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Study area and general observationsFour datasets, equating to four post-whaling timeframes, were used for this study: 1997 (32 years post-whaling), 2003/2004 (38/39 years post-whaling), 2008 (43 years post-whaling) and 2014/2015 (49/50 years post-whaling). Data collection for each timeframe occurred during the annual migration of humpback whales, from breeding grounds in the Great Barrier Reef, to feeding grounds in the Antarctic Ocean. The study site was located off the coast of Peregian Beach (north of Brisbane, in Queensland, Australia), which was approximately one-third of the way along their return migration route. Here, humpback whales were still exhibiting breeding behaviours, such as singing, males joining females as escorts, and males forming competitive groups around a central female. Field work took place in September and October of each year. Generally, the number of migrating groups increased per day to peak during late September and early October. Numbers then gradually fell until the end of the migration.For this study, a group was defined as cluster of whales within approximately 100 m of each other that were diving and surfacing together (as estimated by the land-based visual observers). Groups were constantly changing membership with animals joining and splitting from the group and tend to move at different speeds, and in different directions, whilst making general progress southwards. Groups, unless joining together, were separated by at least 2 km, meaning it was relatively easy to keep a separate track of each group (see below).Acoustic recordings were made from three to five hydrophone buoys moored in 18–28 m of water and arranged in a line or T-shaped array (Fig. 6). Each hydrophone buoy consisted of a surface buoy containing a custom-built pre-amplifier (+20 dB gain) and 41B sonobuoy VHF radio transmitter. A High Tech HTI-96-MIN hydrophone with built-in +40 dB pre-amplifier was suspended approximately 1 m above each buoy’s mooring. Signals were received onshore at a base station 1.5 to 2.5 km away using a directional Yagi antenna and type 8101, four-channel sonobuoy receiver. Singing whales were located by cross-correlating the same song sound arriving at the different hydrophones to determine time-of-arrival differences. These differences, together with an accurate knowledge of the positions of the hydrophones, were then used to determine the most likely location of the singer. Singers generally move slowly and calculating an acoustic position approximately every 10 min produced a detailed track of the singer.Fig. 6: Outline of the study site including the range of visual observations and the position of the acoustic tracking array.Illustrating the study site at Peregian Beach, north of Brisbane, east coast of Australia. The map indicates the position of the land-based station (Emu Mountain) and the acoustic base station along with the position of the 5-buoy hydrophone array. The outline designates the study area. Whales moved in a southerly direction through the area daily. Whale icons illustrate acoustically tracked singing whales (circled in blue) and visually tracked presumed males (black), females (orange), and calves (small black). The 5 km social circle radius for a focal singing (blue circle) and a non-singing (black circle) male are also illustrated. The map is taken from “Google Earth” with permission to print without the need to submit a request (Brand Resource Center | Products and Services – Geo Guidelines (about.google)).Full size imageMigrating groups were tracked visually (7am to 5pm, weather permitting) from a land-based elevated survey point, Emu Mountain (73 m elevation). A theodolite (Leica TM 1100) was used in conjunction with a notebook computer running Cyclopes software (E. Kniest, Univ. Newcastle, Australia) to track the groups in real-time and note group behaviours. The field of view was approximately 20 km in a north/south direction and 10 km offshore (Fig. 6). Humpback whale groups were observed ad libitum and tracked by teams of five people. When whale groups surfaced, the observers called the sighted behaviour, compass bearing, and angle from the group to the horizon (in reticules). Each observation included group identification letter, the time, group size and composition, whether a calf was present, direction of travel, and group location, either by using a binocular reticular measurement or a theodolite measurement. Joining and splitting animals were also noted. A join was defined as one of more animals actively moving towards a group to surface within 100 m and then match the group surfacing times. Examples of this include an individual singing or non-singing whale actively moving towards, and then joining, another individual or group of whales. If more animals subsequently moved in and joined the group, this was termed an additional join to that group. These additionally joined group usually comprised of a female-calf and more than one male escort, or three or more adults, with additional joiners highly likely to be male (21,25,26, supplementary results). On rare occasions a singing whale remained in one place but was joined by another individual. This was termed an additional join given there was no evidence the singer actively moved to join this animal. However, the rarity of these occurrences meant the allocation of this behaviour to additional join, rather than join, had no influence on the results.Some of the migrating animals were biopsied during the day for post-field later sexing. Note biopsied animals were sometimes part of different studies occurring at the field site30,50 and were not necessarily the animals used in this study. However, these biopsy results were used to test assumptions made in this study regarding the sex of joining whales and whales within the observed groups (supplementary results and supplementary note). Weather was noted hourly.Statistics and reproducibilityDefining the proximate effect of male density on individual mating tacticsFor this analysis, a specific period, the 2003/2004 dataset, was chosen as it had the most instances of identified singers and non-singers. Within this timeframe, whales were migrating through the study area at sufficiently low density to avoid confusion. After 2004, it became increasingly difficult to focally follow males.First, for singing males (n = 86), their location within the study area was recorded at the start of singing using the acoustic array. Whilst singing they remained in the same location or meandered slowly within a small area. Non-singing animals that were observed to join a group (n = 31) were assumed to be male (21,25,26,30, supplementary methods and supplementary results). For these joining animals, visual observations were backtracked for 10 to 15 min until they were sighted alone. They were only included in the analysis if they could be definitively backtracked using visual (theodolite) observations, with no opportunity for confusion with other whales in area (i.e., no other whales within 2 km).For each unaccompanied focal male, the number of, and roles, of other presumed males within 5 km radius from the focal whale (Fig. 6) was used as a measure of local male density. The 5 km radius was termed social circle and was chosen as the most likely communication space for their acoustic signals51. For singing focal whales, their social circle was estimated using their location when they began to sing. For non-singing focal males, their social circle was estimated using the backtracked theodolite position to when it was first sighted alone. Next, all groups within the 5 km social circle of the focal whale, along with each group composition (singing animal, lone animal, female and calf pair, female-calf and escort number, adult-only group with the number of adults) were recorded at that timepoint. It was not logistically possible to biopsy and sex all migrating animals, therefore, to estimate the number of males within their social circle several assumptions were made. These assumptions were also tested using a biopsy study carried out in the area (supplementary methods and supplementary results). Female-calf pairs were discounted as it was assumed all adults with a calf were female. It was assumed that female-calf pairs were being escorted by males (21,25,26, supplementary methods and supplementary results). Groups of multiple adults were assumed to be comprised of a likely single female, principal male escort and secondary male escorts or challengers (21,25,26, supplementary methods and supplementary results). Lone animals not involved in any group interactions, and not singing, were given a 70% chance of being male (supplementary note). Animals within adult pairs were given a 70% chance of being male given the likelihood of having a mix of female-male pairs and male-male pairs (21,30, supplementary results and supplementary note).All analysis models were carried out in R (version 3.4.0). The first analysis aimed to determine if the likelihood of first observing the focal individual as a singing or non-singing male was significantly related to local male density, as determined by the number of males within a 5 km radius, termed social circle. Singing whales were allocated a 0 and non-singing whales were allocated a 1. A generalised linear model structure was used, assuming a binomial distribution. Likely males within their social circle were divided into non-singing and singing males (to delineate tactics) and these were included as the two covariates.$${{{{{rm{Singing}}}}}},(0),{{{{{rm{or }}}}}},{{{{{rm{Non}}}}}}{mbox{-}}{{{{{rm{singing}}}}}},(1) sim {{{{{rm{Non}}}}}}{mbox{-}}{{{{{rm{singing}}}}}},{{{{{rm{males}}}}}}, 5,{{{{{rm{km}}}}}}+{{{{{rm{Singing}}}}}},{{{{{rm{whales}}}}}}, 5,{{{{{rm{km}}}}}}$$Each focal male was an independent sample given males were migrating southwards and extremely unlikely to back-track into the study area and therefore be resampled. Significance was set at p  More

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Insect rearingWT B. dorsalis were collected from Haikou, Hainan province, China, in 2008. They were maintained at the Key Laboratory of Entomology and Pest Control Engineering in Chongqing at 27 ± 1 °C, 70 ± 5% relative humidity, with a 14-h photoperiod. Adult flies were reared on an artificial diet containing honey, sugar, yeast powder, and vitamin C. Newly hatched larvae were transferred to an artificial diet containing corn and wheat germ flour, yeast powder, agar, sugar, sorbic acid, linoleic acid, and filter paper.Behavioral assaysDouble trap lure assays were set up to compare the olfactory preferences of gravid and virgin females in a 20 × 20 × 20 cm transparent cage with evenly distributed holes (diameter = 1.5 mm) on the side walls. The traps were refitted from inverted 50-mL centrifuge tubes and were placed along the diagonal of the cage. The top of each trap was pierced with a 1-mL pipette tip, which was shortened to ensure flies could access the trap from the pipette. For the olfactory preference assay with mango, one trap was loaded with 60 mg mango flesh and the other trap with 20 μL MO in the cap of a 200-μL PCR tube. For the olfactory preference assay with 1-octen-3-ol (≥98%, sigma, USA), one trap was loaded with 20 μL 10% (v/v) 1-octen-3-ol diluted in MO, and the other with 20 μL MO. A cotton ball soaked in water was placed at the center of the cage to provide water for the flies. Groups of 30 female flies were introduced into the cage for each experiment, and each experiment was repeated to provide eight biological replicates. All experiments commenced at 10 am to ensure circadian consistency. The number of flies in each trap was counted every 2 h for 24 h. We compared the preferences of 3-day-old immature females, 15-day-old virgin females, and 15-day-old mated females. The olfactory preference index was calculated using the following formula41: (number of flies in mango/odorant trap – number of flies in control trap)/total number of flies.Oviposition behavior was monitored in a 10 × 10 × 10 cm transparent cage with evenly distributed holes on the side walls as above. A 9-cm Petri dish filled with 1% agar was served as an oviposition substrate, and the mango flesh, 10% (v/v) 1-octen-3-ol or MO were added at opposite edges of the dish. We tested the preference of flies for different substrates: (1) ~60 mg of mango flesh on one edge and 20 μL of MO on the other; (2) 20 μL of 1-octen-3-ol on one edge and 20 μL of MO on the other; (3) ~60 mg mango flesh on one edge and 20 μL of 1-octen-3-ol on the other; and (4) ~60 mg mango flesh plus 20 μL 1-octen-3-ol on one side and ~60 mg of mango flesh plus 20 μL MO on the other. The agar disc was covered in a pierced plastic wrap to mimic fruit skin, encouraging flies extend their ovipositor into the plastic wrap to lay eggs. The agar disc was placed at the center of the cage, and we introduced eight 15-day-old gravid females. Two Sony FDR-AX40 cameras recorded the behavior of the flies for 24 h, one fixed above the cage to record the tracks and the other placed in front of the cage to record the oviposition behavior. Based on the results from double traps luring assays, a 3 h duration (6–9 h) of the videos was selected to analyze the tracks and spent time of all flies in observed area (the surface of Petri dish). The videos were analyzed using EthoVision XT v16 (Noldus Information Technology) to determine the total time of all flies spent on each side in seconds and the total distance of movement in centimeters, and the tracks were visualized in the form of heat maps17. The number of eggs laid by the eight flies in each experiment was counted under a CNOPTEC stereomicroscope, and each experimental group comprised 7–16 replicates.Annotation of B. dorsalis OR genesD. melanogaster amino acid sequences downloaded from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) were used as BLASTP queries against the B. dorsalis amino acid database with an identity cut-off of 30%. The candidate OR genes were compared with deep transcriptome data from B. dorsalis antennae42, maxillary palps and proboscis, and other tissues.Cloning of candidate B. dorsalis OR genesHigh-fidelity PrimerSTAR Max DNA polymerase (TaKaRa, Dalian, China) was used to amplify the full open reading frame of BdorOR genes by nested PCR using primers (Supplementary Table 2) designed according to B. dorsalis genome data. Each 25-μL reaction comprised 12.5 μL 2 × PrimerSTAR Max Premix (TaKaRa), 10.5 μL ultrapure water, 1 μL of each primer (10 μM), and 1 μL of the cDNA template. An initial denaturation step at 98 °C for 2 min was followed by 35 cycles of 10 s at 98 °C, 15 s at 55 °C and 90 s at 72 °C, and a final extension step of 10 min at 72 °C. Purified PCR products were transferred to the vector pGEM-T Easy (Promega, Madison, WI) for sequencing (BGI, Beijing, China).Transcriptional profilingTotal RNA was extracted from (i) male and female antennae, maxillary palps, head cuticle (without antenna, maxillary palps, and proboscis), proboscis, legs, wings and ovipositors, and (ii) from the heads of 15-day-old virgin and mated females using TRIzol reagent (Invitrogen, Carlsbad, CA). Genomic DNA was eliminated with RNase-free DNase I (Promega) and first-strand cDNA was synthesized from 1 µg total RNA using the PrimeScript RT reagent kit (TaKaRa). Standard curves were used to evaluate primer efficiency (Supplementary Table 3) with fivefold serial dilutions of cDNA. Quantitative real-time PCR (qRT-PCR) was carried out using a CFX Connect Real-Time System (Bio-Rad, Hercules, CA) in a total reaction volume of 10 µL containing 5 μL SYBR Supermix (Novoprotein, Shanghai, China), 3.9 μL nuclease-free water, 0.5 μL cDNA (~200 ng/μL) and 0.3 μL of the forward and reverse primers (10 μM). We used α-tubulin (GenBank: GU269902) and ribosomal protein S3 (GenBank: XM_011212815) as internal reference genes. Four biological replicates were prepared for each experiment. Relative expression levels were determined using the 2−∆∆Ct method43, and data were analyzed using SPSS v20.0 (IBM).Two-electrode voltage clamp electrophysiological recordingsVerified PCR products representing candidate B. dorsalis OR genes and BdorOrco were transferred to vector pT7Ts for expression in oocytes. The plasmids were linearized for the synthesis of cRNAs using the mMESSAGE mMACHINE T7 Kit (Invitrogen, Lithuania). The purified cRNA was diluted to 2 µg/µL and ~60 ng cRNA was injected into X. laevis oocytes. The oocytes were pre-treated with 1.5 mg/mL collagenase I (GIBCO, Carlsbad, CA) in washing buffer (96 mM NaCl, 5 mM MgCl2, 2 mM KCl, 5 mM HEPES, pH 7.6) for 30–40 min at room temperature before injection. After incubation for 2 days at 18 °C in Ringer’s solution (96 mM NaCl, 5 mM MgCl2, 2 mM KCl, 5 mM HEPES, 0.8 mM CaCl2), the oocytes were exposed to different concentrations of 1-octen-3-ol diluted in Ringer’s solution from a 1 M stock in DMSO. Odorant-induced whole-cell inward currents were recorded from injected oocytes using a two-electrode voltage clamp and an OC-725C amplifier (Warner Instruments, Hamden, CT) at a holding potential of –80 mV. The signal was processed using a low-pass filter at 50 Hz and digitized at 1 kHz. Oocytes injected with nuclease-free water served as a negative control. Data were acquired using a Digidata 1550 A device (Warner Instruments, Hamden, CT) and analyzed using pCLAMP10.5 software (Axon Instruments Inc., Union City, CA).Calcium imaging assayVerified PCR products representing candidate B. dorsalis OR genes and BdorOrco were transferred to vector pcDNA3.1(+) along with an mCherry tag that confers red fluorescence to confirm transfection. High-quality plasmid DNA was prepared using the Qiagen plasmid MIDIprep kit (QIAgen, Düsseldorf, Germany). The B. dorsalis OR and BdorOrco plasmids were co-transfected into HEK 293 cell using TransIT-LT1 transfection reagent (Mirus Bio LLC, Japan) in 96-well plates. The fluorescent dye Fluo-4 AM (Invitrogen) was prepared as a 1 mM stock in DMSO and diluted to 2.5 μM in Hanks’ balanced salt solution (HBSS, Invitrogen, Lithuania) to serve as a calcium indicator. The cell culture medium was removed 24–30 h after transfection and cells were rinsed three times with HBSS before adding Fluo 4-AM and incubating the cells for 1 h in the dark. After three rinses in HBSS, 99 μL of fresh HBSS was added to each well before testing in the dark with 1 μL of diluted 1-octen-3-ol. Fluorescent images were acquired on a laser scanning confocal microscope (Zeiss, Germany). Fluo 4-AM was excited at 488 nm and mCherry at 555 nm. The relative change in fluorescence (ΔF/F0) was used to represent the change in Ca2+, where F0 is the baseline fluorescence and ΔF is the difference between the peak fluorescence induced by 1-octen-3-ol stimulation and the baseline. The healthy and successfully transfected cells (red when excited at 555 nm) were used for analysis. The final concentration of 10−4 M was initially used to screen corresponding ORs, and then to determine the response of screened ORs to stimulation with different concentrations of 1-octen-3-ol. Each concentration of 1-octen-3-ol was tested in triplicate. Concentration–response curves were prepared using GraphPad Prism v8.0 (GraphPad Software).Genome editingThe exon sequences of BdorOR7a-6 and BdorOR13a were predicted using the high-quality B. dorsalis genome assembly. Each gRNA sequence was 20 nucleotides in length plus NGG as the protospacer adjacent motif (PAM). The potential for off-target mutations was evaluated by using CasOT to screen the B. dorsalis genome sequence. Each gRNA was synthesized using the GeneArt Precision gRNA Synthesis Kit (Invitrogen) and purified using the GeneArt gRNA Clean-up Kit (Invitrogen). Embryos were microinjected as previously described20. Purified gRNA and Cas9 protein from the GeneArt Platinum Cas9 Nuclease Kit (Invitrogen) were mixed and diluted to final concentrations of 600 and 500 ng/µL, respectively. Fresh eggs (laid within 20 min) were collected and exposed to 1% sodium hypochlorite for 90 s to soften the chorion. The eggs were fixed on glass slides and injected with the mix of gRNA and Cas9 protein at the posterior pole using an IM-300 device (Narishige, Tokyo, Japan) and needles prepared using a Model P-97 micropipette puller (Sutter Instrument Co, Novato, CA). Eggs were injected with nuclease-free water as a negative control. Injection was completed within 2 h. The injected embryos were cultured in a 27 °C incubator and mortality was recorded during subsequent development.G0 mutants were screened as previously described20. G0 adult survivors were individually backcrossed to WT flies (single pair) to collect G1 offspring. Genomic DNA was extracted from G0 individuals after oviposition using the DNeasy Blood & Tissue Kit (Qiagen). The region surrounding each gRNA target was amplified by PCR using the extracted DNA as a template, the specific primers listed in Supplementary Table 2, and 2 × Taq PCR MasterMix (Biomed, Beijing, China). PCR products were analyzed by capillary electrophoresis using the QIAxcel DNA High Resolution Kit (Qiagen). PCR products differing from the WT alleles were purified and transferred to the vector pGEM-T Easy for sequencing. To confirm the mutation was inherited, genomic DNA was also extracted from one mesothoracic leg of G1 flies using InstaGene Matrix (Bio-Rad, Hercules, CA) and was analyzed as above. To avoid potential off-target mutations, heterozygous G1 mutants were backcrossed to WT flies more than 10 generations before self-crossing to generate homozygous mutant flies.Electroantennogram (EAG) recordingThe antennal responses of 15-day-old B. dorsalis adults to 1-octen-3-ol were determined by EAG recording (Syntech, the Netherlands) as previously reported20. Briefly, antennae were fixed to two electrodes using Spectra 360 electrode gel (Parker, Fairfield, NJ, USA). The signal response was amplified using an IDAC4 device and collected using EAG-2000 software (Syntech). Before each experiment, 1-octen-3-ol and other three volatiles (ethyl tiglate, ethyl acetate, ethyl butyrate) were diluted to 10%, 1% and 0.1% (v/v) with MO to serve as the electrophysiological stimulus, and MO was used as a negative control. A constant air flow (100 mL/min) was produced using a controller (Syntech) to stimulate the antenna. We then placed 10 µL of each dilution or MO onto a piece of filter paper (5 × 1 cm), and the negative control (MO) was applied before and after the diluted odorants to calibrate the response signal. The EAG responses at each concentration were recorded for 15–20 antennae, and each concentration was recorded twice. Each test lasted 1 s, and the interval between tests was 30 s. EAG response data from WT and mutant flies for the diluted odorants were analyzed using Student’s t test with SPSS v20.0.Molecular docking and site-directed mutagenesisThe three dimensional-structures of BdorOR7a-6 and BdorOR13a were modeled using AlphaFold 2.044. The quality and rationality of each protein structure was evaluated online using a PROCHECK Ramachandran plot in SAVES 6.0 (https://saves.mbi.ucla.edu/). AutoDock Vina 1.1.2 was used for docking analysis, and the receptor protein structure and ligand molecular structure were pre-treated using AutoDock 4.2.6. The docking parameters were set according to the protein structure and active sites, and the optimal docking model was selected based on affinity (kcal/mol). Docking models were imported into Pymol and Discovery Studio 2016 Client for analysis and image processing. Based on the molecular docking data, three residues (Asn86 in OR7a-6, Asp320, and Lys323 in OR13a) were replaced with alanine by site-directed mutagenesis45 using the primers listed in Supplementary Table 2. Calcium imaging assays and molecular docking of mutated proteins were then carried out as described above.Statistics reproducibilityAll of the olfactory preference assays, oviposition bioassays, expression profiles analysis, EAG recording assays were analyzed using Student’s t-test (*p  More

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Algal blooms that form because of phytoplankton proliferation have key roles in marine ecology and carbon fixation. When the blooms die, most of the fixed carbon is transferred to higher trophic levels, and a small fraction sinks into the deep sea. Viral infection is one of the causes of bloom termination, but its effect on the fate and flow of carbon in the ocean is unknown. In this study, Vincent et al. perform a mesocosm experiment to analyse the bloom dynamics of the coccolithophore microalga Emiliania huxleyi and the impact of viral infection on surrounding bacterial communities and the carbon cycle. The authors observed that viral infection was not only the main cause of phytoplankton mortality, but it also shaped the composition of free-living bacterial and eukaryotic species in the blooms. On viral infection of E. huxleyi, the authors found a comparable biomass of eukaryotic and bacterial heterotrophic recyclers, as well as increased organic and inorganic carbon release that contributed to carbon sinking into the deep ocean. Altogether, these results highlight the impact of viruses on the microbial communities of blooms and the consequences on carbon cycling. More

The pesticide hexachlorocyclohexane (HCH) is a toxic and persistent contaminant in the environment. Some bacteria and fungi can degrade HCH and its isomers under laboratory conditions. However, in heterogeneous environments, where many different factors are at play, the biodegradation capacity is challenged by the availability of nutrients to support degraders’ growth. As opposed to bacteria, fungi are more adapted to heterogeneous habitats, and in some cases mycelial fungi can facilitate the transport of organic substrates throughout the mycosphere, increasing their availability to promote bacterial contaminant biodegradation. However, how this occurs is not entirely understood. In this study, Khan et al. demonstrate that mycelial nutrients transferred from nutrient-rich to nutrient-deprived habitats promote co-metabolic degradation of HCH by bacteria. The authors incubated a non-HCH-degrading fungus (Fusarium equiseti K3) and a co-metabolically HCH-degrading bacterium (Sphingobium sp. S8) in a structured model ecosystem. Results from 13C isotope labelling and metaproteomics showed that fungal 13C was incorporated into bacterial proteins responsible for HCH degradation, thus illustrating the importance of synergistic fungal–bacterial interactions for contaminant biodegradation in nutrient-poor environments. More