Diversification and Independent Evolution of Troponin C Genes in Insects

Troponin C (TpnC), the calcium-binding subunit of the troponin regulatory complex in the muscle thin filament, is encoded by multiple genes in insects. To understand how TpnC genes have evolved, we characterized the gene number and structure in a number of insect species. The TpnC gene complement is five genes in Drosophilidae as previously reported for D. melanogaster. Gene structures are almost identical in D. pseudoobscura, D. suboboscura, and D. virilis. Developmental patterns of expression are also conserved in Drosophila subobscura and D. virilis. Similar, but not completely equivalent, TpnC gene repertoires have been identified in the Anopheles gambiae and Apis mellifera genomes. Insect TpnC sequences can be divided into three groups, allowing a systematic classification of newly identified genes. The pattern of expression of the Apis mellifera genes essentially agrees with the pattern in Drosophilidae, providing further functional support to the classification. A model for the evolution of the TpnC genes is proposed including the most likely pathway of insect TpnC diversification. Our results suggest that the rapid increase in number and sequence specialization of the adult Type III isoforms can be correlated with the evolution of the holometabolous mode of development and the acquisition of asynchronous indirect flight muscle function in insects. This evolutionarily specialization has probably been achieved independently in different insect orders.Reviewing Editor: Dr. Rüdiger Cerff     

Kekkon5 is an extracellular regulator of BMP signaling

Precise spatial and temporal control of Drosophila Bone Morphogenetic Protein (BMP) signaling is achieved by a host of extracellular factors that modulate ligand distribution and activity. Here we describe Kekkon5 (Kek5), a transmembrane protein containing leucine-rich repeats (LRRs), as a novel regulator of BMP signaling in Drosophila. We find that loss or gain of kek5 disrupts crossvein development and alters the early profile of phosphorylated Mad and dSRF in presumptive crossvein cells. kek5 phenotypic effects closely mimic those observed with Short gastrulation (Sog), but do not completely recapitulate the effects of dominant negative BMP receptors. We further demonstrate that Kek5 is able to antagonize the BMP ligand Glass bottom boat (Gbb) and that the Kek5 LRRs are required for BMP inhibitory activity, while the Ig domain is dispensable in this context. Our identification of Kek5 as a modulator of BMP signaling supports the emerging notion that LIG proteins function as diverse regulators of cellular communication.     

The somatic envelopes around the germ-line cells of polytrophic insect follicles: Structural and functional aspects

The polytrophic ovarioles of three insect species, the fruit fly Drosophila melanogaster, the fungus gnat Bradysia tritici, and the honeybee Apis mellifera, were compared morphologically and with respect to the cytological organization of the peripheral somatic layers. Staining with rhodaminyl-phalloidin revealed differences in the organization of the muscle strands of the epithelial sheath and the microfilament pattern in the basal part of the follicular epithelium (mid-vitellogenic stages). Also, the size of the intercellular space between the follicle cells differed considerably in the three analyzed species. The basement membrane of Drosophila and Bradysia follicles was partially digested using purified collagenase. The observed morphological changes indicated that in both species the basement membrane of the follicular epithelium plays an important role in shaping the follicles. The possible functional significance of the species-specific structural differences is discussed.     

A stereological analysis of developing egg chambers in the honeybee queen,Apis mellifera

Summary A polytrophic ovariole of the queen honeybee, Apis mellifera, is composed of a linear series of increasingly mature egg chambers, each consisting of an oocyte, an interconnected cluster of nurse cells, and a covering layer of follicle cells. This study describes changes in the volume of each of these components, as a function of the position of the egg chamber in the ovariole. An oocyte increases in volume from approximately 8.9 × 10³ m³ to approximately 9.6 × 10⁶ m³ over an average series of 20 egg chambers.     

Tracking down the "head blob": comparative analysis of wingless expression in the developing insect procephalon reveals progressive reduction of embryonic visual system patterning in higher insects

The evolution of larval head morphology in holometabolous insects is characterized by reduction of antennal appendages and the visual system components. Little insight has been gained into molecular developmental changes underlying this morphological diversification. Here we compare the expression of the segment polarity gene wingless (wg) in the pregnathal head of fruit fly, flour beetle and grasshopper embryos. We provide evidence that wg activity contributes to segment border formation, and, subsequently, the separation of the visual system and protocerebrum anlagen in the anterior procephalon. In directly developing insects like grasshopper, seven expression domains are formed during this process. The activation of four of these, which correspond to polar expression pairs in the optic lobe anlagen and the protocerebral ectoderm, has shifted to postembryonic stages in flour beetle and Drosophila. The remaining three domains map to the protocerebral neuroectoderm, and form by disintegration of a large precursor domain in flour beetle and grasshopper. In Drosophila, the precursor domain remains intact, constituting the previously described “head blob”. These data document major changes in the expression of an early patterning gene correlated with the dramatic evolution of embryonic visual system development in the Holometabola.     

Drosophila morgue and the intersection between protein ubiquitination and programmed cell death

In Drosophila, cell survival decisions are mediated by the integrated functions of the Grim-Reaper death activators and Inhibitor-of-Apoptosis-Proteins (IAPs), such as DIAP1, to regulate caspase activities. We recently identified a gene that enhances the actions of the Grim-Reaper proteins and negatively regulates the levels of DIAP1 protein. This gene, morgue, encodes a novel protein that contains both an F box and a ubiquitin conjugase domain. Interestingly, the Morgue conjugase domain lacks the active site cysteine required for covalent linkage to ubiquitin. Morgue could target IAPs and other proteins for ubiquitination and proteasome-dependent turnover by acting either in an SCF ubiquitin E3 ligase complex, or as a ubiquitin E2 conjugase enzyme variant (UEV) in conjunction with a catalytically active E2 conjugase. Morgue is evolutionarily conserved, as a Morgue ortholog was identified from the mosquito, Anopheles gambiae. Elucidation of morgue function should provide novel insights into the mechanisms of ubiquitination and programmed cell death.     

Comparative phylogenetic analyses of Halomonas variabilis and related organisms based on 16S rRNA, gyrB and ectBC gene sequences

Halomonas variabilis and phylogenetically related organisms were isolated from various habitats such as Antarctic terrain and saline ponds, deep-sea sediment, deep-sea waters affected by hydrothermal plumes, and hydrothermal vent fluids. Ten strains were selected for physiological and phylogenetic characterization in detail. All of those strains were found to be piezotolerant and psychrotolerant, as well as euryhaline halophilic or halotolerant. Their stress tolerance may facilitate their wide occurrence, even in so-called extreme environments. The 16S rDNA-based phylogenetic relationship was complemented by analyses of the DNA gyrase subunit B gene (gyrB) and genes involved in the synthesis of the major compatible solute, ectoine: diaminobutyric acid aminotransferase gene (ectB) and ectoine synthase gene (ectC). The phylogenetic relationships of H. variabilis and related organisms were very similar in terms of 16S rDNA, gyrB, and ectB. The ectC-based tree was inconsistent with the other phylogenetic trees. For that reason, ectC was inferred to derive from horizontal transfer.     

The distribution of Paenibacillus larvae spores in adult bees and honey and larval mortality, following the addition of American foulbrood diseased brood or spore-contaminated honey in honey bee (Apis mellifera) colonies

Within colony transmission of Paenibacillus larvae spores was studied by giving spore-contaminated honey comb or comb containing 100 larvae killed by American foulbrood to five experimental colonies respectively. We registered the impact of the two treatments on P. larvae spore loads in adult bees and honey and on larval mortality by culturing for spores in samples of adult bees and honey, respectively, and by measuring larval survival. The results demonstrate a direct effect of treatment on spore levels in adult bees and honey as well as on larval mortality. Colonies treated with dead larvae showed immediate high spore levels in adult bee samples, while the colonies treated with contaminated honey showed a comparable spore load but the effect was delayed until the bees started to utilize the honey at the end of the flight season. During the winter there was a build up of spores in the adult bees, which may increase the risk for infection in spring. The results confirm that contaminated honey can act as an environmental reservoir of P. larvae spores and suggest that less spores may be needed in honey, compared to in diseased brood, to produce clinically diseased colonies. The spore load in adult bee samples was significantly related to larval mortality but the spore load of honey samples was not.     

Comparative studies on the diversity of the edible mushroom Pleurotus eryngii: ITS sequence analysis, RAPD fingerprinting, and physiological characteristics

Verification of Pleurotus eryngii strains was assessed using ITS sequence analysis and RAPD fingerprinting. Sequence analysis of the ITS1–5.8S rDNA–ITS2 region of 24 strains of Pleurotus sp., which consisted of 22 strains of P. eryngii and the control strains P. ostreatus and P. ferulae, demonstrated that the DNA regions share mostly 99 % sequence identity, indicating that sequence-based analysis is not applicable for the verification of closely related mushroom strains. To verify the mushroom strains using RAPD, we amplified DNA fragments from the total cellular DNA of 24 mushroom strains with 18 different random primers, yielding 538 distinct DNA fragments ranging from 200–4000 bp. Analysis of the DNA fragment pattern showed that the 22 P. eryngii strains were clearly distinguished from the control strains P. ostreatus and P. ferulae, and could be categorized into five subgroups. Subsequent physiological studies on the development of fruiting bodies demonstrated the close correlation of the RAPD-based grouping with the phenotypical characteristics of mushroom fruiting bodies.     

Transfer of members of the genus Falcivibrio to the genus Mobiluncus, and emended description of the genus Mobiluncus

It has long been thought that the genera Mobiluncus and Falcivibrio contain the same organisms. Using a polyphasic approach, it was found that Mobiluncus curtisii and Mobiluncus mulieris were the same as Falcivibrio vaginalis and Falcivibrio grandis, respectively. As the genus name Mobiluncus takes precedence, it is proposed that F. vaginalis and F. grandis be transferred to the genus Mobiluncus. In agreement with previous studies, results from phenotypic tests did not support the separation of M. curtisii strains into its two subspecies, M. curtisii subsp. curtisii and M. curtisii subsp. holmesii. Phenotypic complexity within M. curtisii dictates that the species should be treated as a complex until more in-depth analyses of the species have been performed. Phylogenetic analyses, based on 16S rRNA gene sequences, demonstrated that the genus Mobiluncus was associated with Varibaculum cambriense and the two subspecies of Actinomyces neuii, and that A. neuii is only distantly related to Actinomyces sensu stricto.     

A comparative evaluation of phenotypic and molecular methods in the identification of members of the Staphylococcus sciuri group

Members of the Staphylococcus sciuri group (S. sciuri, S. lentus, and S. vitulinus) are coagulase-negative, novobiocin-resistant staphylococci that could be distinguished from other staphylococci on the basis of positive oxidase activity. In the present study, a scheme based on conventional methods and utilization of various carbohydrates was evaluated for the identification of oxidase-positive staphylococci, and validated using two molecular techniques. Of the 173 oxidase-positive staphylococcal tested strains, 161 were identified as S. sciuri, 9 as S. lentus, 2 as S. vitulinus, and one as S. fleurettii by our scheme. The level of agreement with tRNA intergenic length polymorphism analysis (tDNA-PCR) was high (97.5-100% correlation). The accuracy and ease of use of this protocol suggest that it may be useful and valuable in microbiological laboratories for the identification of members of this group.     

Elimination of EVE protein by CALI in the short germ band insect Tribolium suggests a conserved pair-rule function for even skipped

The question of the degree of evolutionary conservation of the pair-rule patterning mechanism known from Drosophila is still contentious. We have employed chromophore-assisted laser inactivation (CALI) to inactivate the function of the pair-rule gene even skipped (eve) in the short germ embryo of the flour beetle Tribolium. We show that it is possible to generate pair-rule type phenocopies with defects in alternating segments. Interestingly, we find the defects in odd numbered segments and not in even numbered ones as in Drosophila. However, this apparent discrepancy can be explained if one takes into account that the primary action of eve is at the level of parasegments and that different cuticular markers are used for defining the segment borders in the two species. In this light, we find that eve appears to be required for the formation of the anterior borders of the same odd numbered parasegments in both species. We conclude that the primary function of eve as a pair rule gene is conserved between the two species.     

携带与非携带松材线虫的松墨天牛miRNA表达谱比较分析

【目的】松墨天牛是松树的重要蛀干害虫,也是林业重大外来入侵种松材线虫的媒介昆虫。虽然松墨天牛和松材线虫互作的化学生态和分子进化机制受到人们的广泛关注,但miRNA等表观遗传因子在天牛—线虫互作中的作用未见报道。【方法】使用illumina HiSeq 2000平台进行miRNA高通量测序,得到4个携带线虫的天牛miRNA库和4个未携带线虫的天牛miRNA库。此外,对鉴定出的miRNA进行了差异表达分析,并对这些miRNA的靶基因进行了GO注释和KEGG通路富集分析。【结果】在携带线虫的天牛表皮、脂肪体、中肠和气管样本中分别鉴定出780、802、617和762个miRNA;在未携带松材线虫的天牛的不同组织样本中分别鉴定出784、723、713和837个miRNA。在携带松材线虫的松墨天牛中,某些已知miRNA表达量会显著升高,如miR-14、miR279和miR-312等。差异表达miRNA的功能大多指向代谢、免疫等方面。【结论】miRNA在松墨天牛和松材线虫互作中起着重要的调控作用。     

一株东方醋酸杆菌分离与其促进果蝇生长发育

【目的】分离与鉴定黑腹果蝇体内醋酸杆菌,并研究其对宿主生长发育的促进作用。【方法】利用醋酸杆菌选择性培养基分离果蝇肠道醋酸杆菌;通过革兰氏染色和16S rRNA基因比对鉴定菌种;肠道定植实验验证共生关系;发育历期和生长速率实验检测其促进果蝇生长作用;免疫荧光染色技术检测肠道细胞增殖;RT-PCR法检测促生长的分子标志物和相关的信号通路。【结果】菌株为东方醋酸杆菌(Acetobacter orientalis),可以持续地定植在果蝇肠道及其培养基中,并且明显促进果蝇的生长。东方醋酸杆菌通过胰岛素信号通路增加肠分裂细胞的数量和促进蜕皮激素的分泌。【结论】东方醋酸杆菌是果蝇的一种共生菌,对果蝇肠道结构和机体发育具有重要的作用。     

Phylogenetic relationships, character evolution and biogeography of southern African members of Zygophyllum (Zygophyllaceae) based on three plastid regions

The plastid coding rbcL and non-coding trnLF regions of 53 of 55 southern African Zygophyllum species were sequenced and used to evaluate the phylogenetic relationships within the southern African representatives of the genus. Published sequences of the same gene regions of Australian, Asian and North African Zygophyllum species were included to assess the relationships of the species from these regions to the southern African species. The addition of Z. stapffii from Namibia, found to be conspecific with Z. orbiculatum from Angola, lead to a greatly resolved tree. The molecular results were largely congruent with a recent sectional classification of the southern African species and supported their subdivision into subgenera Agrophyllum and Zygophyllum. Reconstruction of the character evolution of capsule dehiscence, seed attachment and seed mucilage showed that these characters allowed a division of southern African species into the two subgenera but that this could not be applied to species occurring elsewhere. Other morphological characters were found to vary and unique character combinations, rather than unique characters, were found to be of systematic value in sectional delimitation. The study suggests that repeated radiations from the horn of Africa to southern Africa and Asia and back lead to the present distribution of the taxa in the subfamily Zygophylloideae. Although this study supports some of the recent taxonomic changes in the group, the unresolved relationships between the proposed genera Tetraena and Roepera and those retained as Zygophyllum species suggest that changes to the taxonomy may have been premature.     

Comparative and functional analysis of the rRNA-operons and their tRNA gene complement in different lactic acid bacteria

The complete genome sequences of the lactic acid bacteria (LAB), Lactobacillus plantarum, Lactococcus lactis, and Lactobacillus johnsonii were used to compare location, sequence, organisation, and regulation of the ribosomal RNA (rrn) operons. All rrn operons of the examined LAB diverge from the origin of replication, which is compatible with their efficient expression. All operons show a common organisation of 5'-16S-23S-5S-3' structure, but differ in the number, location and specificity of the tRNA genes. In the 16S-23S intergenic spacer region, two of the five rrn operons of Lb. plantarum and three of the six of Lb. johnsonii contain tRNA-ala and tRNA-ile genes, while L. lactis has a tRNA-ala gene in all six operons. The number of tRNA genes following the 5S rRNA gene ranges up to 14, 16, and 21 for L. lactis, Lb. johnsonii and Lb. plantarum, respectively. The tRNA gene complements are similar to each other and to those of other bacteria. Micro-heterogeneity was found within the rRNA structural genes and spacer regions of each strain. In the rrn operon promoter regions of Lb. plantarum and L. lactis marked differences were found, while the promoter regions of Lb. johnsonii showed a similar tandem promoter structure in all operons. The rrn promoters of L. lactis show either a single or a tandem promoter structure. All promoters of Lb. plantarum contain two or three -10 and -35 regions, of which either zero to two were followed by an UP-element. The Lb. plantarum rrnA, rrnB, and rrnC promoter regions display similarity to the rrn promoter structure of Esherichia coli. Differences in regulation between the five Lb. plantarum promoters were studied using a low copy promoter-probe plasmid. Taking copy number and growth rate into account, a differential expression over time was shown. Although all five Lb. plantarum rrn promoters are significantly different, this study shows that their activity was very similar under the circumstances tested. An active promoter was also identified within the Lb. plantarum rrnC operon preceding a cluster of 17 tRNA genes.     

DNA barcoding for the identification of eight species members of the Thai Hyrcanus Group and investigation of their stenogamous behavior

Eight species members of the Thai Hyrcanus Group were identified based on the intact morphology and molecular analysis (COI barcoding, 658 bp) of F₁-progenies. Five iso-female lines of each species were pooled in order to establish stock colonies. A stenogamous colony of each species was investigated by making 200 and 300 newly emerged adult females and males co-habit in a 30 cm cubic cage for one week. After ovipositon, the spermathecae of females were examined for sperms. The results revealed that Anopheles argyropus, Anopheles crawfordi, Anopheles nitidus, Anopheles pursati, Anopheles sinensis, Anopheles nigerrimus, Anopheles paraliae and Anopheles peditaeniatus yielded insemination rates of 0%, 0%, 0%, 31%, 33%, 42%, 50% and 77%, respectively. Continuous selection to establish stenogamous colonies indicated that An. sinensis, An. pursati, An. nigerrimus, An. paraliae and An. peditaeniatus provided insemination rates of 33–34%, 27–31%, 42–58%, 43–57% and 61–86% in 1, 2, 5, 6 and 20 generations of passages, respectively.     

Biochemical and structural analysis of Gox2181, a new member of the SDR superfamily from Gluconobacter oxydans

Gluconobacter oxydans enable to oxidize sugars and polyols incompletely to corresponding materials with potential industrial applications, containing around 75 putative dehydrogenases. One of these putative dehydrogenases, Gox2181, was cloned and expressed in Escherichia coli BL21 (DE3), and its X-ray crystal structure was determined to a resolution of 1.8 Å. Gox2181 formed a homo-tetramer in the crystal that was coincident with the apparent molecular mass determined in the solution. Gox2181 displayed α/β-folding patterns, the conserved catalytic tetrad of Asn119-Ser147-Tyr162-Lys166, and the NAD-binding pocket, which aligned well with the ‘classical’ type of short-chain dehydrogenase/reductase (SDR) enzymes. Gox2181 was denoted SDR51C based on the SDR nomenclature system. The purified recombinant Gox2181 was demonstrated to be NAD(H)-dependent and active towards a wide range of substrates, including sugar alcohols, secondary alcohols, ketones, and ketoses. Among the substrates tested, Gox2181 displayed preference for secondary hydroxyl or carbonyl groups, showing low K_m values with d-arabitol and butanedione.