"So, naturalists observe, a flea has smaller fleas that on him prey; and these have smaller still to bite ’em; and so proceed ad infinitum."
- Jonathan Swift

June 11, 2019

Pennella instructa

Swordfish are one of the top predators of the ocean. They can swim through the sea at blistering speed, and slash at their prey with their long, flat bill. But no matter how fast you are, there's one thing you can never swim away from - and that's parasites. This is especially the case for big animal like swordfish as their anatomy provides a wide range of different habitat for all kinds of parasites.
They range from sea lice (caligid copepods) that cling to the swordfish's face, to tapeworm larvae which dwell in their muscle, to roundworms that lay eggs under their skin - just to name a few.

Pennella instructa adult with a cyst. From Fig. 4 of the paper

This post will be focused on a study that reported on the occurrence a parasitic copepod - Pennella instructa - on swordfish caught from the north-eastern Atlantic. The researchers in this study visited the fish auction market at Virgo, Spain, during March to September 2011, looking for the presence of P. instructa on swordfish which were brought in by Portuguese and Spanish long line fish boats over that period.

Even though P. instructa is classified as a crustacean, those who are familiar with this blog (and my Twitter feed) would know that when it comes to parasitic copepods, one should abandon any and all preconceptions they might have of what a crustacean is "supposed" to look like. Pennella instructa is shaped vaguely like a toothbrush - a long narrow body that ends with an abdomen covered in a brush-like plume. The adult parasite can grow to about 20 centimetres (or 7 inches) long. It spends its adult life with the lower half of the body protruding from the swordfish, while the front half is anchored deeply in the host's tissue.

Having a parasite that is half-buried in its host's flesh sounds gruesome enough, but P. instructa does something else which elevates it to Cronenberg-level body horror. See, the parasite has not merely stuck its head into the swordfish's flesh and sucking its blood, it is also wrapped in a kind of meat cocoon that the parasite has crafted out of the host's own tissue. Essentially this parasite has sculpted a cosy little bag for itself out of swordfish meat. This parasite-induced cyst is similar to what some other fish parasites, like the fluke that lives on sunfish (Mola mola) gills, can do with their host.

Of the 1631 swordfishes that the researchers looked at, 167 were found to have visible P. instructa infections, though they only occurred in low numbers on each fish, with the most heavily infected fish carrying 4 individual copepods. But being the kind of parasite that it is, even a single P. instructa can have some significant impact on the swordfish's overall health, depending on where it is located. Aside from drinking the host's blood, the meaty cyst that P. instructa forms around itself can put pressure on the surround tissues and organs. The researchers found that while P. instructa can be found all over the swordfish's body, for whatever reasons, most of them prefer the posterior part of the swordfish, mostly in the thick, meaty part of the tail.

It could be that those sturdy tail muscles provide the parasite with a good site to anchor itself in place. Furthermore, that part of the fish's body is made of the powerful muscle which allows the swordfish to propel itself so quickly through the water, thus they'd be constantly supplied with a steady flow of blood which P. instructa can drink from. But this comes at a significant cost to the host, because if the parasite's cyst is located near the vertebrate column - as they would be if they are embedded in the tail - it may affect the fish's nervous system and compromising its swimming ability.

While P. instructa doesn't infect or cause any health issues in humans, a piece of swordfish steak with a big hole through it and a weird worm thing dangling out the side would probably be off-putting to any would-be customers. But perhaps we might want to consider adding P. instructa to the menu?
Pennella balaenopterae - a related copepod which infect whales - is considered to be gastronomic treat by the Inuit people of the Canadian arctic. So instead of seeing them as a pest, perhaps Pennella might be reconsidered as added garnish for your swordfish steak?

Reference:
Llarena-Reino, M., Abollo, E., & Pascual, S. (2019). Morphological and genetic identification of Pennella instructa (Copepoda: Pennellidae) on Atlantic swordfish (Xiphias gladius, L. 1758). Fisheries Research 209, 178-185.

May 8, 2019

Antarctophthirus microchir

Lice are common parasites on birds and mammals. They belong to the order Phthirapteraand this entire order of insects have dedicated themselves to living in the dense forest of feathers and fur on those warm-blooded animals. Aside from a few species of pelagic sea skaters, lice that live on pinnipeds (the group of mammals which includes sea lions, seals, and walruses) and sea birds can be considered as the only group of insects to have successfully made a living out in the open ocean.
Antarctophthirus microchir stages: (a) egg, (b) second-stage larva, (c) adult male, (d) adult female.
Photos from Fig. 4 of the paper

Living on a pinniped poses certain challenges which are unique to that particular environment. Any external parasites of such animals would have to withstand being frequently immersed in saltwater, and not get washed away when these marine mammals propel themselves through the sea. Lice found on birds and land mammals are commonly studied because they are fairly accessible. Studying sea lion lice such as Antarctophthirus microchir and their suite of unique adaptations is another matter.

Just collecting them in the first place is a challenge in itself. How does one collect lice from seals or sea lions? They are large, wild animals, and they spend a lot of their time at sea. Previously, pinniped lice can only be obtained from dead hosts - which is not ideal for a variety of reasons. But a team of researchers have come up with an ingenious but very simple solution - a lice comb, admittedly somewhat a modified one.

In the Chilean city of Valdivia, there is a small "urban" colony of sea lions. Those are a group of sea lions that hang out around the fish markets and piers of the Calle-Calle River and they are used to the presence of people. These sea lions present a valuable opportunity for researchers to study them in more details, including their ectoparasites. To collect lice from those marine mammals, the researchers made a "telescopic lice comb apparatus" - which is basically a lice comb taped to the end of a telescopic metal rod. They selected five individuals on the basis of their skin condition and temperament to try out their new device.

The "telescopic lice comb" being deployed and a close-up of the end of the comb. From Fig.1 and 2 of the paper

They carefully approached the sea lions with their telescopic lice comb and begin combing them for lice. All this took place under the sea lion's terms - when approaching the sea lions, the researchers maintain eye contact and avoid sudden movements, and the sea lions were allowed to inspect the telescopic lice comb before the researchers start applying it to their skin.

Each sea lions were combed for 15-45 mins, starting at their head, then moving further down the body. The researchers never tried to coax the sea lions with food, and they were free to leave if they ever felt uncomfortable about the whole process. And based on how the sea lion reacted to the experience of being combed, they seemed to have thoroughly enjoyed the process, in some cases changing position so that the researchers can scratch their itchier spots.

While the "telescopic lice comb apparatus" seems to have won the sea lions' approval, how well did it work for its original purpose of collecting parasites? Well, the researchers were able to successfully collect live lice from four of the five sea lions they combed, and every life stages of the sea lion louse were present in those samples - eggs, juveniles, and adults - the lot. So they were able to obtain the entire life cycle. And in the process, they were also able to pick up some samples from the sea lions themselves including hair and dandruff.

This opens up all manner of research possibilities into the life and adaptation of these otherwise difficult to access lice. These urban sea lions may have provide science with an opportunity to study an enigmatic parasitic insect, and all that was needed to make the most of it was a modified lice comb.

Reference:
Ebmer, D., Navarrete, M. J., Muñoz, P., Flores, L. M., Gärtner, U., Taubert, A., & Hermosilla, C. (2019). Antarctophthirus microchir infestation in synanthropic South American sea lion (Otaria flavescens) males diagnosed by a novel non-invasive method. Parasitology Research 118: 1353-1361

April 8, 2019

Ixodes holocyclus

There are 14000 known living species of blood-sucking animals, but while drinking blood has become a staple in many different lineages of animals,  some of nature's vampire can be quite picky about which animals they feed on. Even for those that drink from a variety of different animals, they might have preference for certain bouquets of blood over others.
Left: Female (top) and Male (bottom) Ixodes holocyclus, Right: Engorged female after feeding
Photos by Alan R Walker from here and here
Ixodes holocyclus is a species of hard tick native to Australia. It can infect a wide range of different animals including various Australian native marsupials, bird and reptiles. But over the last two hundred years, many other species of mammals have been introduced to the Australian continent, and I. holocyclus has eagerly taken to those new hosts as well. But while I. holocyclus is capable of drinking from both Australian native marsupials and the more recently introduced placental mammals, that does not mean that they are equivalents from the tick's perspective.

A group of researchers in Sydney conducted a study to look at the distribution of I. holocyclus on native and introduced mammals, in particular the long-nosed bandicoots and introduced black rats from areas around the Northern Beaches of Sydney, Australia. They captured these small mammals with cage traps, then briefly inspected them for ticks before letting them go free.

They found that on average, bandicoots had about three to four times as many I. holocyclus as rats, but most of those ticks were found on an unlucky few that were each infected with over 30 ticks. The ticks also distributed themselves different on the bodies of those animals. On the bandicoots, I. holocyclus spread themselves out pretty evenly across the host's body, clinging to the bandicoot's head, legs, belly, flanks, and there were even a few around the genital region. But on the rat they mostly hung around the head and neck region of the animal.

So even though I. holocyclus would happily drink blood from both bandicoots and rats, it seems they would much prefer a bandicoot. Compared with bandicoots which have co-evolved with I. holocyclus for a long time, rats are relatively recent interlopers. So while the ticks can infect them, rats are just not comparable to the native marsupials that they are more used to.

Ticks have specialised mouthparts for clinging to and feeding from their host, and even though I. holocyclus is a generalist that can drink blood from many different animals, its mouth part might not work equally well on them all. So whereas they can comfortably access all areas on the bandicoot, on a rat they stick to the sweet spot around the head to get their fill of blood.

This has important consequences when it comes to quantifying parasite abundance in a given environment. For example, if you are trying to find out about tick abundance in a given region, you might get vastly different results depending on which animals you decide to examine. Parasites are not evenly distributed across the landscape, across hosts, or even across different hosts' bodies. For a tick like I. holocyclus the host's body is an entire landscape in itself, and when in unfamiliar territory, it is better to stick to a well-trodden path.

Reference:
Lydecker, H. W., Etheridge, B., Price, C., Banks, P. B., & Hochuli, D. F. (2019). Landscapes within landscapes: A parasite utilizes different ecological niches on the host landscapes of two host species. Acta Tropica 193: 60-65

March 9, 2019

Mitrastemon yamamotoi

Parasitic plant are among the most enigmatic plants on the planet - they spend most of their life completely out of view until it comes time for them to reproduce. Mitrastemon yamamotoi is one such plant, and it is found in the tropical and subtropical forest of Southeast Asia and Japan. This plant parasitises the roots of the evergreen tree Itajii Chinkapin, and only part of this parasite which is visible to any outside observers are small flowers that poke out from the undergrowth - the rest of the plant is completely embedded within its host's roots.
Top: Male stage (left), Transitional stage (centre), Female stage (right)
Bottom: Some of the insect visitors of M. yamamotoi including (from left to right) hornet, cricket, beetle, cockroach
Top row of photos from Fig 1 of the paper. Bottom row of photos from Fig 2 of the paper.

Mitrastemon yamamotoi is protandrous - which means their flowers go through a male phase before transforming into their female form. This kind of sequential sex change is quite common in the flowers of various plants, but it is also found in many different animals as well.

Aside from plants that spread their pollen haphazardly by wind or water, most flowering plants need pollinators - so what would pollinate this parasitic flower? In New Zealand, short-tailed bats are the pollinator for a parasitic plant called the wood rose (Dactylanthus taylorii). In Central and South America, another parasitic plant - Langsdorffia hypogaea - is pollinated by a range of insects (and possibly birds). So what about M. yamamotoi?

A researchers in Japan embarked on a study to investigate the sex lives of these plants, using both direct observation and via remote camera. The remote camera was rigged to be set off by any movement from animals, however insects are too small to be able trigger the camera, so the researcher did it the old fashion naturalist way. This involved spending many hours each day sitting by the flower clusters, watching for any insect that came by, and using red lamps to continue observations during nighttime.

Throughout the period of study between October 2008 to November 2011, the remote cameras failed to capture any photos of animals visiting the M. yamamotoi flowers - since the cameras can only be set off by comparatively larger animals such as birds and small mammals and it seems that none of them were all that interested in the parasite's flowers.

While the flowers of M. yamamotoi seemed have been snobbed by the feathery and furry beasties, they were rather popular with the creepy crawlies. All manner of insect including wasps, crickets, cockroaches, flies, beetles, and ants visited the flowers. Among those, beetles seem to be particular good pollinators as they would visit multiple flowers in one go, carrying with them pollen from each of the flowers that they had visited. The author of the paper did note that since the study was conducted on the southern part of Yakushima Island, this is near northern end of this parasite's distribution, so in other regions it might be visited by different type of animals.

Parasitic plants are among the most endangered organisms on the planet for most of them we don't know just how endangered they might be. Like other parasites, they are deeply interconnected with the rest of the ecosystem. And while insects like wasps and cockroaches tend to get a bad rap from people, for some organisms, they are a vital lifeline.


Reference:
Suetsugu, K. (2019). Social wasps, crickets and cockroaches contribute to pollination of the holoparasitic plant Mitrastemon yamamotoi (Mitrastemonaceae) in southern Japan. Plant Biology 21: 176-182.

February 14, 2019

Petromyzon marinus (revisited)

Today we're featuring a guest post by Darragh Casey - a student from 4th year class of the Applied Freshwater and Marine Biology' degree programme at the Galway-Mayo Institute of Technology in Ireland. This class is being taught by lecturer Dr. Katie O’Dwyer and this post was written as an assignment about writing a blog post about a parasite, and has been selected to appear as a guest post for the blog. Some of you might remember Dr. O'Dwyer from previous guest post on ladybird STI and salp-riding crustaceans. I'll let Darragh take it from here.

What makes huge sharks jump skywards? Perhaps, the answer to this question is the ancient sea lamprey, Petromyzon marinus.

Image from Figure 1 of this paper
No one is quite sure about what makes the basking sharks of our oceans breach and leap like their predacious cousin, the great white shark. Many theorise this phenomenon is the shark’s action to rid itself of various menacing parasites from their bodies. It could be the case that the annoyingly adapted sea lamprey is proving one rowdy passenger too many, hence, pushing these sharks over the edge, or, in this case, the waterline.

Sea lampreys are one of the most noticeable and common ectoparasites observed on the second largest fish in the sea, the basking sharks. Interestingly, it’s not until the lampreys become adults that they begin to bother larger fish in the ocean, in fact, they don’t even enter the ocean until they’re adults.

Prior to becoming fully metamorphosed they will have spent the last 3 – 5 years of their lives burrowed in the sediment of rivers, filter feeding on organic matter in the water column, and then they transform to become parasitic wanderers. Once they find a suitable host they use their oval shaped sucking mouth and many small teeth to grasp on and feed on the tissues and blood of an unsuspecting donor.

When the victim is the basking shark, the lamprey show their unique abilities to full power. First off, they have to penetrate the hard dermal denticle armour of sharks, which is no mean feat! The next problem they face is the high urea levels in the tissues and the blood of basking sharks. To cope with this potentially toxic level of urea in their host’s blood, the lamprey has a fantastic capability to dispel the urea whilst feeding, using this ability for their survival as described by Wilkie and colleagues. The lamprey also use lamphredin, a chemical in their saliva with anti-clotting properties, to prevent wounds from healing while feeding.

A pair of sea lamprey feeding on a basking shark, from Fig. 1 of this paper
The resulting damage from sea lamprey, especially in great numbers, can be very negative on the basking shark. They deprive the sharks of some of their urea, which is vital for osmoregulation to keep constant pressure in their bodily fluids, and they leave the sharks with open wounds which can become infected, and who knows what could happen then? However, it is more likely, that the sharks, only experience minor lamprey-related health deficiencies.

After a few years, the lampreys will eventually jump ship from their aggravated marine host and return to riverine habitats to find a suitable ally to mate with, spawn, and die soon after. In doing so, they set the foundations for a new generation of lampreys to hassle the basking sharks of the oceans for many years to come.

Are the sea lamprey such a nuisance to these sharks that they decide to momentarily leave the water in an attempt to shake them off? It’s hard to know for certain but one thing is for sure, if blood draining parasitic fish were to latch on to me I would be trying to leave the ocean pretty fast too.

References:
Johnston, EM., Halsey, LG., Payne, NL., Kock, AA., Iosilevskii, G., Whelan, B. and Houghton, JDR. (2018). 'Latent power of basking sharks revealed by exceptional breaching events’. Biology Letters. 14: 20180537

Wilkie, M., Turnbull, S., Bird, J., Wang, Y., Claude, J. and Youson, J. (2004). ‘Lamprey parasitism of sharks and teleosts: high capacity urea excretion in an extant vertebrate relic’. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 138: 485-492.

This post was written by Darragh Casey.

February 4, 2019

Acanthamoeba spp.

Today we're featuring a guest post by Sally O'Meara - a student from 4th year class of the Applied Freshwater and Marine Biology' degree programme at the Galway-Mayo Institute of Technology in Ireland. This class is being taught by lecturer Dr. Katie O’Dwyer and this post was written as an assignment about writing a blog post about a parasite, and has been selected to appear as a guest post for the blog. Some of you might remember Dr. O'Dwyer from previous guest post on ladybird STI and salp-riding crustaceans. I'll let Sally take it from here.

This blog post today is dedicated to all you visually impaired contact lens wearing folk out there! Before I begin, I just want to say that I truly hope all of you adhere to the instructions your optometrist gives you with regards to using contact lenses (washing hands before and after handing them, taking them out while showering/bathing). If not, I’m afraid you are running the risk of meeting my new acquaintance; Acanthamoeba spp., also known as the cornea guzzling free-living protozoa from hell!

Acanthamoeba in its two forms: (A) trophozoite, (B) impenetrable cyst
Image by Jacob Lorenzo-Morales, Naveed A. Khan, and Julia Walochnik, used under CC BY 2.0
Acanthamoeba spp. are microscopic organisms that can be found just about anywhere, from soil to water, to the air we breathe. They are the direct culprits of Acanthamoeba keratitis (AK) a relatively rare but sight-threatening disease which is actually caused by at least eight species of Acanthamoeba: A. castellanii, A. culbertsoni, A. polyphaga, A. hatchetti, A. rhysodes, A. lugdunesis, A. quina, and A. griffin. Ocular trauma and contaminated water are also associated with AK infections but it has been found that contact lens wearing accounts for > 80% of the cases. If found early the infection can be cured, but this gets progressively more difficult the longer it remains untreated. The difficulty lies with the life cycle of the Acanthamoeba species which consists of two stages: the trophozoite and the cyst.

The trophozoite is the vegetative form which feeds on organic matter and ranges in size from 10 to 25µm. When the going gets tough, the tough get going... tough being the trophozoite. When conditions become unfavourable, like under extreme heat or lack of nutrients, the trophozoite transforms itself into a double walled cyst which is almost invincible. The cyst remains unscathed by repeated cycles of freeze-thawing, and incredibly high doses of UV and even GAMMA RADIATION. Cue the Terminator and his infamous catchphrase…. “I’ll be back”.

Characteristics of AK include eye pain, redness, itchiness, and a general feeling of something being stuck in your eye. Sounds like most eye infections, right? One extra feature is the presence of a stromal ring-like infiltrate in the eye. Basically, an ulcer forms on the cornea of the infected eye as a result of the hungry Acanthamoeba. It has been discussed that contact lenses serve as vectors for transmitting Acanthamoeba trophozoites, and to make matters worse studies have shown that wearing lenses results in mild corneal trauma which alters the surface of your eye making it even more susceptible to infection!
Healthy human eye (left) vs infected eye with Acanthamoeba keratitis (right). Arrow indicating stromal ring-like infiltrate.
From Figure 1 of the paper
Scientists have tried to create vaccines to prevent AK by terminating the Acanthamoeba trophozoite or the cyst, but these have proved unsuccessful. However, it was discovered that using a vaccine composed of dead trophozoites stimulates the production of antibodies in the tears, and these block adhesion of the trophozoites to the ocular surface which in turn prevents the development of AK.

Now, before you all go destroying your contact lenses in a panic-stricken state let me inform you that over 30 million Americans wear contact lenses, yet remarkably the incidence of AK in contact lens wearers is less than 33 cases per million. Acanthamoeba species are found in virtually every environmental niche on our planet ranging from thermal springs to solid ice, yet why are AK cases so far and few between? Scientists believe the host’s immune system plays an important role in successful AK infections.

Serological analysis of IgG and tear IgA (both of which are antibodies found in blood) revealed that 50-100% of healthy individuals with no history of AK possessed antibodies against Acanthamoeba antigens. What’s more, the serum IgG and tear IgA levels were significantly lower in patients with AK compared to the cohort of normal individuals with no history of AK, suggesting a prominent role of the mucosal immune system in preventing AK.

In 1939, Winston Churchill referred to Russia as “… a riddle, wrapped in a mystery, inside an enigma” … one might classify Acanthamoeba and the infections it produces in the same way! Although scientists have a clearer understanding of Acanthamoeba keratitis and the parasite which causes it, there is still much to be learned about its cunning and conniving ways.

References:
Neelam S. and Niederkorn J.Y. (2017) Pathobiology and Immunobiology of Acanthamoeba Keratitis: Insights from Animal Models
. The Yale Journal of Biology and Medicine. 90:261-268.

This post was written by Sally O'Meara

January 11, 2019

Polypipapiliotrema stenometra

Corals are host to a wide range of pathogens and one of the most unusual is a type of parasitic fluke which cause the polyps of Porite corals to become pink and puffy. Parasitic flukes (trematodes) have complex life cycles and are known to use a wide variety of different animals as temporary hosts in order to complete their life cycles. The fluke larvae that infect coral polyps complete their life cycle in coral-eating butterfly fishes, and their existence have been known for decades.
Left: taxonomic drawing of an adult Polypipapiliotrema stenometra from Fig. 2 of the paper.
Right: Pink, swollen Porites coral polyps infected with Polypipapiliotrema larvae (photo by Greta Aeby).
For quite a while, they were considered to be just another species within a genus call Podocotyloides, specifically Podocotyloides stenometra. But a recent study by a group of researchers found that not only are these coral-infecting flukes distinctive enough to be placed into its own genus called Polypipalliotrema, but that the flukes which have previously been classified collectively as "Podocotyloides stenometra" is in fact a whole conglomerate of different species, infecting coral polyps far and wide.

In this study, researchers examined 26 species of butterfly fishes collected from the French Polynesian Islands, and O'ahu, Hawai'i, and found 10 species which were infected with Polypipaliliotrema. Upon examining the DNA and the physical features of those flukes, they discovered that what was thought to be a single species turns out to be at least FIVE different species of coral-infected flukes, and there are variations in their geographical distribution.

Butterfly fish species that are found across different locations were sometimes found to have different species of Polypipapiliotrema at each location, so it seems some fluke species were localised to particular island groups. This means there might be more unique species of coral-infected flukes that remain undiscovered and undescribed from other coral reefs around the world.

In order for Polypipalliotrema to complete its life cycle, it needs the host polyp to be eaten by a butterfly fish. While coral polyps are stable food for some fish, they can be small and finicky to handle - you have to be quick and precise in picking the coral polyp lest it retreats back into its skeleton. Also, corals usually occur in vast colonies composing of hundreds and thousands of polyps, so the chances that the infected polyp would be among the ones eaten by a butterfly fish would be quite slim. On top of that, the polyps of Porite is consider to be poor quality food for most coral-eating fishes - their polyps are tiny and quick to retracts into its skeleton - so even fish that feed almost exclusively on coral polyps prefer species other than Porites.

But Polypipalliotrema has a clever way of stacking the odds in its favour, and it does what many parasites do - by manipulating its host. Coral polyps infected with Polypipalliotrema become swollen and bright pink, in complete contrast to the tiny uninfected polyps. Not only does the colouration draws the attention of butterfly fish, the swollen polyp also can't retract into the coral skeleton, making it easier to the butterfly fish pick them up and get more coral flesh for every mouthful.

But why should the butterfly fish eat something that is filled with parasites? Shouldn't they try to avoid parasitised prey, especially when the infected polyps are so easy to distinguish? Since this fluke is commonly found in butterfly fish, it is clear that they make no attempt at avoiding the fluke-laden polpys.

This could be that while Polypipapiliotrema is technically a parasite, it doesn't really harm the fish host that much, and because of what the fluke larvae do to coral polyps, the fish have an easier time getting its meal. As such, the relationship between Polypipapiliotrema and butterfly fishes is closer to a form of mutualism - by altering the coral polyp, the fluke helps butterfly fish get more to eat for less effort, and for its side of the bargain, butterfly fish allows the fluke to complete its life cycle.

Reference:
Martin, S. B., Sasal, P., Cutmore, S. C., Ward, S., Aeby, G. S., & Cribb, T. H. (2018). Intermediate host switches drive diversification among the largest trematode family: evidence from the Polypipapiliotrematinae n. subf.(Opecoelidae), parasites transmitted to butterflyfishes via predation of coral polyps. International Journal for Parasitology 48: 1107-1126.