Microsporidia

by Bill Oehlke

On a couple of occasions this year and last, I have been asked if it is possible that diseases can be transmitted from adult Saturniidae moths to the eggs they produce.

My original answer was, "I don't know, but I do remember reading somewhere that Louis Pasteur is credited with saving the silkworm industry by discovering/identifying the causative agent of pebrine, a microbial disease of silkworms." Pasteur examined adult moths under a microscope and advocated destruction of all eggs deposited by infected females.

I recently learned "In the culture of Antheraea mylitta Drury, a semi-domesticated Tasar Silk Moth, eggs of mother moths infected with Nosema sp., (microsporidian) must be discarded to avert any catastrophe on crops caused by this pathogen. The infected mother moths (pebrine diseased) are detected by a method derived from that used in sericulture (Pasteur, 1870). In this method, the abdomen of an adult is severed with scissors, placed in a small mortar, mixed with water and crushed with pestle. A drop of the smear is placed on a clean slide and examined under a microscope for Nosema sp., spores."

It is now clear to me that Microsporidia can be transmitted from an infected Saturniidae female to her offspring in what is called "vertical transmission".

On November 9, 2002, I received an article from Karen Perizzolo. The article (2001 Microbes and Infection 3, 381-388) was written by

Dr. Alison Dunn and

Dr. Judith E Smith
Senior Lecturer
School of Biology
Leeds University
Leeds LS2 9JT
UK

Short excerpts from Dr. Smith's article appear in red text below. The report was highly technical and dealt with many animal phyla. I have tried to explain implications for rearing Saturniidae. I have written in black text my comments/understandings/explanations.

What are microsporidia?

The following is offered in CancerWEB's On-line Medical Dictionary:
"Common name for members of the protozoan phylum Microspora. It includes some 80 genera parasitizing all classes of vertebrates and many invertebrates, especially the insects. Several genera, such as Encephalitozoon, Enterocytozoon, Nosema, Pleistophora, and Septata have been implicated in the infection of immunocompromised humans."

Here are the excerpts from Dr. Smith's article:

The microsporidia are obligate (able to exist or survive only in a particular environment or by assuming a particular role) intracellular (occurring or situated within a cell or cells) parasites which have diverse life cycles involving both horizontal (insect to insect, usually by ingestion of spores) and vertical (host parent to host offspring) transmission, and parasitise a wide range of vertebrate (including humans) and invertebrate (including lepidoptera) hosts.

Within the microsporidian lifecycle there are two basic morphological forms, the meront or proliferative stage and the spore or transmission stage (Canning and Lom 1986).

The meront develops from a spore, usually ingested by a caterpillar, possibly from the foliage it eats and/or possibly from ingestion of the egg shell when it first emerges, and takes nutrients internally from caterpillar cells. The meronts develop and reproduce at varying rates. Since these organisms are deriving nutrients and energy from their hosts, infected caterpillars usually do not grow as rapidly as non-infected caterpillars.

In some cases the meronts reproduce at an amazing rate and seem to exist in sufficient numbers to result in the sudden death of larvae, usually in the fourth and fifth instars.

Like most spores, the microsporidan spore has a thick, resistant wall which enables it to survive for extensive periods of time, possibly years, outside the body of any potential host.

Creepy, but, it seems the ingested spores have the ablitiy to invade host cells via an extrusion apparatus which literally bores its way through cells membranes, creating a conduit for the movement of sporoplasm from the inside of the spore directly into the living matter of the host cell.

Microsporidian outbreaks may be responsible for the seasonal population crashes associated with many lepidopteran species. The resistant spore, the use of alternate host species in their life cycles and the use of vertical transmission to overwinter in host eggs (eg Canning et al 1985), provide mechanisms for survival through such "crashes".

It is also possible that some microsporidia may have indirect life cycles where different stages have to utilize different hosts.

Horizontal parasite transmission can occur between related or unrelated hosts of the same or different generations and between hosts from the same or different species.

Parasites may be transmitted per os, venereally and by direct invasion through the host epithelium. I believe this means by ingesting (os=opening like a mouth), sexually and during ovipositing, and possibly by wind-blown spores landing on and enterring the skin.

It is likely that the most common type of transmission is by ingestion of spores on foliage. Inside the caterpillar gut, the spores infect the cells, the meronts develop and reproduce, spreading to other cells. Fatty tissues are probably the first to be invaded (hence slower growth), with invasion of critical organs to follow.

If a healthy caterpillar does not become infected until the later stages of its life, it may well pupate and emerge as a moth (since there has been insufficient development of/damage by meronts) carrying spores in and on its body. Colder temperatures (diapausing moth pupae) may well have arrested the growth of meronts, causing them to form spores.

It is possible that these spores may then be transmitted from one generation to the next in what is called vertical transmission where the infection is transmitted from parent to offspring.

I do not know for sure, but here there seem to be several possibilities, none of them very nice:

The spores may be inside the eggs, even while in the female's body;
the spores may be on the eggshells inside the female's body;
the spores may be transmitted during pairing from males to females;
the spores may be transmitted from female body hairs/scales during ovipositing.

Uniparental inheritance imposes unusual selection pressures on host/parasite coevolution which have lead to the evolution of two unusual traits in the microsporidia, male killing and feminisation of the host.

Vertical transmission is mainly uniparental and there will be strong selective pressures for low virulence in the host sex which transmits the infection (females). However, parasites in male hosts are not subjected to the same selective pressures, as male hosts do not transmit the infection vertically (Bandi et al 2000).

Selection may favour parasites which are more virulent in male hosts if this causes an increase in parasite prevalence. Two different strategies of sex-specific virulence occur in the microsporidia. Male killers are benign in female hosts but kill males to release spores for horizontal transmission (Kellen et al 1965).

Feminisers are also benign in female hosts but convert genetic male hosts into functional, phenotypic females which go on to transmit the parasite vertically to the next generation of hosts (Dunn et al 1995).

Crafty little devils, aren't they?? The previous paragraphs suggest that if you are getting mostly female cocoons instead of a healthy 50-50 split, you may have microsporidia which are less virulent (deadly) to females, or the microsporidia may actually be converting the males to females instead of Killing them.

Horizontal transmission.

Horizontal transmission of microsporidian parasites is strongly dependent on parasite burden as spores are released into the environment where they are ingested by the next host. The greater the number of spores produced, the more opportunities of infecting a new host. Meronts divide in these cells and, after 48, hours some differentiate to form early spores characterised by a short polar filament and thin spore wall. The early spores germinate spontaneously to infect adjacent host cells. Other meronts divide and differentiate to form high numbers of thick walled, binucleate spores which have a polar filament with many coils. Massive spore production of binucleatecauses destruction of the host cells to release spores for transmission via the faeces. The high metabolic load and pathogenicity of this parasite lead to reduced host fitness and survival (de Graaf 1994; Canning 1993).

Vertical transmission.

In contrast to horizontal selection is predicted to favour reduced virulence in vertically transmitted parasites (Ewald 1987; Smith & Dunn 1991; Dunn et al 1995). The mechanism of feminisation is unkown. However, breeding studies have shown that 80% of the offspring of infected hosts become female. Although these parasites cause low pathogenicity in the individual host (Terry et al 1997), feminisers have severe implications for the ecology and evolution of the host. Theoretical models show that feminisers may spread through the host population causing sex ratio distortion and even have the potential to drive the population extinct due to lack of males (Hatcher et al. 1999)

In microsporidia with alternate transmission routes, horizontal transmission is associated with high virulence, vertical transmission with low virulence.

Microsporidia with alternating transmission cycles also provide clear evidence for male killing. Male killing parasites cause sex-specific mortality in their hosts. They cause a benign infection in female hosts, but are highly pathogenic in male hosts and lead to mortality during host development (Kellen et al 1965)

Sporogony leads to the production of vertical type spores which infect the gametes. Most females of this F1 generation also have benign infections. However, in F1 generation male hosts (i.e. infected vertically) a horizontal type sporulation sequence occurs. Massive parasite replication in the fat body results in death during the 4th larval instar and subsequent release of spores into the environment for horizontal transmission . Male hosts do not transmit the parasites vertically. Sex specific virulence maximises the opportunities for both vertical (through females) and horizontal (through males) transmission (Hurst 1991).

Conclusions

The role of horizontal transmission is uncontroversial for microsporidia infecting both vertebrate and invertebrate hosts, and this transmission route is associated with massive spore proliferation and pathogenicity to the host. However, there is growing evidence for the importance of vertical transmission in microsporidia which infect invertebrates. This transmission route is associated with spore polymorphism and modified virulence in the form of feminisation and male killing.

What does all this mean?

Naturally occuring microsporidians, infecting Saturniidae, can be transmitted vertically from parents to their offspring via the egg. The microsporidians may be ingested when the emerging larvae consume part of the eggshell and thereby ingest spores, or the microsporidians may already be inside the developing embryos, or both.

Microsporidians may also be transmitted horizontally from one caterpillar to another by contact with feces and by ingesting foliage harbouring spores.

I am not trained in the detection of microsporidians, but they can be seen under a microscope if you know what to look for. Additional information here would be appreciated.

Live moths not sufficiently infected to cause their own demise, apparently survive and can transmit the disease to subsequent generations.

Spores are long lived and can exist outside the caterpillar and moth bodies. It is therefore essential that diseased caterpillars be discarded properly. Letting them fall to ground or re-using rearing cages, jars, tubs, sleeves, etc., that have been exposed to an outbreak is not a good idea, without a thorough scrubbing.

Diseased caterpillars probably should be flushed down a toilet. If you are in the fields or woods and encounter disease, then you probably should have a covered bucket of bleach with you, and infected larvae should be dropped into the bucket. Hands should be washed to avoid contamination of healthy larvae.

I am aware that other breeders have actually used antibiotics to "control" or at least reduce the incidence of "disease". Last year I believe their was an article in Lep. Soc. publication about the use of CIPRO, an antibiotic, to help reduce problems besetting Saturniidae. It is my understanding that you cannot obtain CIPRO legally in Canada without a prescription, and that it is expensive. I do not know laws in the states regarding its availability.

In principle, however, I am against the use of antibiotics because I feel their too frequent use does two things:

1) Aids in successful development of weaker strains of Saturniidae stock;
2) Encourages development of resistant strains of bacteria and possibly viruses and microsporidians.

I advocate proper disposal of infected larvae, thorough cleaning of rearing materials, possible washing of eggs, and maintenance of rearing compartments that are frequently changed/cleaned and are not overcrowded.

I will let you know if I "learn" anything this coming summer. Thanks to Karen for forwarding the response from Dr. Smith. Help from other members would be appreciated.

Hope this doesn't provoke an unwarranted alarm. I suspect major outbreaks are relatively uncommon, probably not as often as bouts of Nuclear Polyhedrosis Virus. Sanitation, proper disposal of diseased insects and avoidance of overcrowding are all good pre-emptive measures.

I asked several people to critique my summary/notes and appreciate the email that came back from Chris Conlan:

Bill,
I am a bit out of my league here to comment on much of this article. What I can tell you is based on my experience working in large scale private insectaries. Both vertical and horizontal transmission occur and it seemed to be more of a problem in some Lep colonies than others.

I have no doubt that it could occur with Saturniids as well. It was also very common for vertical transmission to occur that almost certainly was coming from within the egg. We sterilized the crap out of all our eggs and still an occasional disease would get in and stay put at a low but detectable level.

When nosema showed up I believe we used to maintain as sanitary conditions as possible and treat with a drug called Fumadil(sp.?). I am not certain of the name of the drug as it has been many years (I know it started with an F!). However, it usually took a few generations of treatment to finally push the disease to undetectable levels.

Obviously, if one has the luxury of discarding all stock and starting over that is the best way to go but these things can be lurking in many colonies and show up the minute conditions go sub-optimal.

Antibiotics in rearing do work very well but they must be used from the very start or their value drops off quick. Waiting until symptoms appear to treat will usually still result in heavy losses.

A disease that takes out Hyalophora quickly and efficiently is usually NPV (at least in my experience). Since that is viral, antibiotics would have little effect other than to keep them healthy enough that their resistance to infection or secondary infection would be greater. In my personal rearing I tend to avoid antibiotics unless I have something that I really want/need to get through or a species that is notoriously disease prone. Don't know if this helped at all but if you have other questions just let me know!

Chris

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