Up Close and Personal with the Coffin Fly: Megaselia scalaris (Diptera: Phoridae)

by Edward A. Quinto (CoolBioluminescence) Manila, The Philippines

But see, amid the mimic rout, A crawling shape intrude! A blood-red thing that writhes from out The scenic solitude! It writhes!it writhes!with mortal pangs The mimes become its food, And seraphs sob at vermin fangs In human gore imbued. Outout are the lightsout all! And, over each quivering form, The curtain, a funeral pall, Comes down with the rush of a storm, While the angels, all pallid and wan, Uprising, unveiling, affirm That the play is the tragedy, Man, And its hero, the Conqueror Worm.

The Conqueror Worm by EDGAR ALLAN POE

Through the ages, the worm or maggot has been associated with the inevitability of human death and decay. The worm returns us back to ash from where we came from. Since the worm symbolized the unacceptable end of us all, it has been immortalized in many biblical passages and literary works. Worms of different shapes and sizes found wriggling in carcasses can actually come from different species of Fly belonging to the Order Diptera. This special group of insects are strongly attracted to the stench of decay and contributes in the acceleration of the decomposition of all nonviable organisms. Decomposition is achieved first by the ubiquitous bacteria already present in dead organisms. Among large animals, the biggest concentration of bacteria is found in the large intestine and so this is where decay starts at the moment of death. Bacteria then spread throughout the body of the dead organism and multiply rapidly breaking down proteins to give off the foul smelling gases. Flies and other insects follow next as they are attracted to the dead organism by the rotting smell. There in the dead tissue they lay their eggs that will soon hatch into worms or technically larvae which will then feed voraciously on the putrefying tissues as well as on the teeming bacteria already present. But the kind of fly which is highly widespread as the agent of decay has to be the Coffin Fly. It is a relatively small dark

brown bug, almost identical to the lowly fruit fly, characterized by its rapid incremental jumping movement. Due to its small size, people normally dismiss this fly casually during meals which however would elicit a furious reaction if it were the much bigger and buzzing Blow Fly. Most people probably do not recognize the Coffin Fly as a fly at all. Its strong penetrating ability to locate and hasten the decay of corpses even in seemingly concealed coffin, thus its name, is a big problem for the maintenance and sanitation of cemeteries. In fact, the standard sixfeet below the ground burial became the norm since this is the minimum distance underground that can possibly deter the coffin fly from reaching it to lay eggs and raise maggots. The rapid rate of decay caused by the Coffin Fly is perhaps the only accepted forensic evidence by an animal that can be used to legally estimate the time of death from a criminal act. As a chemist and a microbiologist, the Coffin Fly is the last thing I would want to study because it is not within the scope of my two educational backgrounds. Nevertheless, inadvertent turn of events led me to a thorough study of this unique fly species. As what our beloved Dean, Prof. Carmen Kanapi always say, my venture to study this insect has truly broadened my Biological Culture. I must confess that I have come to know this fly quite well after my study. Soon thereafter, when I accidentally meet this insect at home, in school, in restaurants in Cubao and Trinoma and in grocery stores performing its usual welcoming skips instantly fills me with emotions of admiration and familiarity. I would always thrillingly tell my friends, Look its Megaselia scalaris, the coffin fly. My personal encounter with this fly began during the Dengue Outbreak in Manila in 2007. To help in the fight against Dengue which claims so many innocent lives in the Philippines annually, I tried to develop an innovative lure/trap to kill mosquitoes. Mosquitoes are the carrier of the deadly Dengue Virus. I have known for a long time that I am a beeline target for mosquitoes for the simple reason that I perspire far more than any of my friends and colleagues. Perspiration contains a high concentration of lactic acid, a fermentative product of our cells metabolism of sugars, which evaporates swiftly into the surrounding air from our skin. Mosquitoes rapidly pick up this unique mammalian odour rendering us sitting ducks for these flying deadly blood sucking insects. As a microbiologist, I knew and worked with a special group of bacteria called the Lactic Acid Bacteria (LAB) which feed on the MRS (de Mann, Rogosa and Sharpe) and other related culture media to yield

lactic acid as the main waste product. By the way, many of the LAB are good bacteria that are employed in the commercial fermentative preparation of cheese, yoghurt, sauerkraut and pickles. Still other LAB are edible conferring health and well-being to the consumers popularly known as probiotics. And so like an archetypal scientist, I hypothesize that the LAB fermented MRS medium which contains lactic acid like perspiration should also attract, trap and kill mosquitoes. To test my hypothesis, I inoculated species of Lactic Acid Bacteria (LAB) in the MRS broth. As the LAB grow and thrive in the MRS broth they produce lactic acid at 1.5 to 2.0% (w/v) concentration after 1 to 2 days. This fermented or spent MRS broth culture can now be used as a trap/lure for mosquitoes. With much excitement, I poured the reddish-brown spent MRS broth in the common microbiological glass dishes and installed them in some dark, nook and crannies of our large research complex. Lo and behold after 3 to 4 days I was surprised at the outcome of my experiment. This was what I saw.

I was able to trap only 2 to 3 mosquitoes per dish but another insect got lured at a far greater number. At that time, I presumed that these insects were the common fruit fly: Drosophila malanogaster. They simply piled in by the hundreds in the broth at the end of 5 to 6 days. I was about to discard them all down the drain since they were not what I am after but curiosity got the better part of me and I soon started looking at them under the scanner of a microscope. What I saw startled me and so my up, close, and personal encounter with this insect unfolded more deeply. I would soon spend long hours late into the

night and all alone in the Lab taking hundreds of photos and videos of the various aspects of the life cycle of this insect using my Kodak Digital Camera. Something just hooked me to the biological phenomenon that confronted me. I also know that these insects are the common pests in our microbiology lab. My fellow microbiology teachers and I dont want them in the lab. We fear their presence in the Lab because they can enter into closed agar plates where they will lay their eggs. By the time we examine our plates after the incubation period, we would disappointingly see numerous maggots or worms wriggling inside the agar plates instead of the usual microbial growth. But how come, nutrient agar plates of bacteria like Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia and even Pseudomonas aeruginosa fall prey to the larvae of this fly but the Lactic Acid Bacteria (LAB) agar plates seems to remain maggot infestation free. This observation remains an unanswered question for me. I was simply dumbstruck by what I discovered. What is that long club-like structure protruding from the abdomen of the bigger female fly? I was not aware of the existence of this structure before even from fruit flies. I knew that the bodily structure of all insects is divided into the head, thorax and abdomen. So what is that elongated structure? However, these insects cannot be fruits flies I said to myself, which are smaller and have red eyes not black as shown by these menacing looking flies.

I examined the protruding structure at even higher magnification. OMG, I said to myself with a deep sigh, there were simply so many spikes along the length of this long abdominal structure.

Admitting my lack of experience in biology and entomology, I started asking the help of my biology teaching colleagues. They looked at the structure but none gave a concrete and definitive answer. Some said it could be a mutant or an underdeveloped morphological structure. Desperate, I posted the photo in the internet and requested the help of entomologists for its identity. An angel sent from heaven sent, soon came to my assistance. He is Prof. RHL Disney, Ph.D. of Cambridge Univ., UK. In an email communication he said that the flies that I isolated were not Drosophilidae but Phoridae with the scientific name: Megaselia scalaris commonly known as the Humpbacked Fly, Scuttle Fly or the Coffin Fly. Furthermore, he said that the long structure is the females ovipositor. An ovipositor is a structure used to efficiently lay eggs by a stabbing manner that will embed them into nourishing media like a decaying meat. This embedding process ensures that the eggs will hatch and survive into larvae in a suitable environment. This fly has strong incisive ability to enter and infest agar plate cultures with their squirming maggots even if the plates were wrapped tightly in newspapers or even in tightly coiled plastic bags. It got its common name: coffin fly because it is primarily this fly species that can reached the inside of tightly sealed coffins in cemeteries or those buried deep in the ground. I was so thankful to Dr. Disney for the tremendous help he gave me and he was also so generous to send me his authoritative article entitled Natural History of the Scuttle Fly, Megaselia scalaris

which was published in the prestigious Journal: Annual Review of Entomology. It was a big help because everything about the morphology and life cycle of this insect soon fell into their respective position giving me a complete picture of this fly. And so, I finally knew what I was up with and my months of study of this fly from a microbiological perspective went on farther.

The gross morphology of this fly is quite scary as the entire body: head, thorax, abdomen, wings and legs is completely covered with long and tiny sharp spikes.

Legs

Tip of its spiny legs with sharp hooks and an elongated brush

Head and Thorax

Male Fly

Wing Pattern from a dorsal perspective

Even its pair of wings has numerous tiny spikes

Gross morphology of the male Fly

Characteristics of the Coffin Fly (Diptera:Phoridae) Megaselia scalaris

Also known as the Scuttle Fly/Humpbacked Fly Good penetrating ability to enter seemingly closed containers A female can lay about one thousand eggs in her lifetime Life Cycle from egg to adult can be as short as 7 to 28 days depending on environmental conditions. They feed on almost all decaying matter, plants and animals and at times synthetic substances They cause myasis and can be vector of diseases

Egg-bearing Female

The Eggs of the Coffin Fly

Fertilized Eggs where embryos developed inside

Boat-shaped Eggs with holes for the respiration of the developing embryo inside

Enlarged Boat-shaped Eggs

A first instar larva just coming out from its egg (left side). Eggs are cracked open at the mouth part where the larvae soon emerged similar to a baby chick cracking its egg open starting at its beak. Cracked eggs are transparent and numerously holed because they are empty casings while the opaque egg (right side) still contains the embryo inside. Instar refers to the levels of molting of the larva as it grows bigger similar to the moulting of growing snakes. There are 3 observed levels of moulting for the coffin fly: 1st, 2nd and 3rd instars. The yellowish background of the following photomicrographs indicate that it was taken in the MRS medium where the eggs, larvae and dead adults are found.

Cracked empty eggshells and a newly emerging 1st instar larva

A large 3rd instar larva and a smaller 1st instar larva (left photo). Length of 3rd instar larva is longer than the adult female. Bubble is usually produced by the larva in a liquid medium to keep it afloat.

2nd instar larvae swimming through a viscous spent MRS broth (upper left photo)

The strong 3rd instar larva extended on a glass dish. It moves by means of the repeating cycle of elongation and contraction of its body. It has strong musculature that length of body from the mouth can rise up almost vertically supported by just the remaining of the body to the anal part. Its difficult to follow the rapid movement of the larvae focused under the microscope and photographing it at the same time. It seems to dislike the focused microscopic beam of light shining on it.

A fully developed 3rd instar larva about to pupate. The side arms are well pronounced and the skin covering appears to be turning more transparent.

The larva producing bubbles to help keep it afloat in a liquid medium

Three hungry 3rd instar larvae feeding on the mycelium of Aspergillus niger, a black filamentous fungus (a mold)

A herd of 3rd instar larvae foraging hungrily on the thick and tall stalks of fungal condiophores (small black balls) like cows feeding on thick and tall leaves of grass

Fungi specifically mushrooms are the favourite food of the coffin fly aside from bacteria. This fly is the bane of mushroom growers. However, this study is probably the first time the larvae of this fly has been observed to devour the hyphae of Deuteromycetes inside an agar plate.

Mycelium of Aspergillus niger surrounded by numerous hyphae-feeding larvae of Megaselia scalaris. The mold appears to be unhealthy due to the whitish growth of Bacillus species produced by the larvae which are producing antifungal agents keeping the growth and viability of the mold at bay. Healthy molds have a whitish periphery which is nowhere to be found from this fly infested mold. It must have been already eaten up by the larvae. The larvae seem to have dug a trench around the mycelium of Aspergillus niger.

The internal structure of the larva showing its digestive, respiratory and musculature. The 2 long dark channels from mouth to anus (anterior to posterior) is its respiratory system conducting oxygen throughout the length of the larva.

The glistening and translucent outer covering of the 3rd instar larva which seems to create numerous pustules and spikes on its outer covering

As the larva matures, the side arms appear and the outer glistening appearance seems to turn more transparent which will most likely turn into the dark brown coating of the pupal stage.

What looks like an ejectum emanating from the larvas posterior end

The 3rd instar larva has entered into early pupal stage where the side arms and some spikes are still visible due to its translucent condition. As the pupa ages, it hardens and becomes opaque and it produces 2 horns to conduct breathing. The larvae climb up to the drier portion where they pupate as shown by the larvae inside the flask below.

The Pupal Stage of their Life Cycle at higher magnification

After feeding heavily on bacteria in the spent nutrient broth inside the flask, the sturdy larvae climb up to the drier cotton plug area where they turn brown, stop moving and pupate. After several days, an adult fly will emerge from the pupa and so completing their life cycle.

Larvae emerge from a spent nutrient broth teeming with bacteria where adults laid their fertilized eggs. Once the flies make contact with the broth to lay eggs after being attracted by its smell, bacteria specifically Bacillus species which are so plentiful in the flies are inoculated into the broth. The bacteria in turn multiply and metabolize the broth raising the pH making it more alkaline. The attainment of a pH of 10.3 coincides with the emergence of the larvae. I did not

encounter spent nutrient and MRS broths with pH lower than 10 that cause healthy larvae to emerge. The Bacillus species are responsible for turning the pH alkaline due to their more oxidative form of metabolism. The alkaline pH also makes the biogenic amines which are products of the metabolism of proteins by the Bacillus species, less molecularly charged in the broth rendering them more volatile thereby attracting more flies. The gaseous biogenic amines are the attractant of flies.

The spent nutrient broth above contains 4 adult flies all of which can swim/glide on the broth indicating a change in the broths surface tension property at high alkaline pH of 10.3. For hours they glide effortlessly on the broth where at the same time, larvae are emerging. I posted a video on Youtube under CoolBioluminescence showing the gliding dance of the 4 adults with the background radio DWRJ playing the late Donna Summers MacArthurs Park.

The flies are also particularly attracted to the plate cultures of Bacillus species particularly when the Bacillus species were isolated from them. Even when the plates are kept in lockers or incubators the flies can honed in on these plates to

penetrate them. It seems that the plate culture of bacteria emit unique volatile gases the biogenic amines that invite and attract the flies.

Some of the plate cultures of Bacillus species that were isolated from the microbiome of the mature flies

(Uppermost Photo) Short chain-forming rod shaped Bacillus species with discernible capsule (light violet enclosures) while the (Lowermost Photo) is a very long chain-forming square ended rods which is typical of the morphology of the dreaded Bacillus anthracis. The Bacillus species seem to play a very important role in the development of the fly especially if they were raised from nutrient broths. The pH of the broth starts out at 7 and begins to climb up to 10.3 through the production of biogenic amines like cadaverine and putrescine through the metabolic action of the Bacillus species and it is only in highly alkaline medium that the larvae emerged strong and in large numbers to pupate and finally turn into adults.

Long chain and cross-forming cells of Bacillus species

Other bacterial isolates from the microbiome of the coffin fly were gramnegative endospore-forming rods connected in short chains typical of Paenibacillus species

Photo of a culture agar plate taken from top dish showing intricate column and bubble-like formation of the cultivated microorganism inside.

Photo of a culture plate seen from the bottom dish, showing a hitherto unknown plate culture producing complex growth pattern with twisted columns and walls that connects the upper and lower dishes. It is a probable Paenibacillus species.

The fermented/spent MRS medium of a Lactobacillus species forming whitish sediment composed of cells. Upper red-brown medium containing lactic acid and other potent antimicrobial agents: hydrogen peroxide, diacetyl (2,4-Butadione), short chain fatty acids even lactic acid itself is strongly antibacterial. The broth strongly inhbits the growth of bacteria, yeasts and filamentous fungi.

Some of the lured and dead coffin flies at 1 to 2 days of trapping

Even after 14 days of trapping flies, the fermented MRS medium remained free of bacterial and fungal contamination. There was no sign of the development of foul odor and increased turbidity in the spent MRS broth indicative of spoilage. Water was simply added in small volumes every other day to keep the MRS broth from drying up. It remained free of microbial contamination even after 4 weeks at the end of the experiment. The absence of microbial contamination even though hundreds of flies were trapped each of which are carrying many bacteria mainly Bacillus species must be due to the potent inhibitory activity of the fermented MRS broth. Since heavy bacterial growth did not occur in the spent MRS broth which are the immediate food of the hatching larvae, very few viable larvae emerged or at times none at all from the spent MRS broth.

A statistically significant difference between the numbers of flies trapped in the spent MRS broth from LAB and nutrient broth from E.coli. The spent nutrient broth soon developed maggots at alkaline pH.

The spent MRS broth can also undergo microbial spoilage and soon support the development of larvae only if it is diluted significantly with distilled water. Photo above showed exactly just that. A diluted MRS broth causes the weakening of strength of the inhibitory antimicrobial agents present in it leading to the growth of bacteria and to its subsequent spoilage. Once spoilage occurs due to the proliferation of bacteria, the hatched eggs can now feed on the bacteria which will lead to the growth and emergence of larvae. The powder on the surface of the broth is actually a cluster of eggs of the fly and newly hatched first instar larvae. One can also see several 3rd instar larvae moving to the edge and trying to climb up the walls of the container to look for drier area to pupate. The diluted MRS broth with its acidic pH will also become highly alkaline due to the action of Bacillus species by the time the 3rd instar larvae emerged.

A Sciarid fly showing its ventral parts, it is a relative of the coffin fly and was one of the insects trapped in the spent MRS broth.

Trapped Sciarid and Phorid Flies

Dorsal view of a Sciarid

A side view of a Sciarid

A Sciarid

A mosquito also trapped in the MRS broth

An unknown arthropod trapped

Trapped Ant

In Closing, microorganisms play a major and widespread role in controlling the behaviour of animals. Very recently, a study showed how the protozoan Toxoplasma can affect the behaviour of mice so that it can be transmitted effectively from cat to cat where it resides in the brain. This study also showed how the Bacillus species affect the behaviour of flies and other insect species like cockroaches that are also attracted to the smell of decay and feces. The unique odor comes from various volatile biogenic amines produced by bacteria specifically the Bacillus species. Male and female flies seem to dive and trap themselves deliberately into the broth, a seemingly suicidal act for the fly, to actually inoculate the broths with the microbes they carry. But this sacrificial act will turn out to be more advantageous to the fly for from the death of a few far more will grow and develop into adults.

The few flies that died and have sunk to the bottom of the broth

Far more flies will emerge and live to complete the life cycle

Once the microbes start to grow they change the physico-chemical nature of the broths making them alkaline necessary for the emergence of the 3rd instar larvae. The rapidly multiplying bacteria will serve as food for the 1st instar larvae as well as break proteins down into amino acids and the subsequent conversion of amino acids into amines which are highly basic compounds that will turn the broth alkaline. This chemical change can be monitored using the Triple Sugar Iron (TSI) Agar Test, where a yellow color indicates acidity and red indicates alkalinity. The test tubes below are TSI cultures of several Bacillus species isolated from the gut of the coffin fly showing rapid red slants (alkaline) formation of 4 out of 9 in just one day of incubation indicative of rapid protein metabolism and an increasing alkaline pH. After 48 hours all culture tubes showed red color (alkaline) throughout entire medium.

The pH of the broth inoculated with Bacillus species starts out slightly acidic to neutral and soon increases gradually becoming strongly alkaline. By the time a strongly alkaline pH is reached, the larvae feeding on a rich soup of bacteria would have matured into 3rd instar larvae. Perhaps flies deliberately dive into the broth to inoculate it with their bacteria specifically the Bacillus species. It is by doing so that they can make the broth conducive to the hatching of their eggs and their successful development into adults to complete their life cycle. The death of a few flies in the broth should in turn sustain the propagation of a much bigger number of adults through the emergence of hundreds of larvae later on. It seems that the flies work for this group of bacteria, the Bacillus species: they are attracted to it in agar plates, broths and carcasses, they feed on it, they carry it in their guts and legs because they are protected by it, they help out in their metabolism and they need to inoculate it as well on carrion to help them in the process of decomposition and in sustaining their life cycle. In fact, the Bacillus species are also quite prevalent in the large intestines of mammals where they can start the process of decay upon the death of the animal. As they grow in large numbers they emit biogenic amines like cadaverine and putrescine which inform the flies where they are located and soon flies gravitate to them to pick them up and feed on them as well as to lay eggs. Through the process of feeding, the Bacillus species reaches the gut where they stay there becoming a major symbiotic member of the flies microbiome. In one rare instance of my study, in which hundreds of mature larvae emerged from a slightly acidic but weakened MRS broth due to significant dilution with water, the larvae did not pupate and instantly all died out at the same time by a process of self dissolution. Their bodies simply dissolved completely living traces of whitish substance sticking to the glass walls. None reach the area of the cotton plug to pupate. This observation was also a big surprise for me. How come the mature larvae did not pupate? When I measured the pH of the diluted MRS broth it was only 6.4 which is much lower than my previous observed pH requirement of 10.3. It really seems that a high alkaline pH is needed for the larvae to pupate effectively. Our spent MRS agar plates in the Laboratory where LAB have been grown did not support formation of larvae since no other bacteria particularly the Bacillus species can grow in it due to the presence of potent antimicrobial agents like lactic acid. Since the Bacillus species from the flies cannot grow, the pH remains acidic and so the larvae did not emerged at all.

Which is also true of the LAB fermented MRS broths that I use as trap at the beginning of my study on the coffin fly.

A photo of the diluted MRS broth which at first led to the emergence of hundreds of healthy 3rd instar larvae climbing on the walls of the flask only to die out en masse and dissolving into a whitish substance on the glass walls. The measured pH of this broth was only 6.4 way down lower than the pH of 10.3 needed for the emergence of sturdy larvae which can climb up to the cotton plug to turn brown and to pupate.

And so it answered my question why the LAB MRS agar plates in our laboratory did not underwent larvae infestation simply because there were no bacteria other than the LAB which can serve as food for the hatchlings and which can also make the medium alkaline. The Bacillus species are metabolically important for the coffin fly for providing many hydrolytic enzymes that can help in the breakdown of carbohydrates, proteins, lipids and also the chitin cell wall of fungi as well as supply antibacterial and antifungal agents which are all conducive to the survival and growth of the flies especially during the larval stage. Fungi are destructive contaminant for a larval culture of the fruit fly: Drosophila melanogaster but does not seem to pose a big problem for the larvae of the coffin fly: Megaselia scalaris. This must be to the protection provided by the Bacillus species which can elaborate potent antifungal agents as well as the creation of high alkaline pH. Alkaline pH

is inhibitory to fungi which prefer an acidic pH for their growth. I finally wrapped up the results of my study and presented it as a competing paper during the 37th Annual Convention of the Philippine Society for Microbiology in Boracay. I was so happy that my hard work on the study of Megaselia scalaris won the Best Paper Contest!

From a microbiological perspective, thats the end of my study of the amazing Coffin Fly