Ticks are obligate blood-feeding parasites, meaning they cannot complete their life cycle without obtaining blood from a host. Yet blood is a chemically difficult food. Haemoglobin supplies amino acids and energy, but its haem component can be toxic when released in excess because it can catalyse oxidative reactions that damage cells. Many blood-feeding organisms therefore need specialized systems to capture, transport, store and detoxify haem. Ticks face an additional metabolic constraint: they lack the biochemical pathway needed to manufacture haem from scratch. Unlike organisms that synthesize the molecule de novo, ticks must acquire it from their hosts. Haem is especially important for reproduction, when adult females consume enormous blood meals and produce eggs. The unresolved question was whether young ticks also require a substantial influx of haemoglobin-derived haem during their own feeding and development, or whether they inherit enough from their mothers to bridge the vulnerable larval and nymphal stages.

The team, led by researchers at the Institute of Parasitology of the Biology Centre of the Czech Academy of Sciences, investigated that question using I. ricinus, a widespread European tick and an important vector of pathogens. Their experiments combined membrane feeding, biochemical fractionation, proteomics and gene-silencing approaches. To identify haem-containing material in newly emerged larvae, the researchers separated proteins from larval homogenates using size-exclusion and anion-exchange chromatography, then analysed the resulting fractions with liquid chromatography-tandem mass spectrometry, or LC-MS/MS. These methods allow researchers to determine which proteins are present and to compare their abundance across samples. The analysis showed that host-derived haem was already present in unfed larvae. Rather than floating freely, it was associated with vitellin, the nutrient-storage form of vitellogenin that mothers deposit into developing eggs. In effect, the mother’s blood meal supplied not only the raw material for eggs but also a biochemical inheritance that could support offspring after hatching.

The discovery was tested experimentally by feeding tick larvae and the nymphs that developed from them through an ex vivo membrane-feeding system. Some ticks received blood, while others were offered serum from which haemoglobin had been removed. Serum still contains proteins, salts and other soluble components of blood, but the removal of haemoglobin sharply reduces the supply of dietary haem that would normally be liberated during digestion. Despite that reduction, the immature ticks engorged and moulted. Larvae fed on the haemoglobin-depleted material were able to progress to the nymphal stage, and the resulting nymphs continued developing. Additional comparisons examined adult ticks derived from blood-fed and serum-fed nymphs, helping the researchers assess whether a restricted haemoglobin supply merely delayed development or caused hidden reproductive defects later in life. The results indicated that immature development can tolerate a limited dietary contribution from haemoglobin, consistent with the idea that maternally deposited reserves provide a functional buffer.

That tolerance does not mean haemoglobin is irrelevant. The experiments instead expose a distinction between the quantity of haem available and the digestive process required to obtain nutrients from a blood meal. When dietary proteins were internalized by tick midgut cells but could not be proteolytically processed, ticks failed to feed efficiently. In the tick midgut, specialized digestive cells take up blood proteins and route them into lysosome-like compartments. Within these compartments, proteases progressively cleave large proteins into smaller peptides and amino acids. Cysteine cathepsins are among the principal enzymes involved. Cathepsin B and cathepsin L enzymes are capable of digesting haemoglobin and other host proteins under the acidic conditions of the lysosomal digestive system. If this proteolytic throughput is interrupted, the tick may be unable to convert its blood meal into usable nutrients, regardless of whether the meal contains enough haem.

A broad chemical inhibitor provided the first direct test of this dependency. The researchers used E-64, a compound that inhibits lysosomal cysteine cathepsins, and found that treated ticks could not feed successfully. This result was important because it showed that the phenotype was not simply a consequence of removing one protein or one developmental signal. Instead, it implicated a wider digestive network. The authors then used RNA interference, or RNAi, to reduce the expression of individual cathepsin genes. RNAi works by introducing double-stranded RNA corresponding to a target gene, prompting the cell to destroy matching messenger RNA and thereby lower production of the encoded protein. By separately knocking down cathepsin L1, cathepsin B1 and a long form of cathepsin L, the team could resolve their contributions to feeding, moulting and reproduction. The results showed that individual enzymes were not interchangeable in every context: some affected feeding or moulting in immature ticks, while others also influenced adult reproduction.

The study’s proteomic data offered a broader view of the molecular response to feeding. The researchers compared the midgut proteins of nymphs collected after feeding on bovine blood or serum and examined proteins that bound to haem-affinity beads. Such affinity capture can reveal proteins associated with haem or haem-binding processes, while label-free quantitative proteomics estimates relative protein abundance without attaching chemical tags to every sample. The supplementary datasets included three biological replicates for several comparisons and four replicates for the haem-affinity subproteome. The authors also examined genes responsive to haemoglobin in nymphal and adult-female midguts, as well as the activity of other digestive enzymes such as asparaginyl endopeptidase. Together, these analyses support a model in which the tick’s digestive system is highly organized: haemoglobin is internalized, proteolytically dismantled, and its potentially hazardous haem component is managed through binding and storage pathways. Maternal haem helps young ticks withstand reduced input, but it does not replace the need for an operational digestive system.

The findings could influence the search for ways to control ticks, which transmit bacteria, viruses and parasites affecting people and animals. Traditional tick-control approaches often rely on chemical acaricides, but resistance, environmental persistence and impacts on non-target organisms have increased interest in biological and molecular alternatives. Cysteine cathepsins are attractive candidates because they sit at a critical point in the parasite’s nutritional economy. Inhibiting them could starve ticks at the cellular level by preventing the conversion of host proteins into absorbable nutrients. The RNAi results suggest that targeting a combination of cathepsins might be more effective than disabling a single enzyme, particularly if different cathepsins dominate at different life stages. However, the study does not establish that an inhibitor tested in the laboratory would work as a field treatment, nor does it demonstrate safety for hosts or the environment. Any practical intervention would need to reach the tick, remain active in its gut, avoid harming vertebrate digestive enzymes and account for variation among tick species and populations.

The maternal contribution revealed by the study also changes how researchers may think about tick development. A tick egg is not merely a dormant package waiting for the larva to obtain resources from its first host. It contains a biochemical inheritance assembled from the mother’s blood meal, including haem bound to vitellin and presumably other nutrient reserves. That inheritance may be especially valuable because larvae are the smallest and most vulnerable feeding stage, with limited opportunities to find a host and limited capacity to compensate for nutritional shortfalls. The work was performed with laboratory-reared I. ricinus under controlled conditions, so the balance between maternal reserves and dietary haemoglobin may differ in nature, where temperature, host species, pathogen infection and time between life stages can alter metabolism. Even so, the central result is clear: haemoglobin-derived haem is not an all-or-nothing requirement for immature tick development, whereas lysosomal protein digestion is indispensable for efficient feeding. By separating those two processes, the research exposes a hidden dependency in one of nature’s most successful blood-feeding parasites—and offers a molecular weak point that future tick-control technologies may be able to exploit.