy: The Case of Apanteles opuntiarum (Hymenoptera: Braconidae) to Control Cactoblastis cactorum (Lepidoptera: Pyralidae) in North America.

The cactus moth, Cactoblastis cactorum (Berg) (Lepidoptera: Pyralidae), is an invasive species in North America where it threatens Opuntia native populations. The insect is expanding its distribution along the United States Gulf Coast. In the search for alternative strategies to reduce its impact, the introduction of a natural enemy, Apanteles opuntiarum Martínez and Berta (Hymenoptera: Braconidae), is being pursued as a biological control option. To identify promising areas to intentionally introduce A. opuntiarum for the control of C. cactorum, we estimated the overlap of fundamental ecological niches of the two species to predict their common geographic distributions using the BAM diagram. Models were based on native distributional data for both species, 19 bioclimatic variables, and the Maxent algorithm to calculate the environmental suitability of both species in North America. The environmental suitability of C. cactorum in North America was projected from Florida to Texas (United States) along the Gulf coastal areas, reaching Mexico in northern regions. Apanteles opuntiarum environmental suitability showed a substantial similarity with the calculations for C. cactorum in the United States. Intentional introductions of A. opuntiarum in the actual distribution areas of the cactus moth are predicted to be successful; A. opuntiarum will find its host in an environment conducive to its survival and dispersal.

Inherent variation of functional traits in winter and summer leaves of Mediterranean seasonal dimorphic species: evidence of a 'within leaf cohort' spectrum.

The covariation pattern among leaf functional traits involved in resource acquisition has been successfully provided by the leaf economic spectrum (LES). Nevertheless, some aspects such as how the leaf trait variation sources affect LES predictions are still little investigated. Accordingly, the aim of this paper was to test whether leaf trait variations within different leaf cohorts could alter LES. Improving this knowledge can extend the potential of trait-based approaches in simulating future climate effects on ecosystems. A database on leaf morphological and physiological traits from different leaf cohorts of Cistus spp. was built by collecting data from literature. These species are seasonal dimorphic shrubs with two well-defined leaf cohorts during a year: summer leaves (SL) and winter leaves (WL). Traits included: leaf mass area (LMA), leaf thickness (LT), leaf tissue density (LTD), net photosynthetic rate on area (Aa) and mass (Am) base, nitrogen content on area (Na) and mass (Nm) base. The obtained patterns were analysed by standardized major axis regression and then compared with the global spectrum of evergreens and deciduous species. Climatic variable effect on leaf traits was also tested. Winter leaves and SL showed a great inherent variability for all the considered traits. Nevertheless, some relationships differed in terms of slopes or intercepts between SL and WL and between leaf cohorts and the global spectrum of evergreens and deciduous. Moreover, climatic variables differently affected leaf traits in SL and WL. The results show the existence of a 'within leaf cohort' spectrum, providing the first evidence on the role of leaf cohorts as LES source of variation. In fact, WL showed a high return strategy as they tended to maximize, in a short time, resource acquisition with a lower dry mass investment, while SL were characterized by a low return strategy.

Short-term physiological plasticity: Trade-off between drought and recovery responses in three Mediterranean Cistus species.

Short-term physiological plasticity allows plants to thrive in highly variable environments such as the Mediterranean ecosystems. In such context, plants that maximize physiological performance under favorable conditions, such as Cistus spp., are generally reported to have a great cost in terms of plasticity (i.e., a high short-term physiological plasticity) due to the severe reduction of physiological performance when stress factors occur. However, Cistus spp. also show a noticeable resilience ability in response to stress factors. We hypothesized that in Cistus species the short-term physiological response to stress and that to subsequent recovery can show a positive trade-off to offset the costs of the photosynthetic decline under drought. Gas exchange, chlorophyll fluorescence, and water relations were measured in C. salvifolius, C. monspeliensis, and C. creticus subsp. eriocephalus during an imposed experimental drought and subsequent recovery. Plants were grown outdoor in common garden conditions from seeds of different provenances. The short-term physiological response to stress and that to recovery were quantified via phenotypic plasticity index (PIstress and PIrecovery, respectively). A linear regression analysis was used to identify the hypothesized trade-off PIstress-PIrecovery. Accordingly, we found a positive trade-off between PIstress and PIrecovery, which was consistent across species and provenances. This result contributes in explaining the profit, more than the cost, of a higher physiological plasticity in response to short-term stress imposition for Cistus spp because the costs of a higher PIstress are payed back by an as much higher PIrecovery. The absence of leaf shedding during short-term drought supports this view. The trade-off well described the relative variations of gas exchange and water relation parameters. Moreover, the results were in accordance with the ecology of this species and provide the first evidence of a consistent trade-off between the short-term physiological responses to drought and recovery phases in Mediterranean species.

Geographic distribution suggests that Solenopsis invicta is the host of predilection for Solenopsis invicta virus 1.

Solenopsis invicta virus 1 (SINV-1) was found regularly and prevalently in S. invicta. In sampled locations where S. invicta and S. geminata are sympatric (specifically, Gainesville, FL and Travis, TX), SINV-1 was detected in S. geminata. Conversely, in areas in which S. geminata and S. invicta are allopatric, SINV-1 was not detected in S. geminata; these locations included north Australia (n=12), southern Mexico (n=107), Hawaii (n=48), Taiwan (n=12), and the Johnston Atoll (n=6). A similar relationship was observed for S. richteri. In areas in which S. invicta and S. richteri were sympatric, SINV-1 was detected in the S. richteri population, but in areas in which S. invicta and S. richteri were allopatric, SINV-1 was not detected. These occurrences suggest that S. invicta is the host of predilection, or preferred host for SINV-1, and that the congenerics, S. geminata and S. richteri serve as either accidental, reservoir, or transfer hosts. The minus genome strand of SINV-1 was detected in S. geminata and S. richteri indicating that these species may serve as functional hosts capable of supporting SINV-1 replication. SINV-1 was not detected in S. xyloni regardless of its proximity to S. invicta. These results suggest that SINV-1 may be an example of pathogen spillover or pollution.

Morphological, anatomical and physiological leaf traits of Q. ilex, P. latifolia, P. lentiscus, and M. communis and their response to Mediterranean climate stress factors.

BACKGROUND: Limitations to plant growth imposed by the Mediterranean climate are mainly due to carbon balance in response to stress factors. In particular, water stress associated to high air temperature and irradiance in summer causes a marked decrease in CO2 assimilation. Air temperature sensitivity of photosynthesis (PN) differs from that of leaf respiration (RD). PN often decreases sharply at temperature above its optimum while RD increases exponentially over short term rises in temperature. Nevertheless, the impact of water deficit on RD is still far from clear with reports in literature including decreases, maintenance or increases in its rates. The ratio RD/PN can be considered a simple approach to leaf carbon balance because it indicates the percentage of photosynthates that is respired. RESULTS: The results underline different morphological, anatomical and physiological traits of the evergreen species co-occurring in the Mediterranean maquis which are indicative of their adaptive capability to Mediterranean stress factors. The ratio RD/PN varies from 0.15 ± 0.04 in autumn, 0.24 ± 0.05 in spring through 0.29 ± 0.15 in winter to 0.46 ± 0.11 in summer. The lower RD/PN in autumn and spring underlines the highest PN rates during the favorable periods when resources are not limited and leaves take in roughly three to five times more CO2 than they lose by respiration. On the contrary, the highest RD/PN ratio in summer underlines the lowest sensitivity of respiration to drought. Among the considered species, Quercus ilex and Pistacia lentiscus have the largest tolerance to low winter temperatures while Phillyrea latifolia and Myrtus communis to drought, and Phillyrea latifolia the highest recovery capability after the first rainfall following drought. CONCLUSIONS: The Mediterranean evergreen specie shows a different tolerance to Mediterranean climate stress factors. The predicted global warming might differently affect carbon balance of the considered species, with a possible change in Mediterranean shrublands composition in the long-term. Understanding the carbon balance of plants in water limited environments is crucial in order to make informed land management decisions. Moreover, our results underline the importance of including seasonal variations of photosynthesis and respiration in carbon balance models.

Obstructive sleep apnea and bone mineral density in obese patients.

CONTEXT: Obesity and its co-morbidities may adversely affect bone mineral density (BMD). Obstructive sleep apnea (OSA) is a major complication of obesity. To date, the effects of OSA on BMD in obese patients have been poorly studied. OBJECTIVE: To examine whether the severity of OSA independently correlates with BMD in obese patients. METHODS: One hundred and fifteen obese subjects with OSA (Apnea/Hypopnea Index [AHI] ≥5 events per hour) were included in the study. BMD was measured at lumbar spine, total hip, and femoral neck by dual energy X-ray absorptiometry. Body mass index, lean mass, and representative measures of metabolic syndrome (waist circumference, fasting plasma glucose, blood pressure, HDL-cholesterol, triglycerides) and inflammation (ESR, CRP, fibrinogen) were also evaluated. RESULTS: BMD did not differ among obese individuals regardless of OSA severity. Correlation coefficient analysis for all the covariates showed a lack of association between AHI and BMD that was strongly influenced by age and weight. CONCLUSION: Our study does not support an independent association between AHI and BMD in obese patients. Controlled studies involving a greater number of patients are warranted.

Native ecotypic variation and the role of host identity in the spread of an invasive herbivore, Cactoblastis cactorum.

Environmental niche models (ENMs) have gained enormous popularity as tools to investigate potential changes in species distributions resulting from climate change and species introductions. Despite recognition that species interactions can influence the dynamics of invasion spread, most implementations of ENMs focus on abiotic factors as the sole predictors of potential range limits. Implicit in this approach is the assumption that biotic interactions are relatively unimportant, either because of scaling issues, or because fundamental and realized niches are equivalent in a species' native range. When species are introduced into exotic landscapes, changes in biotic interactions relative to the native range can lead to occupation of different regions of niche space and apparent shifts in physiological tolerances. We use an escaped biological control organism, Cactoblastis cactorum (Berg.), to assess the role of the environmental envelope as compared with patterns of host-herbivore associations based on collections made in the native range. Because all nonnative populations are derived from a single C. cactorum ecotype, we hypothesize that biotic interactions associated with this ecotype are driving the species' invasion dynamics. Environmental niche models constructed from known native populations perform poorly in predicting nonnative distributions of this species, except where there is an overlap in niche space. In contrast, genetic isolation in the native range is concordant with the observed pattern of host use, and strong host association has been noted in nonnative landscapes. Our results support the hypothesis that the apparent shift in niche space from the native to the exotic ranges results from a shift in biotic interactions, and demonstrate the importance of considering biotic interactions in assessing the risk of future spread for species whose native range is highly constrained by biotic interactions.

Quercus ilex L. carbon sequestration capability related to shrub size.

CO(2) sequestration capacity of Quercus ilex L., an evergreen species developing in shrub and forest communities widely distributed in the Mediterranean Basin, was analysed. Experiments were carried out in the period of January to December 2009 on 20 shrubs of different size, growing at the Botanical Garden of Rome. At shrub level, the largest differences concern total photosynthetic leaf surface area per shrub and shrub volume. Shrubs structure significantly contribute to reduce total irradiance and air temperature below the canopy. Leaf mass per area is higher in sun leaves than in shade ones (20 ± 1 and 12 ± 2 mg cm( -2), respectively). Sun leaves are also characterised by the highest leaf thickness (78% higher in sun than in shade leaves), the spongy parenchyma thickness (71% higher in sun than in shade leaves) and the highest adaxial cuticle thickness (7.2 ± 1.2 and 4.7 ± 0.5 µm, respectively). Net photosynthetic rates (P (N)) of sun and shade leaves are the highest in spring, and shade leaves contribute 6% to the whole shrub P (N). Q. ilex CO(2) sequestration depends on shrub size. In particular, the CO(2) sequestration per shrub was 0.20 ± 0.02 Kg CO(2) year( -1) in small shrubs, and it was 75% and 98% lower than in medium and large ones. The highest CO(2) sequestration is measured in spring, decreasing 77% during drought. Q. ilex may play a significant role in mitigating carbon dioxide concentration and lowering air and soil temperature in areas around the Mediterranean Basin.

Exploitation of a high genomic mutation rate in Solenopsis invicta virus 1 to infer demographic information about its host, Solenopsis invicta.

The RNA-dependent RNA polymerase (RdRp) region of Solenopsis invicta virus 1 (SINV-1) was sequenced from 47 infected colonies of S. invicta, S. richteri, S. geminata, and S. invicta/richteri hybrids collected from across the USA, northern Argentina, and northern Taiwan in an attempt to infer demographic information about the recent S. invicta introduction into Taiwan by phylogenetic analysis. Nucleotide sequences were calculated to exhibit an overall identity of >90% between geographically-separated samples. A total of 171 nucleotide variable sites (representing 22.4% of the region amplified) were mapped across the SINV-1 RdRp alignment and no insertions or deletions were detected. Phylogenetic analysis at the nucleotide level revealed clustering of Argentinean sequences, distinct from the USA sequences. Moreover, the SINV-1 RdRp sequences derived from recently introduced populations of S. invicta from northern Taiwan resided within the multiple USA groupings implicating the USA as the source for the recent introduction of S. invicta into Taiwan. Examination of the amino acid alignment for the RdRp revealed sequence identity >98% with only nine amino acid changes observed. Seven of these changes occurred in less than 4.3% of samples, while 2 (at positions 1266 and 1285) were featured prominently. Changes at positions 1266 and 1285 accounted for 36.2% and 34.0% of the samples, respectively. Two distinct groups were observed based on the amino acid residue at position 1266, Threonine or Serine. In cases where this amino acid was a Threonine, 90% of these sequences possessed a corresponding Valine at position 1285; only 10% of the Threonine(1266)-containing sequences possessed an Isoleucine at the 1285 position. Among the Serine(1266) group, 76% possessed an Isoleucine at position 1285, while only 24% possessed a Valine. Thus, it appears that the Threonine(1266)/Valine(1285) and Serine(1266)/Isoleucine(1285) combinations are predominant phenotypes.

Complete genome sequence of an Argentinean isolate of Solenopsis invicta virus 3.

Solenopsis invicta virus 3 (SINV-3) is a recently described positive-strand RNA virus that infects the red imported fire ant, S. invicta. The genome of an Argentinean isolate of Solenopsis invicta virus 3 (SINV-3(ArgSF )) obtained from the Santa Fe region of Argentina was sequenced in entirety. Assembly of nine overlapping fragments yielded a consensus genome sequence 10,386 nucleotides long, excluding the poly(A) tail present on the 3' end (Genbank accession number GU017972). With the exception of the poly(A) tail, the genome length of SINV-3(ArgSF ) was identical to the North American isolate (SINV-3(USDM )). The SINV-3(ArgSF ) genome possessed three major open reading frames (ORFs) (comprised of >or=100 codons) in the sense orientation; SINV-3(USDM ) possessed only two. ORFs 1 and 2 had identical start and stop genome positions for both isolates. Blastp analysis of the translated ORF 1 of SINV-3(ArgSF ) recognized conserved domains for helicase, protease, and RNA-dependent RNA polymerase. These domains and their corresponding positions were identical to those reported for SINV-3(USDM ). ORF 2a, unique to the SINV-3(ArgSF ) genome, was also found in frame 2 and had a canonical start codon located at nucleotide position 8,351 and a stop codon ending at position 8,827. Blastp analysis of the translated amino acid sequence of ORF 2a revealed no significant similarity in the Genbank database. The two SINV-3 isolates exhibited 96.2% nucleotide sequence identity across the entire genome. The amino acid sequences of ORFs 1 and 2 exhibited higher identities (99.0 and 98.2%, respectively) than the corresponding nucleotide regions within the genome. These data indicated that the nucleotide differences between the SINV-3 isolates were largely synonymous. This observation was corroborated by codon substitution rate analysis. Thus, the majority of the SINV-3 codon changes were silent in the two polyproteins, indicating purifying selection pressure on the viral genome.

Multiplex detection of Solenopsis invicta viruses -1, -2, and -3.

Multiplex reverse transcription and polymerase chain reaction (PCR) methods were developed to detect Solenopsis invicta viruses -1, -2, and -3 simultaneously in their host, the red imported fire ant, S. invicta. cDNA synthesis was conducted in a single reaction containing an oligonucleotide primer specific for each virus. Multiplex PCR was subsequently conducted with oligonucleotide primer pairs specific for each virus. The method was specific and sensitive, capable of detecting as few as 500 copies of the viral genomes consistently. Specificity was verified by PCR and amplicon sequencing. The method was evaluated against field-collected samples of ant workers from colonies in Argentina (n=135 ant colonies) and the United States (n=172 ant colonies). The prevalence of each virus in fire ant colonies varied considerably from site to site. A number of colonies exhibited multiple virus infections. However, the multiple SINV infection rate was lower than for single infections. Comparison of viral infection prevalence between S. invicta colonies in Argentina and the U.S. showed no statistical differences, regardless of infection category. This method is anticipated to facilitate epidemiological and related studies concerning the S. invicta viruses in fire ants.