High-Temporal-Resolution In Situ Sensor for Oceanic CO2 Isotope Measurement Enabling Multidimensional Isotope Tracing Analysis (R13C, R18O, and R17O) via Laser Absorption Spectroscopy.

Combined in situ analysis of oceanic CO2 concentrations and diverse C and O isotope characteristics can offer a unique perspective with multiple isotopic tracing dimensions for identifying marine biogeochemical processes. Applying this strategy in marine environments is urgently required, yet it faces inherent challenges in terms of existing analytical methods and instruments, e.g., a lack of in situ sensors, limited detectable isotope variety, and low-temporal-resolution data. Here, we report an underwater in situ dissolved CO2 isotope sensor based on mid-infrared tunable diode laser absorption spectroscopy (MIR-TDLAS) and membrane extraction technology. Through the proposed targeted strategies, the sensor is capable of providing high-temporal-resolution in situ measurement of all monosubstituted isotopes of dissolved CO2 (16O13C16O, 18O12C16O, and 17O12C16O) at marine background concentrations. The sensor is demonstrated to provide comparable precision to that of isotope ratio mass spectrometry. At 400 ppmv, the precision for R13C, R18O, and R17O could achieve 0.084, 0.042, and 0.013‰, respectively, for a 1 s integration time. By enabling a high-frequency in situ analysis in fixed-point time-series field deployment, a 17O anomaly with strong regularity is observed, which is not obvious in 18O and 13C, and therefore, the superiority of the proposed multidimensional in situ isotope tracing strategy is demonstrated. The developed sensor has great potential to open up new prospects for advancing marine carbon research.

Three-dimensional reconstruction based on micro-imaging under wavelength-tunable illumination.

The three-dimensional reconstruction technique has been widely applied across various fields, with imaging serving as a fundamental approach to achieve this reconstruction. In the present study, we employed micro-imaging to realize 3D reconstruction based on the "shape from focus" and the chromatic aberration effect. This approach eliminates the need for sample or imaging lens movement to locate the focal plane for obtaining clear images. Instead, by utilizing tunable illuminance, we can adjust the imaging distance through the chromatic aberration, thereby achieving accurate reconstructions. As a means of verification, a simple system was accordingly constructed with an adjustable illuminance range (500-750 nm) at a magnification of 10× for imaging purposes. The fine reconstruction achieved high precision in micrometers; however, the depth of field emerged as an issue during the reconstruction process. To assess this method, a coin was employed, and the resulting reconstruction bias was determined to be as low as 0.01 mm. These findings indicate that the proposed method is practical for surface reconstruction and its capabilities will be further enhanced through optical design improvements.

Tracking organic matrix in the seashell by elemental mapping under laser-induced breakdown spectroscopy.

As a biogenic calcium carbonate, the seashell plays a crucial role in marine environmental studies. In these studies, it is essential to investigate the composition of the seashell. In this study, we used laser-induced breakdown spectroscopy (LIBS) to analyze the elemental composition of cultured scallop-shell (Patinopecten yessoensis), with a specific focus on examining the organic elements (C, N, O, H) to track the shell organic matrix (SOM). Our findings indicate that the seashell organic layer can be accurately identified by referencing the strong emission of nitrogen or the low signal of calcium. To further confirm the presence of this layer, we employed fluorescence spectroscopy, Raman spectroscopy and FTIR spectroscopy. Correlation analysis revealed a strong connection between LIBS emissions (H, O, CC) and seashell organics, as well as demonstrated the presence of organics in metallic emissions (Si, Ba). However, when we conducted elemental mapping on the shell cross-section, the distribution similarity was observed between the elements N, Ba, and Sr. Based on the correlation of organics and the distribution similarity, it is concluded that barium is an element associated with the SOM. These results highlight the potential of LIBS for organic analysis, which can complement traditional seashell analysis.

Holobiont perspectives on tripartite interactions among microbiota, mosquitoes, and pathogens.

Mosquito-borne diseases like dengue and malaria cause a significant global health burden. Unfortunately, current insecticides and environmental control strategies aimed at the vectors of these diseases are only moderately effective in decreasing disease burden. Understanding and manipulating the interaction between the mosquito holobiont (i.e., mosquitoes and their resident microbiota) and the pathogens transmitted by these mosquitoes to humans and animals could help in developing new disease control strategies. Different microorganisms found in the mosquito's microbiota affect traits related to mosquito survival, development, and reproduction. Here, we review the physiological effects of essential microbes on their mosquito hosts; the interactions between the mosquito holobiont and mosquito-borne pathogen (MBP) infections, including microbiota-induced host immune activation and Wolbachia-mediated pathogen blocking (PB); and the effects of environmental factors and host regulation on the composition of the microbiota. Finally, we briefly overview future directions in holobiont studies, and how these may lead to new effective control strategies against mosquitoes and their transmitted diseases.

Seconds-Scale Response Sensor for In Situ Oceanic Carbon Dioxide Detection.

Research on the transient variation processes of oceanic dissolved CO2 makes significant sense because of the complexity and dynamics of the marine environment. Yet, it is inherently challenging due to the limitation of the response performance of in situ sensors. Here, we report a novel system solution capable of providing high-performance detection with a seconds-scale response, sub-ppmv level precision, and 3000 m rated depth. Through the proposed strategy, we break the limitation of the membrane on the response performance of the sensor and improve it by 2 orders of magnitude to the τ100 of 3.5 s (τ90 = 2.7 s). By taking water temperature and CO2 concentration as the tracer, we succeed in portraying the water mixing process and reveal the microstructure of the concentration variation profile. By enabling in situ detection at an unprecedented response speed, this instrument can provide new insights and prospects into the research on the carbon cycle in deep-sea unstable regions, such as hydrothermal vents and cold seeps.

Quantitation improvement of underwater laser induced breakdown spectroscopy by using self-absorption correction based on plasma images.

Laser-induced breakdown spectroscopy (LIBS) is a practical technique for in-situ detection, but self-absorption effect has been a big issue for quantitative applications of this technique. In presented work, a method was developed to correct self-absorption to improve the quantitation of underwater LIBS. We proposed "relative self-absorption coefficient" as the critical parameter to evaluate self-absorption, and the plasma image was employed as the reference to determine the coefficient value. Based on that, the LIBS detection was successfully corrected by the coefficient to realize quantitative analysis, and the "Dominant Factor-PLS" was used as assistance. The results indicated that our method greatly improved LIBS quantitation in practice. More importantly, the calibration curve was able to be established with high linearity (R2 = 0.9999) to cover a large concentration range (0-103 ppm). It is hoped that our method could be a contribution to developing LIBS as an analytical tool for field measurements.

Development of an Easy-to-Operate Underwater Raman System for Deep-Sea Cold Seep and Hydrothermal Vent In Situ Detection.

As a powerful in situ detection technique, Raman spectroscopy is becoming a popular underwater investigation method, especially in deep-sea research. In this paper, an easy-to-operate underwater Raman system with a compact design and competitive sensitivity is introduced. All the components, including the optical module and the electronic module, were packaged in an L362 × Φ172 mm titanium capsule with a weight of 20 kg in the air (about 12 kg in water). By optimising the laser coupling mode and focusing lens parameters, a competitive sensitivity was achieved with the detection limit of SO42- being 0.7 mmol/L. The first sea trial was carried out with the aid of a 3000 m grade remotely operated vehicle (ROV) "FCV3000" in October 2018. Over 20,000 spectra were captured from the targets interested, including methane hydrate, clamshell in the area of cold seep, and bacterial mats around a hydrothermal vent, with a maximum depth of 1038 m. A Raman peak at 2592 cm-1 was found in the methane hydrate spectra, which revealed the presence of hydrogen sulfide in the seeping gas. In addition, we also found sulfur in the bacterial mats, confirming the involvement of micro-organisms in the sulfur cycle in the hydrothermal field. It is expected that the system can be developed as a universal deep-sea survey and detection equipment in the near future.

Underwater In Situ Dissolved Gas Detection Based on Multi-Reflection Raman Spectroscopy.

The detection of dissolved gases in seawater plays an important role in oceanic observations and exploration. As a potential technique for oceanic applications, Raman spectroscopy has been successfully applied in hydrothermal vents and cold seep fluids, but it has not yet been used in common seawater due to the technique's lower sensitivity. In this work, we present a highly sensitive underwater in situ Raman spectroscopy system for dissolved gas detection in common seawater. Considering the difficulty of underwater degassing and in situ detection, we designed a near-concentric cavity to improve the sensitivity, with a miniature gas sample chamber featuring an inner volume of 1 mL placed inside the cavity to reach equilibrium in a short period of time. According to the 3σ criteria, the detection limits of this system for CO2, O2, and H2 were calculated to be 72.8, 44.0, and 27.7 ppm, respectively. Using a hollow fiber membrane degasser with a large surface area, the CO2 signal was found to be clearly visible in 30 s at a flow rate of 550 mL/min. Moreover, we deployed the system in Qingdao's offshore seawater, and the field test showed that this system is capable of successfully detecting in situ the multiple gases dissolved in the seawater simultaneously.

A Portable Tunable Diode Laser Absorption Spectroscopy System for Dissolved CO2 Detection Using a High-Efficiency Headspace Equilibrator.

Continuous observation of aquatic pCO2  at the ocean surface, with a sensitive response time and high spatiotemporal resolution, is essential for research into the carbon biogeochemical cycle. In this work, a portable tunable diode laser absorption spectroscopy (TDLAS) system for dissolved CO2 detection in surface seawater, coupled with a home-made headspace equilibrator, allowing real time underway measurements, is described. Both the optical detection part and sample extraction part were integrated together into a compact chamber. An empirical equation suitable for this system was acquired, which can convert the concentration from the gas-phase to the aqueous-phase. A monitoring precision of 0.5% was obtained with time-series measurement, and the detection limits of 2.3 ppmv and 0.1 ppmv were determined with 1 s and 128 s averaging time, respectively. Sampling device used in this work was ameliorated so that the response time of system reduced by about 50% compared to the traditional 'shower head' system. The fast response time reached the order of 41 s when the final concentration span was 3079 ppmv. For1902 ppmv, this figure was as short as 20 s. Finally, a field underway measurement campaign was carried out and the results were briefly analyzed. Our work proved the feasibility of the TDLAS system for dissolved CO2 rapid detection.

Normalization of underwater laser-induced breakdown spectroscopy using acoustic signals measured by a hydrophone.

Laser-induced breakdown spectroscopy (LIBS) signals in water always suffer strong pulse-to-pulse fluctuations that result in poor stability of the spectrum. In this work, a spectrum normalization method based on acoustic signals measured by a hydrophone immersed in water was developed and compared with laser energy normalization. The characteristics of the acoustic signals were studied first, and the correlations between the acoustic signals and LIBS spectra were analyzed. It showed that the spectral line intensity has a better linear relationship with the acoustic energy than with the laser energy. Consequently, the acoustic normalization exhibited better performance on the reduction of LIBS spectral fluctuation versus laser energy normalization. Calibration curves of Mn, Sr, and Li were then built to assess the analytical performance of the proposed acoustic normalization method. Compared with the original spectral data, the average RSD_C values of all analyte elements were significantly reduced from 5.00% to 3.18%, and the average RSD_P values were reduced from 5.09% to 3.28%, by using the acoustic normalization method. These results suggest that the stability of underwater LIBS can be clearly improved by using acoustic signals for normalization, and acoustic normalization works more efficiently than laser energy normalization. This work provides a simple and cost-effective external acoustic normalization method for underwater LIBS applications.

PCE3 Plays a Role in the Reproduction of Male Nilaparvata lugens.

Nilaparvata lugens proclotting enzymes (NlPCEs) belong to the clip domain serine protease (clip-SP) family, which is a characteristic protease family in arthropods. NlPCE3 was previously reported to regulate egg production and development in female N. lugens, but its role in male N. lugens is unclear. In the present study, qPCR analysis showed that NlPCE3 was expressed in three different tissues (gut, testis and fat body). RNAi revealed that dsNlPCE3 injection made the male vas deferens thinner and reduced the oviposition level of the females that mated with dsNlPCE3-treated males, causing eggs not to hatch. Furthermore, immunofluorescence staining showed that NlPCE3 was widely expressed in the male internal genitalia. However, after dsNlPCE3 injection, expression of NlPCE3 was diffuse in the male internal genitalia, whose peripheral cells seemed degraded. Overall, these results indicate that NlPCE3 is important for reproduction in male N. lugens.

Development and Field Tests of a Deep-Sea Laser-Induced Breakdown Spectroscopy (LIBS) System for Solid Sample Analysis in Seawater.

In recent years, the investigation and exploitation of hydrothermal region and polymetallic mineral areas has become a hot topic. The emergence of underwater vehicle platforms has made it possible for new chemical sensors to be applied in marine in-situ detection. Laser-induced breakdown spectroscopy (LIBS), with its advantages of rapid real-time analysis, sampling without pretreatment, simultaneous multi-element detection and stand-off detection, has great potential in marine applications. In this paper, a newly more compact and lighter underwater LIBS system based on the LIBSea system named LIBSea II was developed and tested both in the laboratory and sea trials. The system consists of a Nd:YAG single-pulse laser at 1064 nm, a fiber spectrometer, optical layout, a power supply module and an internal environment sensor. The system is encapsulated in a pressure vessel (Φ 190 mm × L 588 mm) with an optical window on the end cap. Experimental parameters of the system including laser energy and delay time were firstly optimized in the laboratory. Then, field test of the system in nearshore was performed with various samples, including pure metal and alloy samples as well as a manganese nodule sample from deep sea, to verify the detection performance of the LIBSea II system. In 2019, the system was deployed on a remotely operated vehicle (ROV) of Haima for deep sea trial, and atomic lines of K, Na, Ca and strong molecular bands of CaOH from a carbonate rock sample were obtained for the first time at depths of 1400 m. These results show that the LIBSea II system has great potential to be used in deep-sea geological exploration.

Laser-induced plasma in water at high pressures up to 40 MPa: A time-resolved study.

The knowledge on the laser-induced plasma emission in water at high pressures is essential for the application of laser-induced breakdown spectroscopy (LIBS) in the deep-sea. In this work, we investigate the spectral features of ionic, atomic and molecular emissions for the plasma in water at different pressures from 1 to 40 MPa. By comparing between the time-resolved spectra and shadowgraph images, we demonstrate that the dynamics of the cavitation bubble at high pressures plays a key role on the characterization of plasma emission. The initial plasma emission depends weakly on the external pressure. As time evolves, the cavitation bubble is more compressed by the higher external pressure, leading to a positive confinement effect to maintain the plasma emission. However, at very high pressures, the bubble collapses extremely fast and even earlier than the cooling of the plasma. The plasma will gain energy from the bubble collapse phase, but quench immediately after the collapse, leading to a sharp reduction in the plasma persistence. These effects caused by bubble dynamics explain well the observed spectral features and are further proved by the temporal evolutions of the plasma temperature and electron density. This work gives not only some insights into the laser-induced plasma and bubble dynamics in high pressure liquids but also better understanding for the application of underwater LIBS in the deep-sea.

Depth Profiling Investigation of Seawater Using Combined Multi-Optical Spectrometry.

Depth profiling investigation plays an important role in studying the dynamic processes of the ocean. In this paper, a newly developed hyphenated underwater system based on multi-optical spectrometry is introduced and used to measure seawater spectra at different depths with the aid of a remotely operated vehicle (ROV). The hyphenated system consists of two independent compact deep-sea spectral instruments, a deep ocean compact autonomous Raman spectrometer and a compact underwater laser-induced breakdown spectroscopy system for sea applications (LIBSea). The former was used to take both Raman scattering and fluorescence of seawater, and the LIBS signal could be recorded with the LIBSea. The first sea trial of the developed system was taken place in the Bismarck Sea, Papua New Guinea, in June 2015. Over 4000 multi-optical spectra had been captured up to the diving depth about 1800 m at maximum. The depth profiles of some ocean parameters were extracted from the captured joint Raman-fluorescence and LIBS spectra with a depth resolution of 1 m. The concentrations of SO42- and the water temperatures were measured using Raman spectra. The fluorescence intensities from both colored dissolved organic matter (CDOM) and chlorophyll were found to be varied in the euphotic zone. With LIBS spectra, the depth profiles of metallic elements were also obtained. The normalized intensity of atomic line Ca(I) extracted from LIBS spectra raised around the depth of 1600 m, similar to the depth profile of CDOM. This phenomenon might be caused by the nonbuoyant hydrothermal plumes. It is worth mentioning that this is the first time Raman and LIBS spectroscopy have been applied simultaneously to the deep-sea in situ investigations.

Detection improvement of laser-induced breakdown spectroscopy using the flame generated from alcohol-solution mixtures.

It has been proved that the detection of laser-induced breakdown spectroscopy (LIBS) could be improved by the flame. In this work, we applied flame enhanced LIBS for the detection of elements in water, while the flame was generated from the mixture of alcohol and aqueous solution. In the measurements, the flame is functioned as an assistance to enhance the LIBS detection, and also worked as a sampling way for the solution. The obtained results indicate that the detection of manganese, calcium, lithium and magnesium were significantly improved by the proposed method. It is found that the flame actually forms an environment for the laser-induced plasma to have higher temperature and lower electron density, as comparing with the plasma underwater. With the method, the quantitative analysis was tried out for the element of manganese, and the internal reference of calcium was used. It is interesting to find that, when mixing with the calcium, the minimum detectable concentration of manganese could be lowered and the intensity was greatly increased. According to the result, it is suggested that the proposed method might be a practical way for liquid detection of LIBS because of the simplicity and the effectiveness.

A Clip Domain Serine Protease Involved in Egg Production in Nilaparvata lugens: Expression Patterns and RNA Interference.

Clip domain serine proteases play vital roles in various innate immune functions and in embryonic development. Nilaparvata lugens proclotting enzymes (NlPCEs) belong to this protease family. NlPCE1 was reported to be involved in innate immunity, whereas the role of other NlPCEs is unclear. In the present study, N. lugens proclotting enzyme-3 (NlPCE3) was cloned and characterized. NlPCE3 contains a signal peptide, a clip domain, and a trypsin-like serine protease domain. NlPCE3 was expressed in all tissues examined (gut, fat body, and ovary), and at all developmental stages. Immunofluorescence staining showed that NlPCE3 was mainly expressed in the cytoplasm and cytomembrane of follicular cells. Double stranded NlPCE3 RNA interference clearly inhibited the expression of NlPCE3, resulting in abnormal egg formation and obstruction of ovulation. These results indicate that NlPCE3 plays an important role in egg production in N. lugens.

Salinity effects on elemental analysis in bulk water by laser-induced breakdown spectroscopy.

The effects of salinity on underwater laser-induced breakdown spectroscopy (LIBS) were investigated with salinities ranging from 2‰ to 50‰. Both spectroscopic and fast imaging techniques were used to observe plasma emission. It was shown that as the salinity increased, emission intensities of the atomic lines increased, while intensities of the ionic lines were suppressed. The signal-to-background ratios of the spectral lines decreased as a function of salinity, but the signal-to-noise ratios changed irregularly with salinity. Image results demonstrated that brighter and longer plasma could be produced at higher salinity with higher plasma temperature and electron density. The calibration curves at different salinities indicated that the high salinity environment did not limit the detection capability of LIBS. The obtained results reveal the significant influences of salinity on underwater LIBS detection, which plays an important role in promoting applications of LIBS in the ocean.

Development of a compact deep-sea Raman spectroscopy system and direct bicarbonate detection in sea trials.

In recent years, Raman spectroscopy techniques have been successfully applied to the area of deep-sea exploration. However, there are still some problems impeding the further application of Raman systems. For example, the large size of an underwater Raman system makes it difficult to deploy on the underwater vehicle. Meanwhile, the sensitivity is often a disadvantage, requiring improvement for detecting more trace components. To solve these problems, a new compact deep-sea in situ Raman spectroscopy system is presented in this paper. The whole system weighs 60 kg and is housed in an L800 mm×ϕ258 mm pressure vessel with an optical window on the front end cap. The main components include a 532 nm Nd:YAG laser, an optics module, a high-throughput spectrograph with 0∼4900 cm-1 spectral range and 8 cm-1 spectral resolution, a TEC-cooled 2000 pixel×256 pixel CCD detector, a PC104 embedded computer, and an electronics module. To evaluate the performance of the newly developed Raman system, systematic experiments have been carried out with solutions in laboratory, and the results have shown that the system limit of detection of SO42- is 0.4 mmol/L. The Raman system has been successfully deployed on a remote-operated vehicle on the Kexue research vessel in June 2015. The typical in situ detection results are presented in this paper, and it is shown that the Raman system is capable of detecting the Raman signal of SO42- and fluorescence of chlorophyll a (chl-a) and chromophoric dissolved organic matter (CDOM) in seawater. With 500 spectra accumulations and some data processing, the Raman signal of HCO3- is obtained. This is the first report of direct measurement of HCO3- by Raman system in in situ experiments. After further optimization, it is hoped to apply the Raman system in seafloor observation networks for long-time carbon cycling research.

Effects of Ambient Temperature on Laser-Induced Plasma in Bulk Water.

Laser-induced breakdown spectroscopy (LIBS) has been successfully applied to ocean exploration, but the changes in marine environmental factors could have an important impact on the LIBS signals. The aim of the research is to investigate the ambient water temperature effects on laser-induced plasma in bulk water. Both the spectroscopic and fast imaging techniques are used to observe the plasma emission with the temperatures in the range of 5-60 ℃. It is shown that as the ambient temperature increases, an obviously increasing trend of emission intensity is observed, both for the atomic and ionic lines of Ca. Higher plasma temperature and electron density can be obtained at higher ambient temperature. The image results demonstrate that hotter and larger plasmas can be produced in water with the increase of ambient temperature. In addition, it is found that the changes of plasma emission and morphologies could be related to the changes of physical property parameters of water such as thermal expansivity and viscosity with ambient temperature. The results suggest that the ambient temperature has great influences on laser-induced plasma, which needs to be taken into account in underwater LIBS measurement, especially on-site marine applications.

Analysis and Modeling Methodologies for Heat Exchanges of Deep-Sea In Situ Spectroscopy Detection System Based on ROV.

In recent years, cabled ocean observation technology has been increasingly used for deep sea in situ research. As sophisticated sensor or measurement system starts to be applied on a remotely operated vehicle (ROV), it presents the requirement to maintain a stable condition of measurement system cabin. In this paper, we introduce one kind of ROV-based Raman spectroscopy measurement system (DOCARS) and discuss the development characteristics of its cabin condition during profile measurement process. An available and straightforward modeling methodology is proposed to realize predictive control for this trend. This methodology is based on the Autoregressive Exogenous (ARX) model and is optimized through a series of sea-going test data. The fitting result demonstrates that during profile measurement processes this model can availably predict the development trends of DORCAS's cabin condition during the profile measurement process.