The juvenile hormone titer of Trichoplusia ni and its potential role in embryogenesis of the polyembryonic wasp CopidosomaFloridanum

The hemolymph juvenile hormone (JH) titer of third through fifth stadia Trichoplusia ni parasitized by the polyembryonic parasitoid, Copidosoma floridanum, was measured by radioimmunoassay and compared to the titers of unparasitized larvae. The JH titer of parasitized larvae fluctuated from 28 pg/μl to undetectable levels. Maximum levels of hormone were present at ecdysis to the fourth and fifth stadium, and at the prepupal stage. Qualitatively, similar fluctuations were observed in unparasitized larvae. However, the titers in unparasitized larvae were much lower than those of parasitized larvae in the third and early fourth stadia, and the titer fell to undetectable levels in the fifth stadium 24 h earlier (48 h) than in parasitized larvae (72 h). Preventing the JH titer from falling during the fourth and fifth stadia by topical application of (RS)-methoprene or JH II had a juvenilizing effect on parasitized T. ni, and inhibited C. floridanum embryo morphogenesis. The effect of exogenous methoprene and JH on C. floridanum development depended on timing of application and dosage. Application of 100 pmol per day of methoprene beginning at 2 h of the host fourth stadium, prior to the large drop in the endogenous JH titer, inhibited morphogenesis in the majority of C. floridanum embryos. Application of methoprene at later times of host development did not inhibit morphogenesis although other developmental alterations were observed. The potential significance of host JH and ecdysteroid titers on polyembryonic development are discussed.     

Sex differences in the protection of host immune systems by a polyembryonic parasitoid

Endoparasitoids have the ability to evade the cellular immune responses of a host and to create an environment suitable for survival of their progeny within a host. Generally, the host immune system is suppressed by endoparasitoids. However, polyembryonic endoparasitoids appear to invade their hosts using molecular mimicry rather than immune system suppression. It is not known how the host immune system is modified by polyembryonic endoparasitoids. Using haemocyte counts and measurement of cellular immune responses, we evaluated modification of the host immune system after separate infestations by a polyembryonic parasitoid (Copidosoma floridanum) and another parasitoid (Glyptapanteles pallipes) and by both together (multi-parasitism). We found that the polyembryonic parasitoid maintains and enhances the host immune system, whereas the other parasitoid strongly suppresses the immune system. Multi-parasitization analysis revealed that C. floridanum cancelled the immune suppression by G. pallipes and strengthened the host immunity. This enhancement was much stronger with male than with female C. floridanum.     

Response of the agarophyte Gelidium floridanum after in vitro exposure to ultraviolet radiation B: changes in ultrastructure, pigments, and antioxidant systems

As a source of agar, the red macroalga Gelidium floridanum is a seaweed of great economic importance. However, it grows in a region exposed to high ultraviolet B radiation (UVBR). Therefore, to study the in vitro effect of UVBR on this plant, apical segments of G. floridanum were cultivated and exposed to photosynthetically active radiation (PAR) at 80?μmol photons m^?2?s^?1 and PAR + UVBR at 1.6?W?m^?2 at 3?h per day for 7?days. The samples were processed for electron microscopy, and agar yield, growth rates, mitochondrial activity, protein levels, chlorophyll a, phycobiliproteins, carotenoids and phenolic compounds, and photosynthetic performance were examined. After 7?days of exposure to PAR + UVBR, G. floridanum experienced ultrastructural damage that was primarily observed in the internal organization of chloroplasts, increased cell wall thickness, as well as increased volume of plastoglobuli and free ribosomes. Moreover, this exposure might have caused photodamage and photoinhibition of photosynthetic pigments (chlorophyll a and phycobiliproteins), leading to a decrease in photosynthetic efficiency, relative electron transport rate, and maximum photosynthetic rate. These phenomena were matched with a corresponding decrease in growth rates and depigmentation, combined with partial necrosis of the apical segments exposed to PAR + UVBR. Additionally, the UVBR-induced damage elicited a marked cellular antioxidant response, possibly as a consequence of free radical generation.     

The polyembryonic wasp Copidosoma floridanum produces two castes by differentially parceling the germ line to daughter embryos during embryo proliferation

Eggs of the polyembryonic wasp Copidosoma floridanum undergo a clonal phase of proliferation, which results in the formation of thousands of embryos called secondary morulae and two castes called reproductive and soldier larvae. C. floridanum establishes the germ line early in development, and prior studies indicate that embryos with primordial germ cells (PGCs) develop into reproductive larvae while embryos without PGCs develop into soldiers. However, it is unclear how embryos lacking PGCs form and whether all or only some morulae contribute to the proliferation process. Here, we report that most embryos lacking PGCs form by division of a secondary morula into one daughter embryo that inherits the germ line and another that does not. C. floridanum embryos also incorporate 5-bromo-2′-deoxyuridine (BrdU), which allows PGCs and other cell types to be labeled during the S phase of the cell cycle. Continuous BrdU labeling indicated that all secondary morulae cycle during the proliferation phase of embryogenesis. Double labeling with BrdU and the mitosis marker anti-phospho-histone H3 indicated that the median length of the G2 phase of the cell cycle was 18 h with a minimum duration of 4 h. Mitosis of PGCs and presumptive somatic stem cells in secondary morulae was asynchronous, but cells of the inner membrane exhibited synchronous mitosis. Overall, our results suggest that all secondary morulae contribute to the formation of new embryos during the proliferation phase of embryogenesis and that PGCs are involved in regulating both proliferation and caste formation.     

Intrinsic interspecific competition between the polyembryonic parasitoid Copidosoma floridanum and solitary endoparasitoid Microplitis demolitor in Pseudoplusia includens

Multiparasitism studies were conducted in the laboratory between the egg-larval polyembryonic parasitoid Copidosoma floridanum (Ashmead) (Hymenoptera: Encyrtidae) and the larval endoparasitoid Microplitis demolitor Wilkinson (Hymenoptera: Braconidae) in the mutual host Pseudoplusia includens (Walker) (Lepidoptera: Noctuidae). Most multiparasitized hosts produced either a C. floridanum brood or died without any parasitoid emergence. Most multiparasitized hosts underwent supernumerary molting; however, multiparasitized hosts that produced a C. floridanum brood exhibited growth characteristics associated with C. floridanum parasitism while multiparasitized hosts that died exhibited growth characteristics more typical of M. demolitor parasitism. Multiparasitized hosts that produced a C. floridanum brood increased in weight, and usually exhibited the behavioral and morphological characteristics associated with initiation of metamorphosis. In contrast, multiparasitized hosts that ultimately died gained little weight and exhibited no characteristics associated with metamorphosis. M. demolitor progeny died in multiparasitized hosts as eggs or first instar larvae, but no direct evidence was found to indicate that physical attack by C. floridanum precocious larvae was responsible for their elimination.

---

Résumé Lors de l'étude au laboratoire du multiparasitisme de Pseudoplusia includens Walker (Lépido. Noctuidae) par le parasitoïde ovolarvaire polyembryonnaire C. floridanum Ashmead (Hyméno. Encyrtidae) et l'endoparasitoïde larvaire M. demolitor Wilkinson (Hyméno. Braconidae), soit les hôtes donnent en majorité C. floridanum, soit ils meurent sans que quoi que ce soit émerge. P. includens non parasité présente 5 stades larvaires; les hôtes parasités par C. floridanum subissent 5 ou 6 stades avant le développement complet du parasitoïde, et les hôtes parasités par M. demolitor muent en stade 5 avant émergence du parasitoïde. Par contre, les hôtes multiparasités muent en stade 7; ceux qui produisent une couvée de C. floridanum ont présenté la croissance caractéristique des hôtes parasités par C. floridanum, tandis que les hôtes multiparasités qui sont morts avaient présenté la croissance caractéristique des hôtes parasités par M. demolitor. La plupart des hôtes multiparasités desquels C. floridanum a émergé ont augmenté de poids et présenté les caractères associés au début de la métamorphose. La descendance de M. demolitor est morte dans les hôtes multiparasités au stade uf ou chenille du premier stade, mais il n'y avait aucum indice d'attaque physique par des larves de C. floridanum qui auraient été responsables de leur élimination.

     

Interspecific competition between the polyembryonic wasp Copidosoma floridanum and the gregarious endoparasitoid Glyptapanteles pallipes

We examined interspecific competition between the egg-larval polyembryonic parasitoid Copidosoma floridanum (Hymenoptera: Encyrtidae) and the gregarious larval endoparasitoid Glyptapanteles pallipes (Hymenoptera: Braconidae). Approximately 72% of multiparasitized Acanthoplusia agnata (Lepidoptera: Noctuidae) produced C. floridanum adults, 14% produced G. pallipes adults, and 14% died without producing any parasitoids. Development was delayed and the rate of weight gain was reduced in multiparasitized hosts that produced C. floridanum compared with singly-parasitized hosts. In contrast, only weight was reduced in multiparasitized hosts that produced G. pallipes adults. Compared with single parasitism, the brood size of wasps emerging from multiparasitized hosts was reduced in both species. The percentage of hosts containing precocious larvae of C. floridanum, which are considered to be soldiers, did not increase in response to parasitization by G. pallipes. However, developmental cessation and death of G. pallipes eggs and larvae may be closely related to the number of coexisting precocious larvae.     

Expanding duplication of the testis PHD Finger Protein 7 (PHF7) gene in the chicken genome

Gene duplications increase genetic and phenotypic diversity and occur in complex genomic regions that are still difficult to sequence and assemble. PHD Finger Protein 7 (PHF7) acts during spermiogenesis for histone-to-histone protamine exchange and is a determinant of male fertility in Drosophila and the mouse. We aimed to explore and characterise in the chicken genome the expanding family of the numerous orthologues of the unique mouse Phf7 gene (highly expressed in the testis), observing the fact that this information is unclear and/or variable according to the versions of databases. We validated nine primer pairs by in silico PCR for their use in screening the chicken bacterial artificial chromosome (BAC) library to produce BAC-derived probes to detect and localise PHF7-like loci by fluorescence in situ hybridisation (FISH). We selected nine BAC that highlighted nine chromosomal regions for a total of 10 distinct PHF7-like loci on five Gallus gallus chromosomes: Chr1 (three loci), Chr2 (two loci), Chr12 (one locus), Chr19 (one locus) and ChrZ (three loci). We sequenced the corresponding BAC by using high-performance PacBio technology. After assembly, we performed annotation with the FGENESH program: there were a total of 116 peptides, including 39 PHF7-like proteins identified by BLASTP. These proteins share a common exon-intron core structure of 8–11 exons. Phylogeny revealed that the duplications occurred first between chromosomal regions and then inside each region. There are other duplicated genes in the identified BAC sequences, suggesting that these genomic regions exhibit a high rate of tandem duplication. We showed that the PHF7 gene, which is highly expressed in the rooster testis, is a highly duplicated gene family in the chicken genome, and this phenomenon probably concerns other bird species.     

Genetic diversity in the Chinese pangolin (Manis pentadactyla) inferred from protein electrophoresis

We examined protein polymorphism of Chinese pangolins (Manis pentadactyla) from Yunnan Province of China, including two forms of three brown and nine dusky Chinese pangolins. Sixty-two genetic loci were screened; 12 loci were found to be polymorphic. The percentage of polymorphic loci (P) is 0.194, the mean individual heterozygosity (H) is 0.078, and the mean number of alleles (A) is 1.258. Furthermore, we calculated the genetic distance (D) between the two forms and found a low level of genetic divergence (D=0.0206) between them, which indicates an almost-indistinguishable divergence at the level of proteins.     

Linkage Relationships in Wild Emmer Wheat, TRITICUM DICOCCOIDES

The linkage relationships in wild emmer wheat, Triticum dicoccoides , between nine enzymatic loci (Mdh-1, Ipo, β-Glu, Pept-1, Pept-3, Est-5, Est-1, 6Pgdh-2 and Hk) and a coleoptile pigment locus (Rc) were investigated. Chromosome locations of genes were inferred from analysis of ditelocentric lines of Triticum aestivum, cultivar Chinese Spring. The loci Mdh-B1 and Hk are linked (lambda = 0.1869) and are most likely located on the chromosome 1B. The loci Pept-B1 and Rc are linked (lambda = 0.2758) and are located on the 6Bq chromosomal arm. Rc also has significant interactions with the loci Pept-3 and Ipo, although there is no significant linkage detectable. The interactions may be a result of epigenetic interactions. Est-1 has only one active product in T. dicoccoides and is most likely located on the 3Ap chromosome arm. No significant interactions were found for the remaining loci.     

Gene-targeted molecular phylogeny,phytochemical profiling,and antioxidant activity of nine species belonging to family Cactaceae

Cactaceae plant family comprises over 130 genera and 2000 species of succulent flowering plants. The genera Mammillaria and Notocactus (Parodia), which have medicinal and nutritional applications as well as aesthetic appeal, are considered to be among the major genera of the family. Several species of both genera show morphological and chemical similarities and diversities according to environmental conditions and genotypes. Here, we assessed the genetic relationships of nine species belonging to two major genera Mammillaria and Notocactus under the family Cactaceae, using two modern gene-targeting marker techniques, the Start Codon Targeted (SCoT) Polymorphism and the Conserved DNA-Derived Polymorphism (CDDP). Besides, we screened the various phytochemicals and evaluated the antioxidant activities of the nine species of cacti. Five out of the 10 SCoT and eight CDDP primers used to screen genetic variations within the nine species yielded species-specific reproducible bands. The entire 156 loci were detected, of which 107 were polymorphic, 26 were monomorphic, and 23 were unique loci. The nine species were categorized into two groups based on the dendrogram and similarity matrix. Phytochemical profiling revealed that sterols, triterpenes, flavonoids, and tannins were found in all the tested species. Additionally, two Notocactus species (N. shlosserii and N. roseoluteus) and one Mammillaria species (M. spinosissima) revealed a considerable antioxidant activity. Our results demonstrated that gene-targeting marker techniques were highly powerful tools for the classification and characterization of the nine investigated species, despite displaying high similarities at both morphological and phytochemical levels.     

Species Concepts in the Cylindrocladium floridanum and Cy. spathiphylli Complexes (Hypocreaceae) Based on Multi-allelic Sequence Data, Sexual Compatibility and Morphology

Much attention has recently been devoted to the delimitation of species units in Cylindrocladium(Cy.). In this regard the present study focuses on the taxa within the unresolved Cy. floridanum and Cy. spathiphylli species complexes. Maximum parsimony analyses of DNA sequences of ITS, β-tubulin and histone regions of rRNA genes, and mating experiments revealed a geographically isolated species of Cylindrocladium in the Cy. spathiphylli(teleomorph:Calonectria spathiphylli) species complex.Cy. pseudospathiphylli sp. nov. (teleomorph:Ca. pseudospathiphylli sp. nov.) is described as a new phylogenetic, biological and morphological species. It is distinguished from Cy. spathiphylli by being homothallic, having smaller macroconidia, and distinct DNA sequences of β-tubulin and histone genes. Similarly, parsimony analysis of a combined data set also indicated several phylogenetic species to exist within Cy. floridanum(teleomorph:Ca. kyotensis). Based on differences in vesicle morphology and conidium dimensions, the Canadian population of Cy. floridanum, formerly known as Cy. floridanum Group 2, is described as Cy. canadense sp. nov., while a further collection from Hawaii is described as Cy. pacificum sp. nov.     

Ballistic seed dispersal inIllicium (Illiciaceae)

The primary mode of fruit dispersal in the woodyMagnoliidae is animal dispersal.Illicium floridanum and other members of the genus have a well development system of ballistic fruit dehiscence. The ballistic dispersal is effective only over short distances, but may be important in propelling the seeds away from the parent tree. The presence of explosive dehiscence mechanisms in relatively primitive angiosperm genera suggests that a diversity of dispersal mechanisms may have been present in early angiosperms.     

Characterization and mapping of simple sequence repeats (SSRs) in Sorghum bicolor

A total of 13 SSR loci were characterized in Sorghum bicolor. Ten of these loci were isolated by screening sorghum genomic AG-enriched libraries with labelled poly(AG)/poly(CT), the other three were derived from database searches. In order to explore the degree of polymorphism detectable in this species by this type of molecular marker, the SSR markers were tested on nine inbred lines of S. bicolor of different geographic origin. PCR analysis on acrylamide gels revealed a high degree of polymorphism (δ_T=0.80). One locus, in particular, allowed the identification of all of the nine inbred lines used in our study. Seven of these SSR markers were mapped, using an existing sorghum RFLP map.     

A genetic linkage map of Nicotiana plumbaginifolia/ Nicotiana longiflora based on RFLP and RAPD markers

We have constructed a genetic linkage map for Nicotiana plumbaginifolia/Nicotiana longiflora (2n= 2x=20), based on the segregation of 69 RFLP and 102 RAPD loci in 99 F₂ plants from the cross N. plumbaginifolia×N. longiflora. The map consists of nine major linkage groups, each containing more than nine marker loci, and spans 1062 cM. Twenty of the RFLP markers were mapped previously to Nicotiana sylvestris (2n=2x=24) chromosomes using monosomic alien addition lines. Taxonomically, N. plumbaginifolia and N. sylvestris belong to the same section, namely the Alatae; however, cytogenetic evidence indicates that they are not closely related. Comparison of the distribution of markers common to both maps suggests that genome reorganization has occurred during the evolution of these two species. Evidence is also presented that genome reorganization may be accompanied by gain and loss of specific classes of DNA sequences in their genomes.     

Compatible invasion of a phylogenetically distant host embryo by a hymenopteran parasitoid embryo

Embryonic invasion into the tissue of genetically different organisms has been known only in mother-embryo interactions of viviparous organisms. Hence, embryonic invasions have been thought to occur only within the same or closely related species. For endoparasitic Hymenoptera, which are oviposited in their host egg but complete their development in the later stages, entry into the host embryo is essential. To date, the entry of these parasitoids is known to be accomplished by either egg deposition directly into the embryo or by the newly hatched larva boring into the embryo. However, Copidosoma floridanum is a polyembryonic parasitoid whose development is characterized by a prolonged embryonic stage, and which lacks a larval form during its host embryogenesis. We have analyzed the behavior and fate of C. floridanum embryos co-cultured with their host embryo in vitro. Here, we show that the morula-stage embryo of C. floridanum actively invades the host embryo. Histological analyses have demonstrated that C. floridanum embryonic invasion is associated with adherent junction to host cells rather than causing an obvious wound on the host cells. These findings provide a novel case of embryonic invasion into a phylogenetically distant host embryo, ensuring cellular compatibility with host tissues.     

Genetic dissection reveals effects of interaction between high-molecular-weight glutenin subunits and waxy alleles on dough-mixing properties in common wheat

The glutenin and waxy loci of wheat are important determinants of dough quality. This study was conducted to evaluate the effects of high-molecular-weight glutenin (HMW-GS) and waxy alleles on dough-mixing properties. Molecular mapping was used to investigate these effects on Mixograph properties in a population of 290 (Nuomai1 × Gaocheng8901) recombinant inbred lines (RILs) from three environments in the harvest years 2008, 2009 and 2011. The results indicated the following: (i) the Glu-A1 and Glu-D1 loci have greater impacts on Mixograph properties compared to the Wx-1 loci and the effects of Glu-D1d and Glu-D1h on dough mixing are better than those of Glu-D1f and Glu-D1new1 in this population; (ii) the interactions between the Glu-1 and Wx-1 loci affected some traits, especially the midline peak value (MPV), and the lack of Wx-B1 or Wx-D1 led to increased MPV for all types of Glu-1 loci; and (iii) 30 quantitative-trait loci (QTL) over nine wheat chromosomes were identified with ICIM analysis based on the genetic map of 498 loci. Eight major QTL and 16 QTL in the Glu-1 loci from the three environments were found. The major QTL clusters were associated with the Glu-1 loci, and also were found in two regions on chromosome 3B and one region on chromosome 6A, which is one of the novel chromosome regions influencing dough-mixing strength. The two QTL for MPV are located around Wx-B1 on chromosome 4A. QMPT-1D.1, QMPI-1D.1 and Q8MW-1D.1 were stable in different environments and could potentially be used in molecular marker-assisted breeding.     

A Genetic Map of Lettuce (Lactuca sativa L.) with Restriction Fragment Length Polymorphism, Isozyme, Disease Resistance and Morphological Markers

A detailed linkage map of lettuce was constructed using 53 genetic markers including 41 restriction fragment length polymorphism (RFLP) loci, five downy mildew resistance genes, four isozyme loci and three morphological markers. The genetic markers were distributed into nine linkage groups and cover 404 cM which may be 25-30% of the lettuce genome. The majority (31 of 34) of the RFLP probes detected single segregating loci, although seven of these may have been homologous to further monomorphic loci. When several loci were detected by a single probe, the loci were generally linked, suggesting tandem duplications. One probe, however, detected loci in three linkage groups suggesting translocations. The five downy mildew resistance genes (Dm1, Dm3, Dm4, Dm5/8 and Dm13), segregating in the Calmar x Kordaat cross, represented each of the four resistance gene linkage groups. Dm5/8 is flanked by two cDNA loci, each located 10 cM away. These flanking markers will be used to study the source of variation in downy mildew genes and are also part our strategy to clone resistance genes.     

Microsatellite Polymorphisms in Cassava Landraces from the Cerrado Biome, Mato Grosso do Sul, Brazil

Using nine microsatellite loci, we investigated genetic structure and diversity in 83 Brazilian cassava accessions, including several landraces, in the Cerrado biome in Mato Grosso do Sul, Brazil. All nine loci were polymorphic, averaging 6.00 alleles per locus. Treating each of seven municipalities as a cassava group or population, they averaged 3.5 alleles per locus, with 97% polymorphic loci, high values for observed heterozygosity (0.32) and gene diversity (0.56). Total genetic variability was high (0.668), and most of this genetic variability was concentrated within municipalities (0.577). Cluster and structure analyses divided accessions into two major clusters or populations (K = 2). Also, a significant genetic versus geographic correlation was found (r = 0.4567; P < 0.0260). Migratory routes in the Cerrado are considered main contributors to the region’s high cassava diversity and spatial genetic structure, amplifying interactions between traditional farmers and the evolutionary dynamics of this crop.     

Effect of Bt-cotton expressing Cry1A(c) on the survival and fecundity of two hymenopteran parasitoids (Braconidae, Encyrtidae) in the laboratory

We examined the effect of Bt-cotton (Event 531) plants expressing the Bacillus thuringiensis δ-endotoxin CryIA(c) on two hymenopteran endoparasitoids, Cotesia marginiventris and Copidosoma floridanum. In the laboratory, parasitized and unparasitized Pseudoplusia includens larvae were reared on foliage from a conventional soybean cultivar (Pioneer 97B61), a conventional cotton cultivar (DPL 5415), or a Bt-cotton cultivar (NuCotn 33B). C. marginiventris developed significantly faster within P. includens larvae feeding on Pioneer 97B61 and DPL 5415 compared to those feeding on NuCotn 33B. C. marginiventris that developed inside P. includens larvae feeding on NuCotn 33B suffered reduced longevity, and females had fewer ova. NuCotn 33B also affected the growth and development of P. includens parasitized with C. floridanum and life history parameters of adult C. floridanum. Parasitized and unparasitized P. includens developed more slowly when they were fed NuCotn 33B and the prepupae weighed less. Survival of parasitized and unparasitized P. includens was lower when larvae were fed NuCotn 33B and some evidence points to higher susceptibility of parasitized caterpillars to intoxication by NuCotn 33B. Fewer C. floridanum adults emerged from hosts fed NuCotn 33B, but pupal weight and adult longevity were unaffected. Analysis comparing the two experiments conducted with C. floridanum suggests that older NuCotn 33B plants (90–120 days after planting) may affect parasitoid development and adult survival less than younger NuCotn 33B plants (60–90 days after planting). Feeding on NuCotn 33B by P. includens affected the survival and development of the two hymenopteran endoparasitoids studied here, and the degree of the effect was similar to that observed with natural resistance found in soybean plants. It remains to be determined if the effects demonstrated here are less than, equal to, or greater than the impact of conventional insecticide applications used in conventional, non-transgenic, cotton.