Hidden in the Gills: Museum Jars Hold Untapped Larval Secrets of Endangered Freshwater Mussels

Deep inside the alcohol-filled jars of natural history museums, some of the world’s most endangered animals are hiding a secret that curators have overlooked for more than a century. Freshwater mussels, the filter-feeding giants of rivers and lakes whose populations are collapsing across the globe, frequently enter collections while carrying thousands of microscopic larvae in their gills. These larvae, unnoticed and unrecorded, have now been identified as a vast and largely untapped scientific resource. A new study published in the journal Natural History Collections and Museomics argues that simply opening up the swollen gills of preserved specimens could fill critical gaps in our knowledge of bivalve evolution, taxonomy and conservation.

The research, led by scientists working with the malacological collection of the Senckenberg Research Institute and Natural History Museum in Frankfurt and the Federal Scientific Center of the East Asia Terrestrial Biodiversity in Russia, focuses on the two radically different larval types produced by freshwater mussels of the order Unionida. Glochidium larvae, the better known of the two, are tiny bivalved parasites that clamp onto the gills and fins of fish, while lasidium larvae are soft-bodied, hookless forms characteristic of a separate superfamily. Both measure between 60 and 400 micrometres, small enough to escape casual observation but packed with diagnostic structures such as shell hooks, dentition and surface microsculpture that can reveal where a species belongs on the tree of life.

The scale of the knowledge gap is striking. In the United States, one of the best-studied freshwater faunas on Earth, 301 Unionida species are recognised, yet 115 of them, roughly 38 percent, lack any documented data on their glochidia. Elsewhere the situation is far worse, and for the lasidium-bearing Etherioidea the descriptions are so sparse that the diagnostic value of particular larval features cannot yet even be assessed. Because gravid female mussels hold their larvae in specialised marsupial gill chambers for weeks to months before release, specimens can be collected, fixed and shelved without anyone noticing that they are carrying the very structures that would settle long-standing taxonomic arguments.

What makes the discovery so compelling is that the larvae are already there, sitting in jars that museums have owned for decades. The presence of larvae is generally not recorded in inventories of global specimen collections, a blind spot the authors describe as unfortunate given how little effort is needed to check. Gravid specimens can usually be recognised by their thickened, swollen gills, and in suitably preserved animals the filling may even darken the gill tissue, signalling that the larvae inside have reached full maturity. Once found, a small sample of larvae can be removed, imaged and documented without destroying the specimen, effectively converting a static museum object into a data-rich research asset.

To prove the point, the team examined a series of samples spanning more than a century of collection history, from specimens of Unio crassus gathered in 1914 to material of Sinanodonta schrenkii collected as recently as April 2024. The results demonstrate both the promise and the peril of working with old material. A series of Pseudanodonta complanata glochidia preserved since January 1910 turned out to be generally fine, with no soft tissue remains, and yielded a beautifully presentable glochidium after 115 years in the collection, complete with a ventral hook bearing spines. By contrast, glochidia of Unio tumidus from 1914 had mostly cracked shells with hooks no longer recognisable, and larvae of Lanceolaria gladiola from 1911 remained fragile and broken inside their egg membranes.

Some failures are more instructive than others. Glochidia of Physunio superbus, received from Malaysia in 1923, had turned a deep brown-red and revealed little under the light microscope. The team tried everything to brighten the objects, including storage in distilled water, detergent solutions, ultrasonic cleaning and sodium hypochlorite from commercial bleach, but none achieved the desired success. The SEM images showed only flat, shapeless and unstable formations that could not be interpreted. Yet even this unsatisfactory material paid dividends: the examination revealed the presence of a larval thread, a feature not mentioned in previous descriptions, although an asymmetrical conical appendage that would have assigned the species to the tribe Contradentini went unnoticed. The lesson is that even degraded samples can provide useful information, and that the condition of century-old material simply cannot be predicted.

From this accumulated experience the authors propose a routine processing standard that any museum could adopt. Soft bodies are first separated from adult shells and labelled individually, following good museum practice that also protects the periostracum from cracking and colour change in alcohol. A fine needle is then used to open the marsupial gill at its thickest central point, where maturation is most even, and a small sample is inspected under the microscope. Mature, fully formed larvae, the infective stage, are characterised by a well-defined outline with a clearly visible shell edge, although in hook-bearing groups the shell edge may appear before the ventral hook has developed, meaning the larva is not yet mature. When ripe larvae are present, a small piece of the filled gill is cut away, the larvae are shaken free in a screw-cap micro-reaction vessel, and documentation begins.

The imaging protocol is deliberately simple. Light microscope photographs at around 40 times magnification capture the shape and size of closed and open shells, the ventral margin where hooks and denticles appear, and the larval thread. For scanning electron microscopy, larvae are cleaned in a freshly prepared 5 percent potassium hydroxide solution for one and a half to two hours, with gill pieces requiring longer treatment than free larvae, then rinsed repeatedly in distilled water until no foam or bubbles remain. A subsample of 10 to 30 larvae is dropped onto a prepared SEM stub in a drop of water and allowed to evaporate, with the authors warning that overdrying disrupts the external microsculpture of the shell. A modern tabletop SEM allows the objects to be displayed without prior sputter coating, revealing the shell hook, the details of dentition on the margin, the thickness of the shell and the external microsculpture at around 200 times magnification.

The systematic payoff of this work is considerable. Larval type alone separates the two great superfamilies of Unionida, with glochidium larvae defining the Unionoidea and lasidium larvae the Etherioidea. Within the glochidium-bearing group, hookless spherical or semi-oval larvae characterise the family Margaritiferidae, modified S-shaped or bicuspid hooks mark the Hyriidae, and hooked subtriangular larvae define the subfamily Unioninae. At finer scales, bilaterally asymmetrical larvae with an appendage distinguish some Contradentini, while axe-head shaped larvae characterise some Lampsilini. In alpha-taxonomy, dimensions, outline shape, proportions, shell thickness, surface microsculpture and the size and distribution of denticles allow genera and sometimes even species to be differentiated, with most features requiring nothing more than a medium-magnification binocular.

The gills of preserved mussels turn out to be miniature ecosystems in their own right. During the inspections the team encountered water mites of the genus Unionicola clinging to the gills of Acuticosta chinensis, mussel-associated leeches and other invertebrate commensals that are rarely found during routine examinations. In one remarkable find, the inner gill of a Sinanodonta schrenkii specimen collected in 2024 contained encapsulated eggs of the bitterling fish Rhodeus sericeus, documenting the famous reproductive partnership in which these fish deposit their eggs inside living mussels. Such incidental discoveries add layers of biological information to specimens that were previously catalogued by shell alone. With freshwater mussel populations in dramatic decline worldwide and any additional data carrying real weight for species conservation, the authors conclude that detecting and presenting mature larvae increases the scientific value of every sample, and that the larval secrets sleeping in museum jars deserve to be woken up.

Subject of Research: Freshwater mussel larvae in natural history collections as an underutilised resource for taxonomy and conservation

Article Title: Freshwater mussel ( Bivalvia , Palaeoheterodonta ) larvae in natural history collections: an underutilised resource

Article References: Nagel, K.-O., & Sayenko, E. M. (2025). Freshwater mussel (Bivalvia, Palaeoheterodonta) larvae in natural history collections: an underutilised resource. Natural History Collections and Museomics, 2, 1-14. https://doi.org/10.3897/nhcm.2.157419

Image Credits: AI Generated

DOI: 10.3897/nhcm.2.157419

Keywords: freshwater mussels, natural history collections, glochidia, larvae, museum specimens, taxonomy, conservation, SEM imaging, bivalves, Senckenberg, Freshwater, mussel