Knockout of Two Cry-Binding Aminopeptidase N Isoforms Does Not Change Susceptibility of Aedes aegypti Larvae to Bacillus thuringiensis subsp. israelensis Cry4Ba and Cry11Aa Toxins

The insecticidal Cry4Ba and Cry11Aa crystal proteins from Bacillus thuringiensis subsp. israelensis (Bti) are highly toxic to Ae. aegypti larvae. The glycosylphosphatidylinositol (GPI)-anchored APN was identified as an important membrane-bound receptor for multiple Cry toxins in numerous Lepidoptera, Coleoptera, and Diptera insects. However, there is no direct molecular evidence to link APN of Ae. aegypti to Bti toxicity in vivo. In this study, two Cry4Ba/Cry11Aa-binding Ae. aegypti GPI-APN isoforms (AeAPN1 and AeAPN2) were individually knocked-out using CRISPR/Cas9 mutagenesis, and the AeAPN1/AeAPN2 double-mutant homozygous strain was generated using the reverse genetics approach. ELISA assays showed that the high binding affinity of Cry4Ba and Cry11Aa protoxins to the midgut brush border membrane vesicles (BBMVs) from these APN knockouts was similar to the background from the wild-type (WT) strain. Likewise, the bioassay results showed that neither the single knockout of AeAPN1 or AeAPN2, nor the simultaneous disruption of AeAPN1 and AeAPN2 resulted in significant changes in susceptibility of Ae. aegypti larvae to Cry4Ba and Cry11Aa toxins. Accordingly, our results suggest that AeAPN1 and AeAPN2 may not mediate Bti Cry4Ba and Cry11Aa toxicity in Ae. aegypti larvae as their binding proteins.

The transcriptional aftermath in two independently formed hybrids of the opportunistic pathogen Candida orthopsilosis

Interspecific hybridization can drive evolutionary adaptation to novel environments. The Saccharomycotina clade of budding yeasts includes many hybrid lineages, and hybridization has been proposed as a source for new pathogenic species. Candida orthopsilosis is an emerging opportunistic pathogen for which most clinical isolates are hybrids, each derived from one of at least four independent crosses between the same two parental lineages. To gain insight on the transcriptomic aftermath of hybridization in these pathogens, we analyzed allele-specific gene expression in two independently formed hybrid strains, and in a homozygous strain representative of one parental lineage. Our results show that the effect of hybridization on overall gene expression is rather limited, affecting ~4% of the studied genes. However, we identified a larger effect in terms of imbalanced allelic expression, affecting ~9.5% of the heterozygous genes in the hybrids. Some of these altered genes have functions related to pathogenicity, including zinc transport and superoxide dismutase activities. Additionally, the number of shared genes with imbalanced expression in the two independently formed hybrids was higher than random expectation, suggesting selective retention. While it remains unclear whether the observed imbalanced genes play a role in virulence, our results suggest that differences in allele-specific expression may add an additional layer of phenotypic plasticity to traits related to virulence in C. orthopsilosis hybrids.ImportanceHow new pathogens emerge is an important question that remains largely unanswered. Some emerging yeast pathogens are hybrids originated through the crossing of two different species, but how hybridization contributes to a higher virulence is unclear. Here we show that hybrids selectively retain gene regulation plasticity inherited from the two parents, and that this plasticity affects genes involved in virulence.

Identification of Aroma Gene (Mutated badh2) and Properties of Aroma on Aromatic BC5F2 Ciherang

Aromatic rice varieties have some weaknesses such as low productivity, and less resistant to pests and diseases. This study aimed to obtain homozygous strain of BC5F2 Ciherang aromatic through the identification of aroma gene (mutated badh2) and properties of the aroma. Ciherang paddy (nonaromatic paddy) was used as the female parent, whereas Mentik Wangi paddy (aromatic paddy) was used as the male parent. The experiment was conducted in BC5F2 because it is expected to generate plants with properties 98.4% close to female parent. The DNA from five strains of paddy plants BC5F2Ciherang X Mentik Wangi was isolated by a modified CTAB method. The concentration of DNA was determined by measuring absorbance at 260 nm wavelength, while its purity was determined from the ratio of the absorbance at a wavelength of 260/280 nm. PCR-based molecular selection was done by using the Bradbury primers. PCR results showed that of the 250 samples, there were 66 samples had DNA fragment of the same size as that of Mentik Wangi, i.e. 257 bp, 67 samples had the same size as the DNA fragment of Ciherang, i.e. 355 bp, and 117 samples had the same size with the both of DNA fragments, i.e. 257 bp and 355 bp. Plants with amplified 257 bp DNA fragment was subjected to leaf aroma test using 1.7% KOH. The results showed that 42 positive samples, out of 66 samples. Samples positive on leaf aroma test were tested again on rice aroma test. Rice aroma test results showed the majority (85.4%) samples that are positive on leaf aroma test is also positive on the rice aroma test.

The polytene chromosomes of Drosophila triauraria and D. quadraria, sibling species of D. auraria

The polytene chromosomes of Drosophila triauraria and D. quadraria, two of the sibling species of D. auraria, were examined. The polytene chromosomes of all three species exhibit very clear homology. Unlike the stock of D. auraria that we studied, D. triauraria and D. quadraria carry heterozygous paracentric inversions. In both species, 2R and 3R are the arms where these inversions are concentrated. In addition, in D. quadraria, the 3L chromosome arm is very complicated because of heterozygous inversions. The mode of inheritance of these rearrangements was studied. A homozygous strain for all chromosome arms of D. triauraria was isolated, while a homozygous strain was obtained only for the arms X, 2L, 3L, and 4 of D. quadraria. Like D. auraria, both species show a large number of inverted tandem duplications in the paired condition, even in the chromosomes of their hybrids. Small deletions were also detected, one of which, in D. triauraria, is homozygous terminal. Hypotheses are discussed concerning the relationships of the species and the existence of inverted tandem duplications.Key words: Drosophila, duplications, inversions, deletions, evolution.

Genetic control of Delia antiqua (Meigen) (Diptera: Anthomyiidae). Sensitivity to diapause interfering with a field-cage experiment using a homozygous chromosomal translocation

A translocation homozygous strain (TT) of Delia antiqua (Mg) was released into a field cage in Wageningen, The Netherlands, together with a standard laboratory strain (++). During the course of the season, the fecundity and fertility of the adults were measured together with the karyotype frequencies (TT+; T+; ++) of the F1 progeny. No selective disadvantage of the translocation karyotypes was observed. However, only four F1 adults emerged in the field cage; therefore a sample of pupae was removed from the cage, and it was shown that nearly 100% of the surviving pupae had entered diapause. This figure was confirmed from the remainder of the pupae. The fertility of eggs from the emerging adults was reduced to 54%, compared with the standard fertility of nearly 90%, but because of the diapause response of the strains used, the effect of this reduced fertility in the field-cage population could not be followed. The reasons for the change in diapause response of the laboratory strains are discussed and suggestions made as to how this could be prevented. The report highlights the importance of quality in control techniques involving translocations.

OBSERVATIONS ON THE RELEASE AND CLEARANCE OF NEUROPHYSIN AND THE NEUROHYPOPHYSIAL HORMONES IN THE RAT

SUMMARY Circulating levels of neurophysin, oxytocin and vasopressin were determined in rats under various physiological conditions using bovine neurophysin as the reference standard. Under resting conditions the mean plasma concentration of immunoreactive neurophysin was 6·2 ± 2·4 ng/ml while oxytocin and vasopressin were undetectable. Neurophysin and vasopressin were released in response to haemorrhage in Wistar and heterozygous Brattleboro rats; in the homozygous strain haemorrhage resulted in a release of oxytocin and neurophysin. Infusion of 0·1 M-calcium chloride into Wistar rats caused a rise in plasma neurophysin, oxytocin and vasopressin. The half-times for the disappearance from the plasma of arginine-vasopressin and neurophysin in the circulation were 1·6 ± 0·8 (S.D.) and 3·4 ± 0·9 min respectively.

EXTRACHROMOSOMAL ELEMENT DELTA IN DROSOPHILA MELANOGASTER. VIII. INSEPARABLE ASSOCIATION WITH SENSITIVE SECOND CHROMOSOME

ABSTRACT The presence of delta is always accompanied by a sensitive second chromosome in Drosophila melanogaster or vice versa. The separability of delta and the chromosome line was investigated in experiments designed to eliminate delta or to suppress its multiplication for a number of generations. Cy/Sb-5 males which transmit a minute amount of delta b (retained by chromosome Sb, kills Sb/Sb and Sb/Sr zygotes) to their progeny were backcrossed for 61 generations to Cy/Pm females which carried no delta. After backcrossing 465 Cy/Sb-5 males were individually examined for retention of delta, and all were found to retain delta b in its latent state. A homozygous strain for the Sr-20B chromosome which retained a minute amount of delta r (retained by chromosome Sr, kills Sr/Sr zygotes) was obtained. 1,252 Cy/Sr-20B females derived from the homozygous strains at the 21st generation were tested individually for delta retention. All females tested were found to retain delta r in its latent state. The delta retention in Sb-5 chromosome lines isolated from Sr-Cy/Sb-5 heterozygous strains which carried delta r but not delta b was examined. The descendant Cy/Sb-5 lines from the heterozygous strains had accumulated their specific delta, delta b. These findings are consistent with those obtained in an earlier study, and lead to the conclusion that delta is associated inseparably with each specific sensitive chromosome.

The Life-cycle of Bacteria. Alternate Asexual and Autogamic Phases

I. Life-Cycle. Colony Formation, Sporing, Papilla Formation.If any bacterium is sown in the usual way from a fluid suspension on to a plate of suitable culture medium, colonies arise from the multiplication of single bacteria. These colonies grow at their margins ar a rate roughly constant for the species. When the colony has reached a certain size, which is again roughly constant for the species, it ceases to grow or continues growing very slowly. Thus for example colonies of streptococci grow to 1 mm. in diameter, those of coliforms to 3 or 6 mm. The colonies do not continue to grow until they have covered the whole plate. This limitation of growth is not due to the using up of the food supply or to the drying of the medium, since, if a second sowing of the same bacterium is made between the arrested colonies, a new crop of colonies arises.1 Twort (1907) obtained lactose fermenting variants ofB. typhosusafter prolonged growth in this sugar. These variations do not however occur frequently and regularly. They are probably examples of mutation from a homozygous strain (Mode A above).2 The variations mentioned take place in culture where their origin from papillae can be seen. It is likely that exaltation of virulence is an adaptive variation of the same kind occurring in a host.