Bacterium‐Expressed dsRNA Downregulates Microsporidia Nosema bombycis Gene Expression

The microsporidia Nosema bombycis is the insect pathogen of pebrine disease severely destructive to sericulture production. Here, we describe the use of Escherichia coli HT115 strain (DE3) to express double‐strand RNAs targeting the gene encoding ADP/ATP protein in N. bombycis. The results showed that dsRNAs deferentially suppressed the gene expression during N. bombycis infection in the silkworm, and the effect waned gradually. Our results, for the first time, provide a tool to utilize the dsRNA expressed by recombinant E. coli to control the pebrine disease of the domestic silkworm.     

Resting Spore Dormancy and Infectivity Characteristics of the Potato Powdery Scab Pathogen Spongospora subterranea

The soil‐borne potato pathogen Spongospora subterranea persists in soil as sporosori, which are aggregates of resting spores. Resting spores may germinate in the presence of plant or environmental stimuli, but direct evidence for resting spore dormancy is limited. A soilless tomato bait plant bioassay and microscopic examination were used to examine features of S. subterranea resting spore dormancy and infectivity. Dried sporosori inocula prepared from tuber lesions and root galls were infective after both short‐ and long‐term storage (1 week to 5 years for tuber lesions and 1 week to 1 year for root galls) with both young and mature root galls inocula showing infectivity. This demonstrated that a proportion of all S. subterranea resting spores regardless of maturity exhibit characteristics of stimuli‐responsive dormancy, germinating under the stimulatory conditions of the bait host plant bioassay. However, evidence for constitutive dormancy within the resting spore population was also provided as incubation of sporosorus inoculum in a germination‐stimulating environment did not fully exhaust germination potential even after 2.4 years. We conclude that S. subterranea sporosori contain both exogenous (stimuli‐responsive) and constitutively dormant resting spores, which enables successful host infection by germination in response to plant stimuli and long‐term persistence in the soil.     

Susceptibility of Nontarget Hosts to Nosema furnacalis (Microsporida: Nosematidae), a Potential Biological Control Agent of the European Corn Borer, Ostrinia nubilalis (Lepidoptera: Pyralidae)

Nosema furnacalis Wenn whose normal host is the Asian corn borer, Ostrinia furnacalis (Guenee), is also pathogenic to the European corn borer, Ostrinia nubilaus (Hübner), making N. furnacalis a candidate biological control agent for O. nubilalis. Before N. furnacalis can be used in classical biological control, its effect on natural enemies of O. nubilalis needs to be assessed. We fed nine predator species and one parasitoid of O. nubilalis either purified N. furnacalis spores or N. furnacalis-infected larvae. Using microscopic examination and a species-specific serological assay within these natural enemies, we determined whether N. furnacalis spores were present and whether the spores had germinated. Of the 424 adult predators fed N. furnacalis spores or N. furnacalis-infected corn horers, only 2 tested weakly positive with the enzyme-linked immunosorbent assay (ELISA), indicating that ingested spores did not germinate in the adult predators. Fifteen of 130 immature predators tested weakly positive with the ELISA, but because relatively few spores were found upon microscopic examination, we concluded there was no evidence of an active N. furnacalis infection in any of the nine predator species tested. In contrast, the parasitoid Macrocentrus grandii Goidanich always tested strongly positive in the ELISA (mean O.D._{405 nm} = 1.36), indicating that sporoplasm antigens were present. Microscopic examination of this parasitoid showed that a high number of spores were present (mean spore number/larvae 1.4 × 10⁷). From these data, M. grandii might become infected with N. furnacalis.     

An estimator of the Opportunity for Selection that is independent of mean fitness

Variation among individuals in number of offspring (fitness, k) sets an upper limit to the evolutionary response to selection. This constraint is quantified by Crow's Opportunity for Selection (I), which is the variance in relative fitness (I = σ²_k/(u_k)²). Crow's I has been widely used but remains controversial because it depends on mean offspring number in a sample (). Here, I used a generalized Wright-Fisher model that allows for unequal probabilities of producing offspring to evaluate behavior of Crow's I and related indices under a wide range of sampling scenarios. Analytical and numerical results are congruent and show that rescaling the sample variance (s²_k) to its expected value at a fixed removes dependence of I on mean offspring number, but the result still depends on choice of . A new index is introduced, Δ_I = Π– E(Î_drift) = Π– 1/, which makes Î independent of sample without the need for variance rescaling. Δ_I has a straightforward interpretation as the component of variance in relative fitness that exceeds that expected under a null model of random reproductive success. Δ_I can be used to directly compare estimates of the Opportunity for Selection for samples from different studies, different sexes, and different life stages.     

Role of Nosema bombycis infected male silk moths in the venereal transmission of pebrine disease in Bombyx mori (Lep., Bombycidae)

Abstract: The role of male moths of silkworm in the spread of pebrine spores (disease) has been studied using appropriate crossings and examining the multiplication of spores in the testis. Sections of infected testis reveal the presence of spores in the peritoneal sheath, spermatids but not in the sperm. A large number of spores was observed in the bursa copulatrix of the uninfected female moth mated with an infected male moth. This is a first record of venereal transmission of pebrine spores in silkworms. Venereally transmitted spores do not contribute to transovarial transmission, but give a false positive indication during mother-moth examination. An alternative method for mother-moth testing has also been discussed.     

Effects of yeast extract on the production of phenylpropanoid metabolites in callus culture of purple basil (Ocimum Basilicum L. var purpurascens) and their in-vitro evaluation for antioxidant potential

Ocimum basilicum L. var. purpurascens is an enriched reservoir of pharmaceutically important compounds with plenty of health and therapeutic attributes such as phenolic acids and anthocyanins. However, the inefficient production of aforementioned metabolites in wild has restricted its commercial utilization. Herein, commercially viable phytochemicals have been enhanced through elicitation of in-vitro cultures of O. basilicum using yeast extract.The impact of various concentrations (YE 1 mg/L,YE 10 mg/L, YE 25 mg/L, YE 50 mg/L, YE 100 mg/L, YE 200 mg/L and YE 400 mg/L) of yeast extract on biomass accumulation, phytochemical production, and antioxidant activities were assessed in callus cultures. Moderate concentration of yeast extract (100 mg/L) enhanced biomass accumulation i.e. fresh weight (FW 216.28 g/L) and dry weight (DW 15.49 g/L) up to 1.5 folds as compared to control (FW 167.14 g/L and DW 10.25 g/L). Similarly, yeast extract (100 mg/L) increased total phenolic and flavonoid contents as well as enhanced antioxidant activities such as ABTS (2,2 azinobis 3-ethylbenzthiazoline-6-sulphonic acid), FRAP (ferric reducing antioxidant power) and DPPH (2,2-diphenyl-1-picryhydrazyl). High performance liquid chromatography (HPLC) analysis was elucidated for further phytochemical investigation. HPLC analysis showed an increase of almost 1.9 folds as compared to control in rosmarinic acid (15.19 mg/g DW), chicoric acid (2.13 mg/g DW), peonidin (2.70 mg/g DW) and cyanidin (1.57 mg/g DW). Likewise, 1.8 fold and 2.4 folds increase was observed in eugenol essential oils (0.25 mg/g DW) and chavicol (0.037 mg/g DW), respectively. For cellular antioxidant activity, reactive oxygen specie or reactive nitogen specie (ROS/RNS) was induced in yeast cells and the effect of O. basilicum callus culture was further investigated in stressed yeast cells. A positive correlation exists between the antioxidant activities, TPC and TFC analysis. In short, these results showed that yeast extract could act as an efficient elicitor to enhance pharmacologically important metabolites in callus cultures of Ocimum basilicum.

Graphical abstract

     

In vitro organogenesis and plant regeneration of Thymus serpyllum L.: an important aromatic medicinal plant

An efficient in vitro propagation system has been developed for the rapid micropropagation of Thymus serpyllum L. (Banajwain), an aromatic medicinal herb from nodal explant on MS medium. Phenolic leaching and high rate of contamination was the most significant problem in establishing in vitro culture of Thymus serpyllum which was overcome by preparing explants in an antioxidant ascorbic acid (1000 ppm) at 6°C for 45 min and addition of the same antioxidant (50 mgl⁻¹) to the MS medium. The frequency of shoot production was influenced by different cytokinins (Kn, BAP, and Kn + BAP) and 95.56% shoot induction was observed when MS medium was supplemented with 1.0 + 2.0 mgl⁻¹ (Kn + BAP). The maximum average number of shoots 16.93 ± 2.15 and average length (3.98 ± 0.55) was recorded when MS medium have 0.5 + 2.0 mgl⁻¹ (Kn + BAP). The in vitro regenerated microshoots were rooted on MS and half strength MS medium and there was significant difference in root induction on both media under the influence of auxins (IAA, IBA, and NAA). The maximum average number (11.67 ± 3.03) and average root length (3.88 ± 0.71) was reported in half MS medium having 1.0 mgl⁻¹ IBA. The complete regenerated plantlets were acclimatized under growth chamber before transferring to the earthen pots and showed 90% survival.

Graphical abstract

     

Estimating occupancy of Chinese pangolin (Manis pentadactyla) in a protected and non‐protected area of Nepal

Chinese pangolin is the world's most heavily trafficked small mammal for luxury food and traditional medicine. Although their populations are declining worldwide, it is difficult to monitor their population status because of its rarity and nocturnal behavior. We used site occupancy (presence/absence) sampling of pangolin sign (i.e., active burrows) in a protected (Gaurishankar Conservation Area) and non‐protected area (Ramechhap District) of central Nepal with multiple environmental covariates to understand factors that may influence occupancy of Chinese pangolin. The average Chinese pangolin occupancy and detection probabilities were  ± SE = 0.77 ± 0.08;  ± SE = 0.27 ± 0.05, respectively. The detection probabilities of Chinese pangolin were higher in PA (  ± SE = 0.33 ± 0.03) than compared to non‐PA (  ± SE = 0.25 ± 0.04). The most important covariates for Chinese pangolin detectability were red soil (97%), food source (97.6%), distance to road (97.9%), and protected area (97%) and with respect to occupancy was elevation (97.9%). We recommended use of remote cameras and potentially GPS collar surveys to further investigate habitat use and site occupancy at regular intervals to provide more reliable conservation assessments.     

Grading cartilage damage with diffuse reflectance spectroscopy: Optical markers and mechanical properties

Osteoarthritis (OA) is one of the most common joint diseases worldwide. Unfortunately, clinical methods lack the ability to detect OA in the early stages. Timely detection of the knee joint degradation at the level of tissue changes can prevent its progressive damage. Here, diffuse reflectance spectroscopy (DRS) in the NIR range was used to obtain optical markers of the cartilage damage grades and to assess its mechanical properties. It was observed that the water content obtained by DRS strongly correlates with the cartilage thickness (R = .82) and viscoelastic relaxation time (R = .7). Moreover, the spectral parameters, including water content (OH-band), protein content (CH-band), and scattering parameters allowed for discrimination between the cartilage damage grades (10⁻⁴ < P ≤ 10⁻³). The developed approach may become a valuable addition to arthroscopy, helping to identify lesions at the microscopic level in the early stages of OA and complement the surgical analysis.     

Applying the polio model to New Zealand: The contingent role of socioeconomic status in infectious disease mortality

Objectives

The traditional polio model depicts a positive relationship between the disease and socioeconomic status (SES), with higher SES associated with exposure to the poliovirus beyond infancy/early childhood and a higher mortality rate. This study uses occupation data from non-Māori New Zealand death registrations and the Census to assess evidence for differential mortality by SES during the polio epidemics of 1916, 1924–25, and 1936–37.

Materials and Methods

Polio deaths were classified by father's occupation using the Caversham Project scheme, which includes nine collapsible urban occupational strata and codes rural occupations separately. Evidence for differential mortality and differences in age at death between rural and urban areas and among urban occupational strata was assessed using nonparametric Mann–Whitney U, Kruskal–Wallis, Jonckheere-Terpstra, and chi-square tests.

Results

There was limited evidence of either differential mortality or differences in age at death among collapsed urban strata, mainly in 1924/25, only partially meeting the expectations of the traditional polio model. For 1916 only, the rural group had a higher proportion of deaths (χ² (1, n = 95) = 8.341, p = 0.0039, φ = 0.30)) and higher age at death (U = 828.00, p = 0.026, r = −0.23).

Discussion

These results suggest that rural New Zealand was more isolated from poliovirus infection prior to 1916, but thereafter rural and urban groups faced more similar exposure patterns and mortality—at least among non-Māori. The degree to which occupational categories reflect conditions of exposure requires further research.

     

Thermo-optic measurements and their inter-dependencies for delineating cancerous breast biopsy tissue from adjacent normal

The histopathological diagnosis of cancer is the current gold standard to differentiate normal from cancerous tissues. We propose a portable platform prototype to characterize the tissue's thermal and optical properties, and their inter-dependencies to potentially aid the pathologist in making an informed decision. The measurements were performed on 10 samples from five subjects, where the cancerous and adjacent normal were extracted from the same patient. It was observed that thermal conductivity (k) and reduced-scattering-coefficient (μ'_s) for both the cancerous and normal tissues reduced with the rise in tissue temperature. Comparing cancerous and adjacent normal tissue, the difference in k and μ'_s (at 940 nm) were statistically significant (p = 7.94e-3), while combining k and μ'_s achieved the highest statistical significance (6.74e-4). These preliminary results promise and support testing on a large number of samples for rapidly differentiating cancerous from adjacent normal tissues.     

Quantification of airborne Aspergillus fumigatus spores in rabbit houses by real-time polymerase chain reaction

To accurately quantify airborne Aspergillus fumigatus (A. fumigatus) spores in rabbit houses, the real-time polymerase chain reaction (real-time PCR) and culture-based counting method (CCM) were employed to determine the airborne A. fumigatus spore concentrations. The results showed that, of the three rabbit houses (A, B, and C), the average concentrations of airborne A. fumigatus spores determined by real-time PCR were 3.0 × 10³, 3.3 × 10³, and 1.5 × 10³ spores/m³ air, respectively, while those determined by CCM were 2.5 × 10², 2.8 × 10², and 1.1 × 10² colony-forming unit/m³ air (CFU/m³ air), respectively, i.e., the former concentration was 12–14 times higher than the latter one. Therefore, the conventional CCM underestimated the concentrations of airborne fungal spores, and it is insufficient to determine the microbial aerosol concentration and evaluate the health risk only using CCM.     

Issue Information

Lake Inle, a pristine lake in Myanmar, is known to harbor a number of endemic aquatic species and is a home to an enigmatic cyanobacterium Oscillatoria kawamurae, which was first reported in Lake Biwa (Japan). Macroscopic trichome (upper left), microscopic image (upper middle). Photos by Phong San (Lake Inle) and Nanda Kyaw Thu (O. kawamurae). See Thu et al. in this issue. Cover picture from: Article link here

     

Age and growth of the bull shark (Carcharhinus leucas) around Reunion Island,South West Indian Ocean

Sharks exhibit varied demographic strategies depending on both the species and the population location, which make them more or less vulnerable to fishing. Accurate evaluation of local age and growth parameters is therefore fundamental for the sustainable management of their stocks. Although demographic parameters have been assessed for bull shark (Carcharhinus leucas) populations in several locations of the world, this information is missing so far around Reunion Island, in the south-west Indian Ocean. To fill this gap of knowledge, age and growth data were gathered from the vertebrae of 140 individuals of C. leucas (77 females and 63 males, mostly adults) fished around the island between 2012 and 2019. After verification of the annual deposition of growth band pairs on these structures using relative marginal increment analysis on 40 individuals, band pairs were counted along the vertebral centrum for each individual. Thanks to this approach, growth was shown to significantly differ between male and female C. leucas around Reunion Island, with respective von Bertalanffy growth model equations of L_t =  and L_t = . Indeed, the females of the species fished in this area were significantly (P < 0.001) larger than local males, with an estimated difference in size of ~16.1 cm at 20 years old. They also apparently reach older ages, with an estimated maximum age of 33.50 years, against 29.75 years only for the males. The estimated size at birth around the island is larger than elsewhere in the world, varying from 92.30 to 100.00 cm depending on the method used. These results confirm that the population of C. leucas around Reunion Island exhibits a K-selected strategy, which makes it highly vulnerable to fishing pressure.     

A Microsporidian Infection in Phoronids (Phylum Phoronida): Microsporidium phoronidi n. sp. from a Phoronis embryolabi

Microsporidia‐like spores (2.0–3.0 × 1.3–1.5 μm) were discovered upon examination of histological sections taken from Phoronis embryolabi Temereva, Chichvarkhin 2017 found inhabiting burrows of shrimps Nihonotrypeae japonica (Decapoda, Callianassidae) from the Sea of Japan, Russia. Ultrastructural examination of spores revealed one nucleus and a uniform polar filament of 7–11 coils. Representatives of the phylum Phoronida have never been recorded as hosts of microsporidia. Parasites developed in vasoperitoneal tissue and caused formation of multinucleate syncytia. Basing on unique host and fine morphology, we assign the novel finding to Microsporidium phoronidi n. sp. and place provisionally in the collective genus Microsporidium.     

Application of different downstream processing methods and their comparison for the large-scale preparation of Bacillus thuringiensis var. israelensis after fermentation for mosquito control

Bacillus thuringiensis var. israelensis, a gram positive, spore-forming bacillus, produces parasporal crystal protein during sporulation, which is toxic in the mosquito larvae gut. An efficient downstream processing method for separating the spore crystal complex (SCC) from the fermented broth of B. thuringiensis var. israelensis is required to achieve maximum mosquitocidal activity. The different downstream processing methods, viz., tangential flow ultra-filtration, continuous centrifugation and acid precipitation were compared for their efficiency in separating SCC from broth obtained from a pilot-scale fermentor (100 l capacity). Among the three downstream processing methods, tangential flow ultra-filtration yielded the maximum amount of biomass (53.3 g/l), maximum number of spores (2.30 × 10¹⁸ CFU/ml) and highest level of larvicidal activity (LC₅₀ 28 nl/ml) against Aedes aegypti Bora-Bora strain followed by continuous centrifugation and acid precipitation methods.     

Raman spectroscopy of blood and urine liquid biopsies for ovarian cancer diagnosis: identification of chemotherapy effects

Blood plasma and serum Raman spectroscopy for ovarian cancer diagnosis has been applied in pilot studies, with promising results. Herein, a comparative analysis of these biofluids, with a novel assessment of urine, was conducted by Raman spectroscopy application in a large patient cohort. Spectra were obtained through samples measurements from 116 ovarian cancer patients and 307 controls. Principal component analysis identified significant spectral differences between cancers without previous treatment (n = 71) and following neo-adjuvant chemotherapy (NACT), (n = 45). Application of five classification algorithms achieved up to 73% sensitivity for plasma, high specificities and accuracies for both blood biofluids, and lower performance for urine. A drop in sensitivities for the NACT group in plasma and serum, with an opposite trend in urine, suggest that Raman spectroscopy could identify chemotherapy-related changes. This study confirms that biofluids' Raman spectroscopy can contribute in ovarian cancer's diagnostic work-up and demonstrates its potential in monitoring treatment response.     

Monitoring the acoustic activity of an aquatic insect population in relation to temperature,vegetation and noise

1. Acoustic population monitoring is a noninvasive method that can be deployed continuously over long periods of time and at large spatial scales. One of the newly discovered threats acting on biological diversity is anthropogenic noise. High levels of anthropogenic noise occur in aquatic environments, yet their effects on animals living in freshwater habitats have very rarely been investigated.
2. Here, we used acoustic monitoring and automatic detection to assess the acoustic activity of a population of a soniferous freshwater insect.
3. The sounds emitted by the corixid Micronecta scholtzi were recorded in a Mediterranean pond with an array of 12 hydrophones. An automatic analysis based on a measure of the amplitude found in the frequency band of M. scholtzi was developed to assess the level of acoustic activity. We used functional linear models, accounting for the periodicity of the calling behaviour, to estimate the possible effect of temperature, vegetation and a noise due to an immersed engine.
4. The automatic analysis was validated as an efficient method to measure the acoustic activity. The monitoring revealed a clear 24-hr pattern in the acoustic activity of M. scholtzi and three peaks of activity during the morning. Functional linear models revealed negative effects of both temperature and vegetation and showed that an engine noise, played back for 2 hr during the night, elicited an increase in the level of acoustic activity of the population. Moreover, a cross-correlation procedure showed that noise delayed the acoustic activity of the population.
5. Our results suggest that acoustic survey and automatic detection are efficient methods to monitor the acoustic activity of an insect population especially in response to an anthropogenic disturbance.