No fitness costs are induced by Spiroplasma infections of Aphis citricidus reared on two different host plants / Custo adaptativo induzido por Spiroplasma em Aphis citricidus em duas plantas hospedeiras

Abstract Aphids can harbor several secondary symbionts that alter important aphid-related ecological traits, such as defense against natural enemies, heat tolerance and host plant utilization. One of these secondary symbionts, Spiroplasma, is well known in Drosophila as a sex modulator and by interacting with the host immune system. However, little is known on the effects of Spiroplasma on aphids, such as its influence on the host immune defense against fungi and on host plant utilization. Aphid infections by Spiroplasma are known to be low and few aphid species were reported to be infected with this secondary symbiont, however aphids belonging to the genus Aphis in neotropical regions show high infection rates by Spiroplasma. Thus, we investigated the association of Spiroplasma with the tropical aphid Aphis citricidus through comparative biology experiments on two host plants with different nutritional value to the aphid. We demonstrate Spiroplasma induced no significant fitness costs to A. citricidus on either host plant as no changes in the fitness traits we assessed were observed. Spiroplasma infection only induced sutle changes on host longevity and fecundity. Therefore, we concluded Spiroplasma established a neutral interaction with A. citricidus under the selection pressure we tested, and argue on stress conditions that could better demonstrate the role of Spiroplasma in A. citricidus bioecology and associated costs involved.

Resumo Pulgões podem abrigar vários simbiontes secundários que alteram características ecológicas importantes, como defesa contra inimigos naturais, tolerância ao calor e utilização da planta hospedeira. Um desses simbiontes secundários, Spiroplasma, é estudado em Drosophila por alterar a razão sexual e interagir com o sistema imunológico do hospedeiro. No entanto, pouco se sabe sobre os efeitos do Spiroplasma em pulgões, como sua influência na defesa contra fungos entomopatogénicos ou na utilização da planta hospedeira, por exemplo. Spiroplasma foi identificado em poucas espécies de pulgão e geralmente em baixa densidade, contudo pulgões pertencentes ao gênero Aphis em regiões neotropicais apresentam altas taxas de infecção por Spiroplasma. Assim, investigamos a associação entre Spiroplasma e o pulgão neotropical Aphis citricidus por meio de biologia comparativa em duas plantas hospedeiras com diferentes valores nutricionais para o pulgão. Spiroplasma não causou custo adaptativo significativo para A. citricidus em ambas planta hospedeira. A infecção por Spiroplasma induziu apenas pequenas mudanças na longevidade e fecundidade do hospedeiro. Portanto, concluímos que Spiroplasma estabeleceu uma interação neutra com A. citricidus sob ausência de pressão de seleção. Entretanto, discutimos sobre condições de estresse que possam demonstrar o papel de Spiroplasma na bioecologia de A. citricidus e os possíveis custos envolvidos.

No fitness costs are induced by Spiroplasma infections of Aphis citricidus reared on two different host plants.

Aphids can harbor several secondary symbionts that alter important aphid-related ecological traits, such as defense against natural enemies, heat tolerance and host plant utilization. One of these secondary symbionts, Spiroplasma, is well known in Drosophila as a sex modulator and by interacting with the host immune system. However, little is known on the effects of Spiroplasma on aphids, such as its influence on the host immune defense against fungi and on host plant utilization. Aphid infections by Spiroplasma are known to be low and few aphid species were reported to be infected with this secondary symbiont, however aphids belonging to the genus Aphis in neotropical regions show high infection rates by Spiroplasma. Thus, we investigated the association of Spiroplasma with the tropical aphid Aphis citricidus through comparative biology experiments on two host plants with different nutritional value to the aphid. We demonstrate Spiroplasma induced no significant fitness costs to A. citricidus on either host plant as no changes in the fitness traits we assessed were observed. Spiroplasma infection only induced sutle changes on host longevity and fecundity. Therefore, we concluded Spiroplasma established a neutral interaction with A. citricidus under the selection pressure we tested, and argue on stress conditions that could better demonstrate the role of Spiroplasma in A. citricidus bioecology and associated costs involved.

Diversity of the Most Commonly Reported Facultative Symbionts in Two Closely-Related Aphids with Different Host Ranges.

Richness and abundance of facultative symbionts vary strongly with aphid species and genotype, symbiont strain, host plant, biogeography, and a number of abiotic factors. Despite indications that aphids in the same ecological niche show similar levels of facultative symbiont richness, existing reports do not consider the potential role of host plants on aphid microbial community. Little is known about how oligophagy and polyphagy may be influenced by secondary symbiont distribution, mainly because studies on secondary symbiont diversity are biased towards polyphagous aphids from the Northern Hemisphere. Here, we demonstrate the richness and abundance of the most common aphid-associated facultative symbionts in two tropical aphid species, the oligophagous Aphis (Toxoptera) citricidus (Kirkaldy) (Hemiptera: Aphididae) and the polyphagous Aphis aurantii (Boyer de Fonscolombe) (Hemiptera: Aphididae). Aphis citricidus is restricted to Citrus sp. host plants and closely related genera, whereas A. aurantii successfully exploits a wide variety of host plants from different families. Both were collected in the same ecological niche and our data basically indicated the same richness of secondary symbionts, but the abundance at which secondary symbionts occurred was very distinct between the two species. Spiroplasma was the most abundant facultative symbiont associated with A. citricidus and A. aurantii in the ecological niche studied. Single and multiple secondary symbiont infections were observed, but diversity of multiple infections was particularly high in A. citricidus. We discuss our findings and suggest hypotheses to explain causes and consequences of the differences in secondary symbiont diversity observed between these two aphid species.

Seasonal Polyphenism and Behavioral Variations of Ceroplastes glomeratus Peronti (Hemiptera: Coccidae).

The wax scale Ceroplastes glomeratus Peronti shows seasonal behavioral and phenotypic differences. Individuals develop gregariously during autumn, whereas solitarily in spring. Besides such behavioral differences, spring ("isolated form") and autumn ("aggregated form") will also produce different morphotypes. We provide additional data on the morphological data of the species based on the microscopic and macroscopic characteristics of adult females and of first-instars of the two adult morphotypes of C. glomeratus.

No fitness costs are induced by Spiroplasma infections of Aphis citricidus reared on two different host plants

Abstract Aphids can harbor several secondary symbionts that alter important aphid-related ecological traits, such as defense against natural enemies, heat tolerance and host plant utilization. One of these secondary symbionts, Spiroplasma, is well known in Drosophila as a sex modulator and by interacting with the host immune system. However, little is known on the effects of Spiroplasma on aphids, such as its influence on the host immune defense against fungi and on host plant utilization. Aphid infections by Spiroplasma are known to be low and few aphid species were reported to be infected with this secondary symbiont, however aphids belonging to the genus Aphis in neotropical regions show high infection rates by Spiroplasma. Thus, we investigated the association of Spiroplasma with the tropical aphid Aphis citricidus through comparative biology experiments on two host plants with different nutritional value to the aphid. We demonstrate Spiroplasma induced no significant fitness costs to A. citricidus on either host plant as no changes in the fitness traits we assessed were observed. Spiroplasma infection only induced sutle changes on host longevity and fecundity. Therefore, we concluded Spiroplasma established a neutral interaction with A. citricidus under the selection pressure we tested, and argue on stress conditions that could better demonstrate the role of Spiroplasma in A. citricidus bioecology and associated costs involved.

Resumo Pulgões podem abrigar vários simbiontes secundários que alteram características ecológicas importantes, como defesa contra inimigos naturais, tolerância ao calor e utilização da planta hospedeira. Um desses simbiontes secundários, Spiroplasma, é estudado em Drosophila por alterar a razão sexual e interagir com o sistema imunológico do hospedeiro. No entanto, pouco se sabe sobre os efeitos do Spiroplasma em pulgões, como sua influência na defesa contra fungos entomopatogénicos ou na utilização da planta hospedeira, por exemplo. Spiroplasma foi identificado em poucas espécies de pulgão e geralmente em baixa densidade, contudo pulgões pertencentes ao gênero Aphis em regiões neotropicais apresentam altas taxas de infecção por Spiroplasma. Assim, investigamos a associação entre Spiroplasma e o pulgão neotropical Aphis citricidus por meio de biologia comparativa em duas plantas hospedeiras com diferentes valores nutricionais para o pulgão. Spiroplasma não causou custo adaptativo significativo para A. citricidus em ambas planta hospedeira. A infecção por Spiroplasma induziu apenas pequenas mudanças na longevidade e fecundidade do hospedeiro. Portanto, concluímos que Spiroplasma estabeleceu uma interação neutra com A. citricidus sob ausência de pressão de seleção. Entretanto, discutimos sobre condições de estresse que possam demonstrar o papel de Spiroplasma na bioecologia de A. citricidus e os possíveis custos envolvidos.

Exchange of natural enemies for biological control: is it a rocky road?-the road in the Euro-Mediterranean region and the South American common market.

The access and benefit sharing (ABS) regulations from the Convention on Biological Diversity (CBD) for the use of natural resources became an important issue because the biodiversity of developing countries was heavily accessed and unilaterally exploited by pharmaceutical and seed companies. However, natural enemies used for biological control are living and unmodified genetic resources which cannot be patented and have been treated as resources such as drugs, seeds, or other commercial products. Consequently, the ABS requirements have limited not only the use of natural enemies but also the positive effects that scientifically supported biological control strategies have on the society, the environment, and the economy, reducing problems of pesticide residues, water and soil contamination, and non-target effects. During the last several years, the biological control scientific community has faced new and extremely complicated legislation dictated by a high and diverse number of governmental agencies at different levels, making the access to natural resources for biocontrol purposes a rocky road. Society at large should be aware of how the strict ABS regulations affect the use of natural enemies as biological resources to secure food production, food safety, and global environmental protection. We discuss in here the current difficulties derived from CBD for the exchange of natural enemies taking as example the Euro-Mediterranean region, Argentina, and Brazil to demonstrate how long and diverse are the steps to be followed to obtain the required permits for access and exportation/importation of natural enemies. We then argue that the public visibility of biocontrol strategies should be increased and their benefits highlighted in order to persuade legislators for the development of a less bureaucratic, more expedient, and more centralized regulatory frame, greatly favoring the practice and benefits of biological control. We finally propose a general framework in which ABS issues should be dealt in ways to attend the CBD, but also to make the use of natural resources for the biological control of pests to secure food production and security a possible alternative.

The Identity of Specimens of the Anastrepha fraterculus Complex (Diptera, Tephritidae) with Atypical Aculeus Tip.

Several specimens collected in Paraguay along with Anastrepha fraterculus (sensu lato) have an aculeus tip similar to species from the fraterculus complex, but the teeth of the aculeus of these specimens are poorly defined. As Anastrepha species identification is based mostly on subtle differences in the aculeus tip, we studied these specimens with atypical aculeus tips (with poorly defined teeth) that slightly differs from the aculeus tip of species of the fraterculus complex (with well-developed blunt teeth), to determine if this is due to intraspecific variation or if it can characterize a full species. The Paraguayan specimens were separated in six groups under stereomicroscope according to variation in their aculeus tip. Specimens within each group were studied by means of morphometrics (traditional and geometric) and gene sequence analysis (COI and ITS1). Morphometric analyses were significant, but no clear groups were formed by the discriminant analyses of the aculeus and wing, and the COI and ITS1 sequence analysis clustered specimens with all six aculeus variations. Therefore, the subtle morphological differences observed in the aculeus tip of Paraguayan specimens are intraspecific variations and the Paraguayan specimens were more genetically closely related to Anastrepha sp. 3 from the fraterculus complex.

Using population genetic methods to identify the origin of an invasive population and to diagnose cryptic subspecies of Telchin licus (Lepidoptera: Castniidae).

Telchin licus, the giant sugarcane borer, is an important pest species of sugarcane in northeast Brazil. Four subspecies of Telchin licus are recognized in Brazil based on their geographic distribution and subtle differences in wing colour pattern. Some taxa are morphologically indistinguishable, and their accurate identification is key to their efficient control. Mitochondrial genes sequences (cytochrome oxidase I and subunit 6 of the nicotinamide adenine dinucleotide dehydrogenase) were applied to delimit taxonomic entities of T. licus, and to infer the origin of a newly established population in the state of São Paulo. The molecular data indicated that specimens sampled at different regions in Brazil are morphologically cryptic but genetically isolated entities, and at least three subspecies were assigned to the sampled localities. These data also suggested that the population collected from the state of São Paulo must have a common origin with populations from northeast Brazil, which corroborate the hypothesis that ornamental plants infested with larvae of T. licus might have been transported from the northeast to the southeast regions.

Molecular characterization of Wolbachia strains associated with the invasive Asian citrus psyllid Diaphorina citri in Brazil.

Wolbachia is a symbiont intensively studied due to its ability to interfere with their host's reproduction, and it has been recently proposed as an alternative tool to control insect pests or vectors of diseases. The Asian citrus psyllid Diaphorina citri is an important pest of citrus since it vectors the bacterium that causes the "Huanglongbing" disease in citrus. The frequency and diversity of Wolbachia associated with D. citri is unknown, limiting the utilization of Wolbachia as an alternative strategy for insect management. Thus, we aimed to determine the natural rate of infection, to characterize the Wolbachia strains associated with this psyllid by "multilocus sequencing typing" (MLST) and wsp analysis, and to verify the association of the symbiont to particular genotypes of the host. Analysis indicated Wolbachia infects 100 % of all specimens tested from all 15 sampled populations. MLST revealed the occurrence of five new sequence types (STs) of Wolbachia, while analysis based on the wsp sequences indicated only four different types of Wolbachia. ST-173 was predominant, while the remaining STs were population specific. Analysis of the host-symbiont relationship did not reveal any particular association of Wolbachia and haplotypes or a decrease in nucleotide diversity of D. citri in populations in which more than one ST was recorded. The consequences of the diversity of STs reported are still unknown, but the fact that Wolbachia infection is fixed and that there is one ST with a broad distribution highlights the use of this symbiont as an alternative strategy to control D. citri.

Genetic variability and demographic history of Heliothis virescens (Lepidoptera: Noctuidae) populations from Brazil inferred by mtDNA sequences.

Intra- and inter-population genetic variability and the demographic history of Heliothis virescens (F.) populations were evaluated by using mtDNA markers (coxI, coxII and nad6) with samples from the major cotton- and soybean-producing regions in Brazil in the growing seasons 2007/08, 2008/09 and 2009/10. AMOVA indicated low and non-significant genetic structure, regardless of geographical scale, growing season or crop, with most of genetic variation occurring within populations. Clustering analyzes also indicated low genetic differentiation. The haplotype network obtained with combined datasets resulted in 35 haplotypes, with 28 exclusive occurrences, four of them sampled only from soybean fields. The minimum spanning network showed star-shaped structures typical of populations that underwent a recent demographic expansion. The recent expansion was supported by other demographic analyzes, such as the Bayesian skyline plot, the unimodal distribution of paired differences among mitochondrial sequences, and negative and significant values of neutrality tests for the Tajima's D and Fu's F(S) parameters. In addition, high values of haplotype diversity (H) and low values of nucleotide diversity (π), combined with a high number of low frequency haplotypes and values of θ(π)<θ(W), suggested a recent demographic expansion of H. virescens populations in Brazil. This demographic event could be responsible for the low genetic structure currently found; however, haplotypes present uniquely at the same geographic regions and from one specific host plant suggest an initial differentiation among H. virescens populations within Brazil.

Ovary development and maturation in Nezara viridula (L.) (Hemiptera: Pentatomidae).

Ovary development and maturation of Nezara viridula (L.) were evaluated by examining ovariole morphology and the alterations in the biochemical (protein synthesis related to reproduction) composition of the hemolymph. Quantitative and qualitative protein analyses were performed and ovary structural alterations for the pre-reproductive and reproductive stages were recorded. Total concentration of proteins in female hemolymph gradually increased until the end of the pre-mating stage, remaining unaltered thereafter. Proteins linked to reproduction (vitellogenins) appeared in the hemolymph 10 days after adult emergence and indicated the end of the pre-mating stage. After mating, total protein concentration in the hemolymph was lower compared to virgin females; vitellogenin levels were similar during most of the observation period. Oocyte development and maturation were gradual and age dependent. Ten-day-old females had chorionated oocytes ready for fertilization. Mating did not stimulate oocyte development in N. viridula, but the lack of mating activity appeared to have stimulated oocyte resorption in 17-day-old females.

Inorganic elements in the fat bodies of Diatraea saccharalis (Lepidoptera: Crambidae) larvae parasitized by Cotesia flavipes (Hymenoptera: Braconidae).

Koinobiont parasitoids use several strategies to regulate the host's physiological processes during parasitism. Although many aspects of host-parasitoid interactions have been explored, studies that attempted to assess the effects of parasitism on the availability of inorganic elements in the host are virtually nonexistent. Therefore, we aimed to evaluate the effects of parasitism on the concentrations of inorganic elements in the fat bodies of larvae of Diatraea saccharalis (Lepidoptera: Crambidae) during the development of the parasitoid Cotesia flavipes (Hymenoptera: Braconidae), by using total reflection X-ray fluorescence (TXRF). TXRF analysis allowed comparisons of the changes in the availability of the elements P, S, K, Ca, Cr, Fe, Ni, Cu, and Zn in the fat body tissues of D. saccharalis larvae parasitized by C. flavipes. Overall, the concentration of inorganic elements was higher early in parasitoid development (1 and 3days after parasitism) compared to non-parasitized larvae, but much lower towards the end of parasitoid development (7 and 9days after parasitism). Ca, K, and S were reduced after the fifth day of parasitism, which affected the total abundance of inorganic elements observed in the fat bodies of the parasitized hosts. The regulatory mechanisms or pathological effects related to the observed variation of the host inorganic elements induced by the parasitoid remain unknown, but there might be a strategy to make these elements available to the parasitoid larvae at the end of their development, when higher metabolic activity of the host fat body is required to sustain parasitoid growth. The observed variation of the host's inorganic elements could also be related to the known effects of parasitism on the host's immune response.

Host regulation and release of parasitism-specific proteins in the system Toxoneuron nigriceps-Heliothis virescens.

The braconid wasp Toxoneuron nigriceps induced qualitative and quantitative changes in the protein composition of the moth Heliothis virescens host hemolymph. Total protein concentration was found to be higher in parasitized host 4 days after parasitism as compared to control hosts, mainly due to changes in a particular group of proteins. Host proteins with a molecular mass of 173 and 72 kDa were found in higher levels in the hemolymph of parasitized larvae as control hosts approached pupation, while an 80 kDa peptide was found in reduced concentration in the hemolymph of parasitized hosts. Levels of these three peptides were maintained throughout parasitoid development, while two of them (173 and 72 kDa) were cleared from the host hemolymph close to pupation. Besides the regulation of host proteins, three parasitism-specific proteins (PSPs) were released into the host hemolymph. Two of them (PSP1-MW=116 kDa, pI=6.3; PSP2-MW=114 kDa, pI=6.2) first appeared in the hemolymph of parasitized hosts soon after pupation of control host and increased in concentration as the parasitoid developed. The third PSP (PSP3-MW=56 kDa, pI=5.8) was produced towards the end of parasitoid larval development, close to parasitoid egression. Database searches based on the amino acid composition and amino terminal sequence of PSP1 and PSP2 did not produce any significant matches, while PSP3 was identified as a putative chitinase. Incubation of host derived tissues, parasitoid larvae and teratocytes in 35S conditioned media suggested PSPs were a product of teratocytes. The role of the regulation of host proteins and release of PSPs by teratocytes for the successful development of T. nigriceps are discussed.

Differential gene expression during wing morph differentiation of the ectoparasitoid Melittobia digitata (Hym., Eulophidae).

Melittobia digitata is an ectoparasitoid of solitary bees and wasps that displays a trade-off between reproduction and dispersion through the development of two wing morphs (long and short wing morphs (LWM and SWM)). The morph differentiation of this species is an exceptional adaptation to maximize host exploitation and habitat colonization, and an understanding of the mechanisms underlying this developmental process will shed light on how nutrients or environmental elicitors alter regulatory pathways leading to physiological and metabolic changes resulting in such drastic developmental rearrangements. Here we describe the differential gene expression between SWM and LWM larvae of M. digitata in order to unravel the molecular mechanisms controlling the morph differentiation in this minute parasitoid and pinpoint the pathways involved in the regulation of this developmental process. The suppression subtractive hybridization (SSH) methodology was used to isolate differentially expressed genes using mRNA populations collected soon after morph development commitment. Dot blot analysis of 384 clones from a forward SSH library identified approximately 200 differentially expressed clones, including those transcripts present in very low abundance. Further DNA sequence analysis of a sub-population of 42 clones revealed 31 putatively unique transcripts, from which 5 were further analyzed by Northern blot analysis and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The complete cDNA of one of these transcripts, a putative metalloprotease, was fully sequenced and is described. The role of the putative differentially expressed genes during the wing morph differentiation of M. digitata is discussed.

Estimating parasitoid immature mortality by comparing oviposition and pupal development of trichogramma galloi Zucchi and T. pretiosum riley on natural and factitious hosts

Mortality during the immature development of T. galloi and T. pretiosum was estimated on UV-killed and live eggs of a factitious and a natural host, respectively. A staining technique was used to determine the actual parasitization of UV-treated eggs and was compared with the number of parasitoids that emerged per host egg (detectable parasitization). Effects of temperature as a factor of mortality during the immature development of both parasitoids on the factitious host was also assessed. The actual and detectable parasitization of live hosts was measured by recording both the parasitization behavior and the number of eggs where a parasitoid developed successfully. Our data show that mortality during immature development of both parasitoids may occur in live eggs of the natural host. No such mortality was observed when parasitoids developed on UV-killed eggs of the factitious host. Possible causes of parasitoid immature mortality and the effects of using UV-treated eggs of factitious hosts in estimating the parasitism capacity of Trichogramma in field conditions are discussed

Sensilla on the antenna and ovipositor of the parasitic wasps trichogramma galloi Zucchi and T. pretiosum Riley (Hym., Trichogrammatidae).

The understanding of the stimuli perceived by these parasitoids to accept and exploit a potential host may support the development of artificial hosts for their in vitro rearing. The most common structures involved in the host selection process in parasitoids are the antenna and the ovipositor. Sensilla present on these structures are involved in host recognition and acceptance, and the identification of which kind of stimulus (physical or chemical) is perceived by the sensilla could provide data about the host characteristics that elicit the parasitism behavior. The antenna and ovipositor of the wasps Trichogramma galloi and T. pretiosum were studied to identify their sensillar structure and their possible functions in the host selection process, as a support to the development of an artificial host for the in vitro rearing of these parasitoids. Seven sensilla and one setiform structure were present on the antenna of both parasitoid species, with a mixed, chemo-, or mechanoreception function. The ovipositor has four different sensilla and their role in the host evaluation is suggested.