Category: 7. Science

Kovalev, EIPOD Postdoctoral Fellow at EMBL Hamburg’s Schneider Group and EMBL-EBI’s Bateman Group, is a physicist passionate about solving biological problems. He is particularly hooked by rhodopsins, a group of colorful proteins that enable aquatic microorganisms to harness sunlight for energy.

“In my work, I search for unusual rhodopsins and try to understand what they do,” said Kovalev. “Such molecules could have undiscovered functions that we could benefit from.”

Some rhodopsins have already been modified to serve as light-operated switches for electrical activity in cells. This technique, called optogenetics, is used by neuroscientists to selectively control neuronal activity during experiments. Rhodopsins with other abilities, such as enzymatic activity, could be used to control chemical reactions with light, for example.

Having studied rhodopsins for years, Kovalev thought he knew them inside out – until he discovered a new, obscure group of rhodopsins that were unlike anything he had seen before.

As it often happens in science, it started serendipitously. While browsing online protein databases, Kovalev spotted an unusual feature common to microbial rhodopsins found exclusively in very cold environments, such as glaciers and high mountains. “That’s weird,” he thought. After all, rhodopsins are something you typically find in seas and lakes.

These cold-climate rhodopsins were almost identical to each other, even though they evolved thousands of kilometres apart. This couldn’t be a coincidence. They must be essential for surviving in the cold, concluded Kovalev, and to acknowledge this, he named them ‘cryorhodopsins’.

Rhodopsins out of the blue

Kovalev wanted to know more: what these rhodopsins look like, how they work, and, in particular, what color they are.

Color is the key feature of each rhodopsin. Most are pink-orange – they reflect pink and orange light, and absorb green and blue light, which activates them. Scientists strive to create a palette of different colored rhodopsins, so they could control neuronal activity with more precision. Blue rhodopsins have been especially sought-after because they are activated by red light, which penetrates tissues more deeply and non-invasively.

To Kovalev’s amazement, the cryorhodopsins he examined in the lab revealed an unexpected diversity of colors, and, most importantly, some were blue.

The color of each rhodopsin is determined by its molecular structure, which dictates the wavelengths of light it absorbs and reflects. Any changes in this structure can alter the color.

“I can actually tell what’s going on with cryorhodopsin simply by looking at its color,” laughed Kovalev.

Applying advanced structural biology techniques, he figured out that the secret to the blue color is the same rare structural feature that he originally spotted in the protein databases.

“Now that we understand what makes them blue, we can design synthetic blue rhodopsins tailored to different applications,” said Kovalev.

Next, Kovalev’s collaborators examined cryorhodopsins in cultured brain cells. When cells expressing cryorhodopsins were exposed to UV light, it induced electric currents inside them. Interestingly, if the researchers illuminated the cells right afterwards with green light, the cells became more excitable, whereas if they used UV/red light instead, it reduced the cells’ excitability.

“New optogenetic tools to efficiently switch the cell’s electric activity both ‘on’ and ‘off’ would be incredibly useful in research, biotechnology and medicine,” said Tobias Moser, Group Leader at the University Medical Center Göttingen who participated in the study. “For example, in my group, we develop new optical cochlear implants for patients that can optogenetically restore hearing in patients. Developing the utility of such a multi-purpose rhodopsin for future applications is an important task for the next studies.”

“Our cryorhodopsins aren’t ready to be used as tools yet, but they’re an excellent prototype. They have all the key features that, based on our findings, could be engineered to become more effective for optogenetics,” said Kovalev.

Evolution’s UV light protector

When exposed to sunlight even on a rainy winter day in Hamburg, cryorhodopsins can sense UV light, as shown using advanced spectroscopy by Kovalev’s collaborators from Goethe University Frankfurt led by Josef Wachtveitl. Wachtveitl’s team showed that cryorhodopsins are in fact the slowest among all rhodopsins in their response to light. This made the scientists suspect that those cryorhodopsins might act like photosensors letting the microbes ‘see’ UV light – a property unheard of among other cryorhodopsins.

“Can they really do that?” Kovalev kept asking himself. A typical sensor protein teams up with a messenger molecule that passes information from the cell membrane to the cell’s inside.

Kovalev grew more convinced, when together with his collaborators from Alicante, Spain, and his EIPOD co-supervisor, Alex Bateman from EMBL-EBI, they noticed that the cryorhodopsin gene is always accompanied by a gene encoding a tiny protein of unknown function – likely inherited together, and possibly functionally linked.

Kovalev wondered if this might be the missing messenger. Using the AI tool AlphaFold, the team were able to show that five copies of the small protein would form a ring and interact with the cryorhodopsin. According to their predictions, the small protein sits poised against the cryorhodopsin inside the cell. They believe that when cryorhodopsin detects UV light, the small protein could depart to carry this information into the cell.

“It was fascinating to uncover a new mechanism via which the light-sensitive signal from cryorhodopsins could be passed on to other parts of the cell. It is always a thrill to learn what the functions are for uncharacterised proteins. In fact, we find these proteins also in organisms that do not contain cryorhodopsin, perhaps hinting at a much wider range of jobs for these proteins.”

Why cryorhodopsins evolved their astonishing dual function – and why only in cold environments – remains a mystery.

“We suspect that cryorhodopsins evolved their unique features not because of the cold, but rather to let microbes sense UV light, which can be harmful to them,” said Kovalev. “In cold environments, such as the top of a mountain, bacteria face intense UV radiation. Cryorhodopsins might help them sense it, so they could protect themselves. This hypothesis aligns well with our findings.”

“Discovering extraordinary molecules like these wouldn’t be possible without scientific expeditions to often remote locations, to study the adaptations of the organisms living there,” added Kovalev. “We can learn so much from that!”

Unique approach to unique molecules

To reveal the fascinating biology of cryorhodopsins, Kovalev and his collaborators had to overcome several technical challenges.

One was that cryorhodopsins are nearly identical in structure, and even a slight change in the position of a single atom can result in different properties. Studying molecules at this level of detail requires going beyond standard experimental methods. Kovalev applied a 4D structural biology approach, combining X-ray crystallography at EMBL Hamburg beamline P14 and cryo-electron microscopy (cryo-EM) in the group of Albert Guskov in Groningen, Netherlands, with protein activation by light.

“I actually chose to do my postdoc at EMBL Hamburg, because of the unique beamline setup that made my project possible,” said Kovalev. “The whole P14 beamline team worked together to tailor the setup to my experiments – I’m very grateful for their help.”

Another challenge was that cryorhodopsins are extremely sensitive to light. For this reason, Kovalev’s collaborators had to learn to work with the samples in almost complete darkness.

Ants, with their unique genes, outnumber people by roughly 2.5 million to one. Their combined dry mass, about 12 million tons of carbon, rivals one‑fifth of humanity’s weight on land.

An international research team has compared 163 ant genomes to show how these insects turned cooperative living into an evolutionary engine. They reshuffled DNA while guarding key caste genes for more than 100 million years.

Genetic clues to colony life

Dr. Lukas Schrader of the University of Münster helped coordinate the project and still sounds amazed by its scope.

Charles Darwin once fretted over sterile workers, calling them a “special difficulty” because natural selection seemed unable to favor individuals that never breed.

Inclusive‑fitness theory, formalized in 1964, solved the logic by showing that workers spread their genes by helping sisters.

The new data add genetic proof to that idea: worker‑specific gene clusters stayed almost identical across lineages, hinting that any mutation hurting brood care was swiftly purged.

Ant colonies behave like bodies

Biologists label an ant colony a superorganism because its members behave like cells of one body.

The new dataset spans army ants with millions of workers, species such as Camponotus japonicus whose queens dwarf their tiniest laborers more than 100‑fold, and even parasites that have lost workers altogether.

Researchers sequenced 145 species from 25 countries and folded in 18 earlier genomes to reach chromosome‑level quality for 17 of them. That’s no small feat when many ants are smaller than a comma.

“The publication is a milestone in our understanding of the molecular and genetic foundations of ants and probably also other social insects such as honeybees,” said Schrader.

Across the tree, queen and worker blueprints sit side by side. Yet workers never hatch reproductive organs because development is rerouted by hormones and gene‑regulation circuits embedded in the shared DNA.

Ants keep critical survival genes

The study tracked synteny, the order of genes along a chromosome. Whole blocks had flipped, fused, or fractured at a rate up to four times that seen in vertebrates. Ant groups with the fastest breakage spawned the most species.

Even so, 970 tiny gene clusters, street blocks in the genetic city, remained frozen across 80 percent of species. Many code for metabolism and caste traits, suggesting that breaking them would cripple colony function.

One conserved block houses two vitellogenin genes vital for queen egg yolk and sits unchanged in 148 genomes. Another links fatty‑acid enzymes to worker‑biased expression, underlining how diet and labor intertwine.

Holding those modules steady while the surrounding landscape rearranged let ants explore new lifestyles without losing the caste machinery that keeps colonies alive.

Hormones decide jobs and stability

A single molecule can tip a larva toward royalty or toil. Juvenile hormone has long been that switch, and gene copies for the enzyme JHAMT rise in species with extreme queen‑worker size gaps. 

Insulin and MAPK signaling join the act. In the jumping ant Harpegnathos, blocking MAPK with the drug trametinib makes workers grow larger, echoing lab findings that this pathway expands ovaries when workers become egg‑laying gamergates. 

The new comparison shows MAPK genes under intensified selection in lineages where workers can still replace a queen, but relaxed selection where caste roles are rock‑solid.

That fits the idea that plastic colonies need fine hormonal tuning, while rigid ones lock their switches.

Hormone receptors for juvenile hormone and insulin sit inside conserved synteny islands. This hints that the entire endocrine toolkit rode through deep time as a connected package.

Ant genes shift with colony size

Bigger colonies and steeper queen‑worker dimorphism marched together in evolution; both correlate with trails, trophallaxis, and worker polymorphism.

Genes tied to brain development, such as GCM and the muscarinic receptor mAChR‑A, show worker‑type biased activity and signs of adaptive change in species sporting soldiers beside tiny foragers.

Where workers lost ovaries altogether, selection on oogenesis genes like otu relaxed, but those same genes stay under pressure in species whose workers can still lay male eggs.

Social parasites flip the pattern. Workerless inquilines shed odorant‑receptor genes and rack up chromosomal breaks, mirroring their narrow ecological niche and tiny population sizes.

Ant genes explain social evolution

Many themes, hormonal control, preserved gene neighborhoods, break‑induced innovation, also shape honeybees, wasps, and higher termites. Ants simply had a 150‑million‑year head start, offering a living time machine for social evolution.

Knowing which genes stay linked during caste splits could aid synthetic‑biology efforts that aim to engineer division of labor in microbes or even tissues.

The study also reminds us that nature can be both flexible and conservative: colonies reinvent chromosome layouts yet keep critical circuits intact, a balance worth emulating in adaptive technologies.

Ants may be tiny, but their genomes read like manuals on how cooperation rewires life. Future research will test whether the same genetic choreography repeats whenever individual interests yield to collective success.

The study is published in the journal Cell.

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Red Sprite

NASA astronaut Nichole Ayers, currently stationed on board the International Space Station, shared an incredible image of a sprite — a rare weather phenomenon that’s triggered high above the clouds by “intense electrical activity in the thunderstorms below.”

The image shows the rare electrical discharge in the shape of a starkly red, upended umbrella, hovering high over a brewing storm, like some sort of occult tower from “Lord of the Rings.”

“Just. Wow,” Ayers tweeted. “As we went over Mexico and the US this morning, I caught this sprite.”

Being hundreds of miles above the surface of the Earth gave Ayers the perfect vantage point to watch the stunning event unfold.

“We have a great view above the clouds, so scientists can use these types of pictures to better understand the formation, characteristics, and relationship of [Transient Luminous Events] to thunderstorms.”

Seven Up

According to NASA, sprites can appear at altitudes of around 50 miles, which is far higher than where thunderstorms form. They often appear mere moments after lightning strikes, forming spiny tendrils of red plumes.

The otherworldly phenomenon, which was first officially observed in 1989 photographs, is still poorly understood. Scientists have yet to uncover how and why they form.

In 2022, NASA launched a “citizen science project,” dubbed “Spritacular,” to crowdsource images of the TLEs. According to the project’s official website, over 800 volunteers have joined the effort, and 360 observations have been collected across 21 countries.

Other TLEs include elves, an acronym for “emission of light and very low frequency perturbations due to electromagnetic pulse sources,” and jets, a type of cloud-to-air discharge that can appear as blue tendrils.

“While sprites [and other TLEs] may appear delicate and silent in the upper atmosphere, they are often linked to powerful, sometimes devastating weather systems,” University of Science and Technology of China PhD student and TLE expert Hailiang Huang told National Geographic last week.

“Understanding them not only satisfies our curiosity about the upper atmosphere, but also helps us learn more about the storms we face here on Earth,” he added.

Best of all, studying TLEs could even help us learn about distant planets: NASA’s Juno mission found evidence of sprites and elves in the atmosphere of Jupiter as well.

More on sprites: NASA Crowdsourcing Investigation of Otherworldly “Sprites” in Sky

Would you believe that the probiotics found in yogurt could help make batteries safer? A team of scientists at Binghamton University proved that it’s possible, Interesting Engineering reported.

The researchers managed to build a battery using a paper material that dissolves in water and

probiotic bacteria. That’s right: The same organisms that boost your gut health after drinking a smoothie were engineered to produce electricity using a special electrode.

The result was a power source that can basically self-destruct after a set amount of time without harming its surroundings, Interesting Engineering explained.

A big problem with batteries is that many contain toxic chemicals. After they are used, this pollution often enters soil and water through landfills and can end up posing dangers to human health.

Yet with the researchers’ solution, clean power can flow where it needs to flow, and afterward, no one gets hurt. Their model can currently run between four and 100 minutes before it cleanly and safely destroys itself. All that remains are helpful microbes, Interesting Engineering reported.

Watch now: Does clean energy really cause blackouts?

This kind of battery is part of a field called transient electronics, which is all about fuel cells that are not made to last. Instead, they are designed to disintegrate, much like a device from a “Mission: Impossible” movie, Interesting Engineering noted.

That might sound impractical, but there are actually all kinds of useful homes for these batteries that save time and money. For example, they make medical implant procedures simpler and safer, improve sustainable environmental sensors, and make disposable electronics cleaner and secure, per Interesting Engineering.

“Whenever I made presentations at conferences, people would ask: So, you are using bacteria? Can we safely use that?” explained Maedeh Mohammadifar, who developed the original dissolvable battery during her time as a graduate student. She affirmed that the selected probiotics were common and safe to use, according to the outlet.

The full research findings are published in the journal Small.

Join our free newsletter for weekly updates on the latest innovations improving our lives and shaping our future, and don’t miss this cool list of easy ways to help yourself while helping the planet.

The peak of summer is approaching for those of us in the Northern Hemisphere, but as we prepare for more sunshine and sweltering temperatures, our planet is spinning at its farthest point from the sun.

On Thursday at 3:55 p.m. ET, our planet reached what’s called the aphelion — the most distant point in its orbit around the sun, roughly 3 million miles farther away than when it’s closest.

This happens every year in early July, which might sound backward. If we’re farthest from the sun, shouldn’t it be cooler?

People tend to associate proximity with warmth, so it seems natural to assume the seasons are caused by changes in how far Earth is from the sun. But the planet’s distance has little to do with it.

The real reason for seasonal temperature changes lies in the fact that Earth is tilted.

Our planet spins at an angle — about 23.5 degrees — which means different parts of the globe receive more (or less) sunlight depending on the time of year. In July, the Northern Hemisphere is tilted toward the sun, bringing longer days and higher sun angles that lead to more direct sunlight — all of which produce summer-like heat.

In contrast, the shape of Earth’s orbit plays only a minor role. Although it’s slightly oval-shaped rather than perfectly circular, the difference between our closest and farthest points from the sun is relatively small.

Right now, Earth is about 3.1 million miles farther from the sun than it is in early January when it reaches perihelion, its closest point. Compared to its average distance of 93 million miles, that’s only about a 3.3% difference.

Because sunlight spreads out as it travels, even a relatively small change in distance results in about a 7% drop in the amount of solar energy reaching the planet. That’s tiny compared to the effect of Earth’s tilt.

Just how big is the difference? Let’s look at a few examples.

In cities like Houston, New Orleans and Phoenix — near 30 degrees north in latitude — the amount of solar energy reaching Earth’s atmosphere in summer is more than double what those cities receive in winter.

Farther north, around 40 degrees, the seasonal swing is even more dramatic. Cities like New York, Denver and Columbus see solar energy climb from about 145 watts per square meter in winter to 430 in summer — nearly a 300% difference.

So, while it’s true that Earth is receiving less energy from the sun right now, that detail barely registers compared to the power of the planet’s tilt. A slight angle in Earth’s spin does far more to shape our seasonal patterns than a few million miles of extra distance ever could.

In the end, it’s not how close we are to the sun that makes summer feel like summer — it’s how we’re angled toward it.

In 2006, Pluto was famously demoted from a planet to a dwarf planet. It remains the most famous dwarf planet today, but there are others in our solar system, including potentially hundreds that haven’t been discovered yet.

But what, exactly, is a dwarf planet? And how many dwarf planets are there?

Introduction

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) challenges clinical management and global public health. The main issue is the extremely limited antibiotic treatment options, which makes CRKP infections difficult to treat and threatens patient outcomes and the healthcare system.¹ Carbapenem resistance in K. pneumoniae mainly results from carbapenemases (β-lactamase enzymes), particularly Klebsiella pneumoniae carbapenemase (KPC; class A serine enzymes) and New Delhi metallo-β-lactamase (NDM; metallo-β-lactamases requiring zinc).² What is even more alarming is that K. pneumoniae strains co-existing with blaKPC and blaNDM can obtain or spread extra antimicrobial resistance genes, such as extended-spectrum β-lactamase (ESBL) genes, fluoroquinolone resistance genes, tetracycline resistance genes and aminoglycoside resistance genes. This results in a high level of resistance to most of the routinely employed antibiotics, creating serious obstacles for therapeutic treatment.³

Healthcare-associated infections remain a significant challenge, particularly due to the increasing prevalence of multidrug-resistant (MDR) organisms. KPC-producing and NDM-producing K. pneumoniae (KPC-Kp and NDM-Kp) are undoubtedly concerning pathogens, characterized by limited treatment options, high mortality rates, and the capacity to trigger outbreaks in healthcare settings.⁴ A previous investigation of an NDM outbreak indicated that the losses caused by ward closures, temporary admission restrictions, or delayed discharges due to such outbreaks were enormous.⁵

In this study, we identified a multidrug-resistant K. pneumoniae strain (KP3T58) isolated from a clinical patient, exhibiting resistance to nearly all antibiotics except polymyxin. Whole-genome sequencing (WGS) revealed the coexistence of bla_KPC-2 and bla_NDM-5 alongside three critical plasmids. Through conjugation assays, we confirmed the transferability of these high-risk genetic determinants and further investigated the virulence phenotype of KP3T58. This work comprehensively characterizes a clinical CRKP co-producing KPC and NDM, highlighting the urgent threat posed by such dual-carbapenemase strains.

Materials and Methods

Bacterial Isolates and Case Report

A 70-year-old male was admitted to the intensive care unit (ICU) of The Second Affiliated Hospital of Xiamen Medical College due to brainstem hemorrhage. Upon admission, chest CT findings indicated that the patient concurrently suffered from chronic bronchitis and emphysema. During hospitalization at this tertiary care center, K. pneumoniae was detected in the patient’s sputum. To control the infection, piperacillin – tazobactam was initiated at a dosage of 4.5 grams every 12 hours. However, after three weeks of treatment, sputum culture following bronchoscopy still yielded K. pneumoniae, with the isolate showing intermediate susceptibility to piperacillin-tazobactam (MIC = 16 μg/mL). Therapy was subsequently changed to ceftazidime (1 gram every 8 hours) and linezolid (0.6 gram every 8 hours) for one week. Due to deteriorating pulmonary status, meropenem (2 grams every 8 hours) was administered. During meropenem treatment, bronchoalveolar lavage fluid culture was positive for a CRKP isolate (KP3T58). This isolate exhibited resistance to ceftazidime-avibactam, tigecycline, and carbapenems. The patient died of respiratory failure caused by severe pulmonary infection three weeks after KP3T58 detection. Figure 1 summarizes the microbiological details, timeline, and antibiotic regimens.

Figure 1 The patient’s treatment and infection timeline.

Antimicrobial Susceptibility Testing

Isolates were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS, BioMérieux, France). Antimicrobial susceptibility testing was performed using the Vitek-2 system (Vitek-AST-N334/N335 cards), with ceftazidime-avibactam (CZA) susceptibility determined by broth microdilution. Tigecycline breakpoints followed FDA criteria (susceptible ≤2 mg/L, intermediate 4 mg/L, resistant ≥8 mg/L); polymyxin breakpoints followed EUCAST 2023 standards (https://www.eucast.org); all other breakpoints adhered to CLSI M100 guidelines. Escherichia coli ATCC 25922 served as the quality control strain.

Whole-Genome Sequencing and Bioinformatics Analysis

Genomic DNA from KP3T58 was extracted using the Qiagen DNA extraction Kit (Qiagen, Germany). Genome sequencing was performed on the PacBio HiFi and Illumina NovaSeq 6000 platforms. Long-read data (third-generation sequencing) was assembled using hifiasm (v0.19.5). The assembly was error-corrected using pilon (v1.24) and the clean short-read (second-generation) sequencing data. We used Kleborate (https://github.com/katholt/Kleborate/) for multilocus sequence typing (MLST) and serotype analysis.⁶ ResFinder 4.6.0 was used to identify chromosomal mutations and acquired resistance genes.⁷ PlasmidFinder (v2.1) identified plasmid replication origins, resistance genes, and virulence factors.⁸ OriTfinder analyzed plasmid conjugative and mobilizable capabilities.⁹ Additionally, VRprofile was used for analysis and annotation of insertion sequences (ISs) and transposons (Tns).¹⁰

Multilocus Sequence Typing (MLST)

MLST was performed on the first two K. pneumoniae isolates (KP3P34 and KP3R15) recovered from patient sputum samples. PCR amplification and sequencing of seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) were conducted as previously described.¹¹ Allele numbers and sequence types (STs) were assigned using the Pasteur Institute’s Klebsiella pneumoniae MLST database (http://bigsdb.pasteur.fr/klebsiella/).

Phylogenetic Analysis

Genomic sequences and metadata of 35 K. pneumoniae strains (34 public isolates from China co-harboring blaKPC and blaNDM, plus clinical strain KP3T58) were obtained from NCBI. Core genome SNP (cgSNP) analysis was performed using Parsnp v1.2 with KP3T58 as the reference genome, and the resulting phylogenetic tree was visualized and annotated via iTOL (https://itol.embl.de/).

Comparative Genomic Analyses

Sequence alignment was performed using BLASTn. For plasmid comparison, Proksee (https://proksee.ca/) was employed used to generate circular maps comparing KP3T58 plasmids with other representative plasmids. The genetic environments surrounding antibiotic resistance genes were investigated using Easyfig (version 2.25). Nucleotide sequences were aligned using ClustalW in Jalview 2.11.4.0. Amino acid sequence alignment of Tet(A) was performed using ESPript 3.0.¹²

Conjugation Assay

Conjugation assays assessed transfer of resistance plasmids from K. pneumoniae KP3T58 (donor) to E. coli EC600 (recipient). Donor and recipient strains, grown to logarithmic phase, were mixed (1:1 ratio), centrifuged (8,000g, 1 min), and resuspended in 20 µL of 10 mM MgSO₄. The mixture was spotted onto Luria-Bertani (LB) agar and incubated overnight at 37°C. Serial dilutions were plated on LB agar supplemented with selective antibiotics: tetracycline (10 mg/L; tet(A)), bleomycin (10 mg/L; ble), gentamicin (15 mg/L; rmtB), and for the recipient, rifampicin (600 mg/L).

Transconjugants were identified by MALDI-TOF MS. The presence of tet(A), rmtB, bla_NDM-5, and bla_KPC-2 in transconjugants was confirmed by PCR using primers listed in Table 1. Conjugation frequency (CF) was calculated as: CF = [Number of transconjugants (CFU/mL)] / [Number of donor cells (CFU/mL)].

Table 1 Oligonucleotides for PCR

Plasmid Stability Testing and Growth Assays

Plasmid stability in transconjugants was evaluated as described previously.¹³ Fitness was assessed by growth curve analysis. Transconjugants and the recipient strain were cultured overnight in LB, diluted to an OD600 of 0.01, and incubated at 37°C for 24h. OD600 was measured every 30 minutes.¹⁴

Serum Killing Assay

To evaluate the capacity of strains to withstand serum-mediated killing, a serum resistance assay was conducted following previously published procedures.¹⁵ Briefly, mid-log phase bacterial cells at a concentration of colony-forming units (CFU) per milliliter were combined with normal human serum, sourced from healthy human volunteers, in a 1:3 ratio. The mixture was then incubated at 37°C for 2 hours. Subsequently, after serial dilution, the bacteria were plated onto LB agar and incubated overnight at 37°C to enumerate the viable bacteria. Informed consent was obtained from the donors prior to using their serum.

Quantitative Siderophore Production Assay

To assess the capacity of bacterial supernatants to chelate iron, the researchers employed the chrome azurol S (CAS) assay in accordance with the standardized procedures.¹⁶ Briefly, 1 μL of stationary-phase, iron-chelated cultures was placed onto CAS plates. After incubation at 37°C for 48 hours, the formation of orange halos was used as an indicator to detect siderophore production.

Capsule Quantification

To evaluate the mucoviscosity of K. pneumoniae KP3T58, uronic acid extraction and quantification were carried out following a previously reported protocol.¹⁵ Specifically, an overnight culture in LB medium underwent dilution at a ratio of 1:100 into fresh medium and was incubated at 37°C for 6 h. Then, 500 μL of the culture was combined with 100 μL of 1% Zwittergent 3–12 detergent. The mixture was heated at 50°C for 20 min and subsequently centrifuged at 13,000×g for 5 min. Next, 300 μL of the supernatant was mixed with 1.2 mL of absolute ethanol and centrifuged again at 13,000×g for 5 min. The obtained pellet was dried and resuspended in 200 μL of sterile water. Subsequently, 1.2 mL of tetraborate solution (12.5 mM sodium tetraborate in sulfuric acid) was added. The solution was incubated at 100°C for 5 min, followed by rapid cooling on ice for a minimum of 10 min. Finally, 20 μL of hydroxyphenyl reagent was added. After a 5 – minute incubation at room temperature, the optical density (OD) was measured at 520 nm.

G. Mellonella in vivo Infection Model

To assess the pathogenicity of K. pneumoniae strains KP3T58, Galleria mellonella infection assays were conducted following established protocols.¹⁷ First, the caterpillars were stored at 4°C; those weighing between 150 and 200mg were then carefully selected. Two groups were established: a treatment group and a control group. The treatment group was inoculated with 10 μL of a bacterial suspension at a concentration of 1 × 10⁶ colony-forming units (CFU)/mL, while the control group received 10 μL of normal saline.

Each treatment group consisted of at least 30 caterpillars, which were evenly divided into three Petri dishes. All setups were kept at 37°C. Caterpillar survival rates were documented through daily observations over a three-day period.

Statistical Analysis

Data analyses were conducted using GraphPad Prism 8.0.2 software. The results were presented with a two-tailed non-parametric Student’s t-test. For the survival data obtained from in vivo and in vitro experiments, the Log Rank test (Mantel-Cox) was employed for analysis. P-values < 0.05 were considered significant.

Nucleotide Accession Number

The complete genome sequence of K. pneumoniae KP3T58 has been deposited in the GenBank database of the National Center for Biotechnology Information (NCBI), with the accession number PRJNA1206428.

Result

K. Pneumoniae KP3T58 Was a MDR Strain

K. pneumoniae KP3T58 exhibited high-level resistance to ceftazidime/avibactam and tigecycline, carbapenems (ertapenem, meropenem, and imipenem), β-lactam inhibitors (amoxicillin/clavulanic acid,piperacillin/tazobactam, ticarcillin/clavulanic acid, cefoperazone/sulbactam), β-lactam antimicrobials (cefuroxime, ceftazidime,ceftriaxone, cefepime, aztreonam), aminoglycosides (amikacin, tobramycin), quinolones (ciprofloxacin, levofloxacin), tetracyclines (doxycycline, minocycline), it exhibited susceptibility solely to colistin (Table 2).

Table 2 Antimicrobial Drug Susceptibility Profiles

Genomic Characteristics of K. Pneumoniae KP3T58

WGS analysis using Kleborate typed strain KP3T58 as sequence type ST11 and capsule type KL64. Hybrid assembly with Circos revealed a circular chromosome of 5,534,638 bp (accession no. CP177330) with a GC content of 57.0%. Strain KP3T58 carried 23 resistance determinants associated with its MDR phenotype (Table 3). Chromosomal resistance genes included bla_SHV-11, aadA2, and qacE. Notably, three point mutations were identified in the OmpK37 porin (I70M, I128M, N230G), and seven mutations were detected in the transcriptional repressor AcrR (P161R, G164A, F172S, R173G, L195V, F197I, K201M), which regulates the OqxAB efflux pump. Virulome analysis showed that KP3T58 carried multiple virulence-associated factors, such as iron uptake systems (yersiniabactin and Ent siderophore), type 1 and type 3 fimbriae, capsule, and type 6 secretion systems (T6SS-I). However, the isolate lacked multiple virulence genes involved in siderophore biosynthesis, including iucABCD, iroBCD, and rmpA.

Table 3 Genomic Information of the K. Pneumoniae KP3T58

Plasmid Characteristics of K. Pneumoniae KP3T58

Genomic analysis indicated that plasmid pKP3T58_1 (323,738 bp, CP177331), classified as an IncFIB(K)/IncFII(pKP91)/IncR plasmid with a GC content of 52%. It carried multiple resistance genes, including tet(A),qnrS1,bla_CTX-M-14,bla_TEM-1,bla_LAP-2,aph(3”)-Ib and aph(6)-Id (Table 3). OriTfinder analysis indicated that pKP3T58_1 was conjugative, as it contained a complete conjugative apparatus (oriT site, relaxase, type-4 secretion system (T4SS), and type-4 coupling protein (T4CP)).

The resistance gene bla_NDM-5 was located on plasmid pKP3T58_2 (108,400 bp, CP177332), an IncI1-I type plasmid. This plasmid also contained the sul1 and ble drug-resistance genes. Bioinformatics analysis further confirmed pKP3T58_2 as aconjugative plasmid.

Plasmid pKP3T58_3 (56,083 bp, CP177333), assigned to the IncFII type, exhibited a 52% GC content and contained four resistance genes: bla_{KPC – 2}, rmtB, bla_{TEM – 1B}, and bla_{CTX – M – 65}. In contrast to pKP3T58_1 and pKP3T58_2, pKP3T58_3 lacked autonomous conjugative ability, due to an incomplete conjugation system, specifically the absence of oriT, a relaxase, and T4CP.

Plasmid pKP3T58_4 (11,970 bp, CP177334), with a GC content of 56%, was devoid of resistance or virulence genes and belonged to the ColRNAI type.

MLST Confirms ST11 Clonal Persistence

MLST analysis assigned both carbapenem-susceptible isolates (KP3P34, KP3R15) and the carbapenem-resistant isolate KP3T58 to ST11 (Pasteur scheme), confirming clonal persistence within the patient.

KP3T58 Clusters Within Dominant Epidemic ST11 Clade

Phylogenetic analysis of 34 K. pneumoniae strains co-harboring blaKPC and blaNDM carbapenemase genes from nationwide surveillance and the clinical isolate KP3T58 revealed that KP3T58 clustered within the dominant epidemic ST11-KL64 clade, indicating it is a prevalent ST11-KL64 clone circulating in China (Figure 2).

Figure 2 Phylogeny of 35 K. pneumoniae isolates co-harboring blaKPC and blaNDM based on core genome SNP analysis. The tree includes 34 public isolates from China and the clinical strain KP3T58. Annotations indicate multilocus sequence typing (MLST; ST), capsular serotype (KL), Chinese provinces of origin, and distributions of antimicrobial resistance genes and virulence genes.

Comparative Genomic and Linear Comparison

Comparative analysis showed that plasmid pKP3T58_ 1 had 80–84% coverage and 99.9–100% identity with punnamed1 (CP040176.1) of K. pneumoniae strain 2e from Chongqing, p82_1 (CP101547.1) of KP82 from Yunnan, and pKP309 (CP089881.1) of KP309 from Shanghai, all isolated from within China (Figure 3A).

Figure 3 Comparative analysis of pKP3T58_ 1, pKP3T58_ 2 and pKP3T58_ 3 with other reference plasmids. (A) Genome alignment was performed with pKP3T58_ 1(CP177331), plasmid unnamed1 (CP040176.1), p82_1 (CP101547.1) and pKP309 (CP089881.1). (B) Alignment of the genetic environment surrounding blaTEM-1B with pF16KP0064-1(CP052173.1) and plasmid unnamed2(NZ_CP061963.1). (C) Nucleotide and amino acid sequence alignments between the tet(A) of pKP3T58_1 and the wild – type. (D) Genome alignment was performed with pKP3T58_ 2(CP177332), pZYST1C2 (NZ_CP031615.1), pKP11 – 2 (OW848878.1), and pKP – NDM – 5 (NZ_CP084746.1). (E) Comparison of the genetic environment surrounding blaKPC−2.

Further analysis revealed pKP3T58_1 contained two large antibiotic resistance gene clusters.The bla_TEM-1B gene, together with other resistance genes (sul2,aph(3”)-Ib,aph(6)-Id,bla_LAP-2,qnrS1), formed a 17,803-bp antimicrobial resistance (AMR) region. The genomic context upstream of bla_TEM-1B was homologous to plasmid pF16KP0064-1 (CP052173.1) from Seoul, South Korea. A similar genetic environment for qnrS1 was observed when comparing pKP3T58_1 with the JNK002 plasmid unnamed2 (NZ_CP061963.1) (Figure 3B). The presence of multiple insertion sequences (ISs) and recombinase genes suggested this AMR region likely arose from successive insertion and recombination events. Furthermore, comparative analysis identified a point mutation (Leu294Val) in the tet(A) gene of pKP3T58_1 (Figure 3C).

Comparative analysis of plasmid pKP3T58_2 revealed potential evolutionary pathways for antibiotic resistance gene acquisition.pZYST1C2 (NZ_CP031615.1) from Heilongjiang, China, pKP11-2 (OW848878.1) from Catalan, Spain, and pKP-NDM-5 (NZ_CP084746.1) from Zhejiang, China, respectively, exhibited 80–98% coverage and 99.9–100% identity with pKP3T58 _ 2 (Figure 3D).

Structural analysis of the bla_KPC-2 locus identified ISkpn27 and ISkpn6 flanking the gene upstream and downstream, respectively. Notably, this resistance cassette was entirely embedded within an IS26-bounded region. A complete T4SS was detected downstream of bla_KPC-2, containing essential conjugation genes such as traA, traB, traE, traK, and traM (Figure 3E).

The Non-Conjugative Plasmid pKP3T58_3 Was Mobilized with the Assistance of Conjugative Plasmid pKP3T58_2

Given the three key resistance plasmids and their potential for interbacterial transfer, we assessed the dissemination risk of antimicrobial resistance associated with strain KP3T58. Conjugation assays demonstrated that conjugative plasmids pKP3T58_1 and pKP3T58_2 could transfer individually or together to E. coli EC600. Notably, pKP3T58_2 exhibited an exceptionally high conjugation frequency (1.1 × 10⁻² – 1.8 × 10⁻²). Contrary to expectations, pKP3T58_3 (lacking complete conjugative elements) was mobilized from KP3T58 to E. coli EC600 at a lower frequency (4.2 × 10⁻⁶ – 5.8 × 10⁻⁵) with the help of the conjugative helper plasmid pKP3T58_2. Antimicrobial susceptibility profiles of recipient cells and transconjugants are summarized in Table 1. PCR and agarose gel electrophoresis confirmed the presence of resistance genes in all transconjugants.

Plasmid stability assays showed that all KP3T58-derived plasmids were stably maintained in transconjugants during serial passages. Furthermore, E. coli EC600 harboring these drug-resistant plasmids showed no significant growth defect, ensuring their persistent maintenance in bacterial populations.

In vitro and in vivo Virulence of KP3T58

Subsequently, we aimed to investigate whether KP3T58 possesses hypervirulent traits. For this purpose, K. pneumoniae strain HS11286 (classical strain, ST11)¹⁸ served as the virulence-negative control, while K. pneumoniae NTUH-K2044 (ST23, KL1)¹⁹ was used as the virulence-positive control.

Quantitative siderophore production assays revealed that KP3T58 produced significantly fewer siderophores (13 mm halo diameter) than the positive control NTUH-K2044 (22 mm), comparable to the negative control HS11286 (12 mm) (Figure 4A).

Figure 4 The virulence phenotypes and levels of KP3T58. (A) Siderophores production determined by CAS agar plate. (B) The production of capsule measured based on uronic acid levels. (C) The survival rate (%) evaluated by serum resistance assay. (D) The survival curves of G. mellonella infected by KP3T58, NTUH-K2044 and HS11286. Note:NS (normal saline). Unpaired two-sided Student’s t-tests were performed for uronic acid production and the survival rate in the serum resistance assay. ***P < 0.001; ****P < 0.0001. A log – rank (Mantel–Cox) test was employed for the assessment of the survival curves. A significant difference (P<0.0001) was observed between these groups.

Capsule quantification via uronic acid measurement demonstrated that KP3T58 produced less uronic acid than NTUH-K2044, but slightly more than HS11286 (Figure 4B). Similar trends were observed in serum resistance assays (Figure 4C).

Pathogenicity assessment using the Galleria mellonella infection model showed that KP3T58-infected larvae exhibited a 63% survival rate at 72 hours post-infection, significantly lower than the negative control HS11286 (96%), but substantially higher than the positive control NTUH-K2044 (6%) (Figure 4D).

Discussion

Over the past decade, the global prevalence of MDR K. pneumoniae, particularly carbapenem-resistant variants (CRKP), has escalated significantly, posing a critical public health challenge.^20,21 To elucidate the origin of such high-risk strains, we investigated the clonal and epidemiological context of KP3T58. MLST analysis confirmed that both carbapenem-susceptible isolates (KP3P34, KP3R15) and the carbapenem-resistant isolate KP3T58 from the same patient belong to ST11. This suggests that resistance in KP3T58 likely evolved through acquired resistance mechanisms—such as plasmid acquisition or horizontal gene transfer—rather than new clonal invasion. The ST11 clone demonstrated prolonged persistence within the host, developing resistance under antibiotic pressure. Phylogenetic analysis of 35 K. pneumoniae strains co-harboring KPC and NDM carbapenemases from nationwide surveillance further revealed that KP3T58 clustered within a predominant epidemic subclade characterized by co-production of KPC and NDM carbapenemases in ST11-KL64 clones circulating in China. This clustering pattern, combined with the prevalence of ST11-KL64 among dual-carbapenemase producers in the national dataset, suggests that the ST11-KL64 lineage may serve as a key epidemiological vehicle for the acquisition and dissemination of the KPC-NDM co-production phenotype in China. Its dominance positions KP3T58 within a high-risk transmission network potentially driven by this successful clone.

KP3T58 exhibited near-pan-resistance to clinically used antibiotics, including ceftazidime-avibactam (CZA) and tigecycline. CZA, a β-lactam/β-lactamase inhibitor combination effective against KPC-producing CRKP, is compromised by NDM enzymes due to their zinc-dependent hydrolysis mechanism, which avibactam cannot inhibit.^22–24 Critically, unlike KPC-variant-mediated CZA resistance, NDM production does not restore carbapenem susceptibility. Moreover, the stable CZA resistance phenotype in such strains may facilitate horizontal dissemination of resistance genes within bacterial populations.

Plasmid pKP3T58_1, harboring three replicons (IncFIB, IncFII, and IncR), circumvented incompatibility through its multi-replicon configuration, enabling stable coexistence with other plasmids. This large conjugative plasmid carried multiple resistance genes, including qnrS1, tet(A), and diverse β-lactamases. The qnrS1 gene was embedded within a complex AMR region (ΔIS3–ISKpn19) alongside other determinants (IS5075-aph(3”)-Ib-aph(6)-Id- bla_TEM-1-IS26-bla_LAP-2). Comparative analysis with plasmids pF16KP0064-1 and pJNK002 indicated that this region arose from successive insertion and recombination events. Functionally, qnrS1 acquisition elevated levofloxacin MICs in transconjugants, while chromosomal acrR mutations (mediating fluoroquinolone resistance via RND efflux pump dysregulation) synergistically enhanced resistance.²⁵ Although qnrS1 alone confers low-level resistance, it expands the mutant selection window, promoting high-level resistance emergence.^26,27

Tigecycline resistance in KP3T58 likely stems from efflux pump upregulation. While rpsJ mutations and tet(A) variants are established tigecycline resistance mechanisms,^28–31 comparative analysis excluded rpsJ alterations. However, we identified a Leu294Val substitution in tet(A), which—combined with RND efflux activity—may explain the reduced tigecycline susceptibility in transconjugants. Prior studies corroborate that such mutations diminish tigecycline sensitivity and potentiate MDR phenotypes.³²

Conjugation assays revealed exceptionally high transfer efficiency of pKP3T58_2 (IncI1-I), consistent with its phylogenetic clustering within an epidemic subclade and suggesting conserved dissemination mechanisms. Comparative genomics indicated high sequence similarity with plasmid pKP-NDM-5 from Zhejiang Province, implying regional spread of this plasmid lineage. In East Asia, bla_KPC-2 frequently localizes to IncFII-type plasmids,³³ creating an ecological niche for co-dissemination with pKP3T58_2. Critically, KPC-NDM co-production confers elevated resistance to carbapenems and CZA, demanding urgent surveillance of pKP3T58_2-like plasmids.

The blaKPC gene resided on an IncFII-type plasmid, the primary vector for bla_KPC-2 spread in ST11.³⁴ Structural analysis revealed a unique genetic environment flanked by ISKpn27 and IS26, with a truncated ISKpn6 and an incomplete recombinase gene upstream. Notably, no ΔTn3 homolog was detected downstream; instead, a complete T4SS was present. This architecture—distinct from predominant Tn1721 transposons in Chinese isolates—highlights how IS accumulation enhances plasticity around bla_KPC-2, facilitating carbapenem resistance dissemination.^35,36

Previous studies define mobilizable plasmids as those incapable of autonomous transfer due to defective conjugative machinery, yet capable of horizontal transfer via “hitchhiking” with helper conjugative plasmids. Conventional knowledge dictates that such plasmids retain, at minimum, an oriT site to enable co-transfer. In our conjugation assays, plasmid pKP3T58_3—containing only a partial T4SS—was successfully co-transferred with the conjugative plasmid pKP3T58_2. This unconventional finding expands the mechanistic paradigm of plasmid dissemination. Notably, mobilizable plasmids may possess greater dissemination potential and broader host ranges than conjugative plasmids, potentially attributable to fewer protospacer sequences vulnerable to CRISPR-Cas targeting.^37,38 Consequently, the observed co-transfer of pKP3T58_3 raises significant concerns regarding the accelerated dissemination of antimicrobial resistance genes.

Although KP3T58 lacked hallmark hypervirulence genes (iucABCD, iroBCD, rmpA) and assays confirmed atypical hypervirulence, its yersiniabactin system poses substantial risks. This siderophore enhances respiratory colonization and pneumonia development, potentially enabling acquisition of additional virulence determinants.^39,40 While reports of carbapenem-resistant hypervirulent K. pneumoniae (hv-CRKP) are increasing,¹⁷ most global CRKP infections remain opportunistic healthcare-associated infections (HAIs).⁴¹ High-risk groups (neonates, elderly, immunocompromised) in ICUs—where CRKP mortality reaches 48.9%⁴²—are particularly vulnerable. Critically, the within-host evolution of resistance in ST11 underscores the need for decolonization protocols targeting susceptible carriers pre-resistance emergence. Antibiotic pressure concurrently drives convergence of MDR and hypervirulence, exacerbating therapeutic challenges.⁴³

Conclusion

In summary, this study systematically characterized the resistome of the clinical isolate and conducted in-depth genomic analysis of key resistance gene contexts. MLST analysis confirmed clonal persistence of the ST11 lineage within the host, while phylogenetic positioning revealed KP3T58’s clustering within a dominant epidemic subclade circulating in China. Critically, we experimentally demonstrated that mobilizable plasmids retaining only a T4SS—despite lacking core conjugative machinery—can achieve horizontal transfer when assisted by conjugative plasmids. This discovery fundamentally reshapes our understanding of plasmid dissemination mechanisms. Given these novel insights into plasmid transmission, coupled with the high-risk epidemiological context of the strain, and considering the potential role of the ST11-KL64 clone as a major disseminator of the KPC-NDM co-production phenotype in China, we conclude that the spread of CRKP co-producing KPC and NDM carbapenemases demands enhanced surveillance. Targeted monitoring of this high-prevalence, genetically adaptable lineage may be critical for understanding and interrupting the transmission dynamics of dual-carbapenemase resistance.

Ethics

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Second Affiliated Hospital of Xiamen Medical College (2025010). Written informed consent was obtained from the deceased patient’s next-of-kin for the publication of this case report and associated data.

Author Contributions

All authors made a significant contribution to the work reported, whether in the conception, study design, execution, acquisition of data, analysis, and interpretation, or in all these areas, took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This work was supported by the Joint Special Fund for Applied Basic Research of Kunming Medical University (No. 202201AY070001-182).

Disclosure

The authors report no conflicts of interest in this work.

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In the wild, survival often depends on looks. Some insects evolve disguises that trick their predators into keeping a safe distance. One of the most fascinating examples of this is Batesian mimicry, where harmless species mimic dangerous ones.

Predators that are wary of painful stings, or toxic bites, learn to avoid the warning patterns on the bodies of these potential prey species.

But some other species evolve to copy these signals, despite being completely harmless themselves. This strange dance of deception shapes the appearance of many species worldwide.

Recently, researchers at the University of Nottingham pushed this idea further. They explored how advanced 3D printing technologies can unravel mysteries surrounding mimicry. Their results revealed surprising insights about evolution, predators, and survival strategies.

This study not only shines a light on insect mimicry but also offers a deeper look at the forces that mold nature’s endless forms.

How predators judge insect disguises

The Nottingham team, led by Dr. Tom Reader and Dr. Christopher Taylor, decided to take an experimental leap. They created life-size, 3D-printed models of insects to study how predators respond to different levels of mimicry.

By controlling every aspect of these models – such as shape, color, size, and patterns – they crafted accurate representations of real species. These included wasps that are known for their stings, and hoverflies that are famous mimics of wasps.

This approach allowed the scientists to explore a central question: Why do some mimics look almost identical to their models, while others resemble them only vaguely?

With 3D printing, they could manipulate every trait precisely and test how predators react to slight variations. This marked a significant step beyond previous studies that relied on natural specimens alone.

“In our study, we are asking a question about how evolution works and what determines where evolution reaches at a particular point in time,” Dr. Reader said.

“Our experiments looked at the competing influences which might ultimately shape what organisms look like. Insects and mimicry offer a powerful and accessible way to investigate questions that are relevant across the entire tree of life,” he explained.

Color and shape matter

The team employed cutting-edge imaging tools to scan real wasps and hoverflies. Then, using advanced morphing software, they modified these images to create insects with varying degrees of accuracy in terms of mimicry.

These experiments allowed the researchers to demonstrate the potential for using modern 3D imaging, along with computer morphing, to design insect models that displayed many different combinations of colors and patterns, shapes and sizes.

“The models enabled us to ask ‘what-if’ questions about these insects. What if they were better mimics because their color was more wasp-like?” said Dr. Taylor.

“It allowed us to play around with the insect’s appearance in a way you can’t with real specimens,” he said. “Which meant we could ask a much broader range of questions about what it is that makes a good or bad mimic.”

This experimental flexibility opened new doors for exploring mimicry. The researchers could fine-tune every trait independently and combine them in unexpected ways.

Their key aim was to understand how much precision a mimic needs in order to avoid being eaten. They also wanted to see why poor mimics still manage to survive in nature.

Birds demand better insect disguises

In their experiments, the scientists presented their models to real predators in controlled settings. Their main subjects were wild birds, particularly great tits, which rely heavily on sight to identify prey.

The results showed that birds responded strongly to variations in mimicry. They focused mainly on color and size, ignoring finer pattern details.

The birds were quick learners. They soon began to avoid models that looked more like wasps, even if those models were still rewarding to eat.

Interestingly, the team discovered that intermediate mimics, those blending traits of two different wasp models, gained no extra protection. Birds seemed to prefer clear signals over mixed ones, demanding accurate mimicry.

Sloppy mimics still survive

While birds proved strict judges, invertebrate predators reacted differently. The team tested crab spiders, jumping spiders, and mantises alongside birds to compare predator responses.

Invertebrates showed less concern about precise mimicry. They tolerated models with poor resemblance and attacked more broadly.

This revealed an important insight: invertebrates impose weaker pressure on mimics to evolve perfect disguises. Some insects can survive despite their sloppy mimicry if their main threats come from these predators.

This creates a fascinating evolutionary tension. Birds push for more accurate mimics, but invertebrates allow greater variation to persist.

How evolution shapes insect disguises

A major breakthrough in this study was the creation of an adaptive landscape for mimicry. By using their 3D printed models, the team could systematically map how changes in traits affected predator decisions.

They designed smooth transitions between models, allowing them to visualize shifts in predator responses as traits varied. The experiments showed that the landscape was steep for color and size, traits that birds select strongly for, but flatter for other features like pattern.

This landscape explained why some mimics evolve to look highly accurate, while others remain imperfect. It all depends on the types of predators in their environment.

In bird-heavy areas, precise mimicry becomes essential. In areas dominated by invertebrates, imperfect mimics can survive without major risks.

Simulating future disguises

3D modeling and visualization tools allow researchers to create life-size, full-color models of potential past or future hoverflies and then test them with real predators like birds and spiders to see how they respond to those traits.

“As an evolutionary biologist, you are constantly trying to understand something that happened in the past, and without a time machine you can’t know how a hoverfly ended up like it did, ” Dr. Reader said.

This study not only revealed the subtle pressures shaping insect disguises today but also hinted at how those forces may have operated throughout history.

By bridging technology and biology, the team has crafted a remarkable tool for studying evolution. Their work helps explain how nature fine-tunes survival strategies and keeps evolving in unexpected ways.

Their experiments brought new clarity to questions that have puzzled scientists for decades, showing how insect mimicry remains one of nature’s most astonishing tricks.

The study is published in the journal Nature.

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