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Why are my mealworms dying?

New thread for trouble-shooting mealworm deaths
@kerri: "These are the possibly dead meal worms I mentioned earlier... or did they just moult? I have heaps of these black dry ones at this stage, I can tell some are just shells, others... not so sure."
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@jena: "@kerri Yea, those (black ones) are looking quite dead. Did they all die together like that on that, what is it, potato? Or did you put them there for examination? I wonder why you're getting so many dead ones (we've never had 'heaps of dry black ones') - it could be a number of factors like too hot/not enough humidity, or something they're eating."
@kerri: "Hi @jena, great to see someone new joining the conversation. Yes, I did put those ones aside to watch them, so I can see now that most or all of them are whole dead worms, so perhaps the ones that look like shells got eaten at. The shell cast offs look quite different (not black but thin & crumpled. I have many such deaths now (50-80?) and think I may have let them get too cool overnight (it's very hot by day here in Australia). I've added a heat matt (small) and am watching the temp at different times (don't want to cook them either!) Their food is fine, oat substrate (pre-frozen to sterilise) and carrot or potatoes. That leaves humidity which I imagine will come alongside the extra warmth. I would love to hear about what you are doing @jena, What you're breeding, how long for, what you have learned etc."
@jena: "@Kerri The shell/castoffs are just that - shells. In other words, old skins from molting - totally normal, the mealworms grow too large for their skin, they leave the old behind (appearing lighter in color and more energetic afterward) and grow new. This happens many times during their lifecycle. How cool do they get overnight? We've left ours outside in relatively cold temps for extended periods of time (45 - 60's), and the only effect we've seen is a prolonged larval and pupal stages. In my experience, too much heat/desiccation are more often the culprit than coldish weather.

To answer your question on what I'm up to: I'm a Tiny Farms team member, am currently breeding crickets and mealworms, but have experience with a few other species like wax worms, roaches, and silkworms. Been at it for a couple years now."
@kerri: "Hi @jenna, - In Aus we use Celsius so I have to convert. Good advice though, I doubt it's the cool air (we have horrific raging fires here because of the hot summer) . I'll look into humidity, but how can you create that if it is cooler than say, 80 degrees F.? My heat mat is now keeping them at about 85 F (about 30 C) - Do you reckon that's OK? This is my first batch so I'm fairly clueless! . I am also breeding my first batch of crickets and after adding some warmth have found them crowding towards the heat... so that must be the right thing there. No deaths in crickets that I know of so far."

Comments

  • Hi @kerri, I think 85F should be fine if the humidity is high enough. It would be great if you could share the humidity once you find out, untiI then, I would experiment with giving them more moisture sources (carrot, etc.) and see if they fare better..
  • Hey @kerri, what temperature are they reaching during the day? They might be having trouble if they're getting too hot.

    If the air is very dry, you could try boosting the humidity by putting an open container of water in their habitat - tall enough that they can't climb up the sides and drop in.
  • edited February 2014
    I'll do that with the water - great idea. I put mesh over the top of the cricket 'humidifier' to make sure they don't jump in and drown... however, is there a nifty way to measure humidity? :-c Naw, not getting too hot, only up to 88 F tops (rarely that).
  • Hi @jenna, I didn't realise till now that you are a co-founder... love that page telling each other who we are.
  • @kerri in general we use these, fairly inexpensive thermometer/ humidity gauge to keep a general sense of the humidity levels in and around our habitats. They're available at most hardware stores, and some drug stores, although may be least expensive online.

    http://www.amazon.com/AcuRite-613-Indoor-Humidity-Monitor/dp/B0013BKDO8/ref=sr_1_5?ie=UTF8&qid=1392177610&sr=8-5&keywords=thermometer+hydrometer
  • Hi @andrew, I checked the product reviews and they're pretty bad - even after drilling holes in it to modify many said it was out of whack... any others you could recommend?
  • We've never had trouble with ours (we have about 5) and that one linked has pretty overwhelmingly high reviews (over 1000 5 star reviews). The other sensors we use are run with Arduinos, probably more involved than you're looking for. I would imagine any off-the-shelf humidity sensor will be good enough to keep tabs on your little friends :)
  • Oh, OK. Probably depends on what links you tap on... I'll believe you!!! I'll get two. :D
  • Reactive oxygen & larvae particulars. The larvae grow due to their cellular power plants called mitochondria, which are organelles inside cells. The way mitochondria are supposed to work to end up producing ATP molecules is they oxidize ("burn", oxidative phosphorylation) a type of raw material molecule by moving an electron(s) along in something similar to a chain of transportations of the electron....It is along the way, in stages of the electron transport chain, that the oxygen involved can be spun off as different forms (species) of reactive oxygen species (ROS). Technically the mitochondrial ROS are generated at staging areas made up of special enzymes (a complex of enzymes) along shuttling route; ROS are put out at Complex I & Complex III of the electron transport chain. Not all ROS ,hydrogen peroxide (H2O2) included is generated by mitochondrial activity, but it is very significant....To keep the electron going along the chain of stages & into a subsequent "complex" the mitochondria organelle's own membranes (they have an inner & outer membrane) toggle proton's + charge across a mitochondrial membrane for the boost. Larvae growth is so dramatic because as their insulin like protein/insulin levels rise this insulin causes less proton's to "leak" out of place & the downstream effect is to increase the amount of raw material for making ATP to be oxidized; which means the larvae get lots of ATP to use for development growth....Insulin raises H2O2 as a side effect. One of the other ROS generated is called "super oxide", an unstable configured oxygen molecule that "accepted" an extra electron. This super oxide can only stay inside the mitochondria, where it is potentially problematic; therefore super oxide is phase shifted into H2O2 (using MnSOD, not discussed here) which then is capable of diffusing out of the mitochondria....Once the larvae ramp up development of their "fat body" this means they are generating a higher amount of super oxide; & subsequently they cope with high levels of H2O2. There is an additional positive affect for larvae due to the fact there is also H2O2 coming into circulation from the insulin signal that triggers another downstream signal cascade; this signal engages what is termed "Akt" inside a cell & when Akt roused into action it in turn activates one of the controllers of making fat cell.... An integral part of ROS signalling is the nitric oxide molecule, which is also made as part of the mitochondrial oxygen dynamic. Nitric oxide, having an unpaired electron, usually counterbalances the super oxide(extra electron) effects; & indirectly modulates H2O2 levels. When larvae need lots of ATP the milli-voltage of their mitochondrias' membrane changes to keep a high proton forcing going on, which speeds the electron through distinct staging complexes, causing lots of oxygen be acted on, instigating reactive super oxide levels to go up , causing (normally) a drop in nitric oxide restriction (inhibition) of super oxide that forms H2O2 & by H2O2 shifting out of the way the mitochondria can function full on despite making ROS....Larval mitochondria age & over time mitochondrial membranes lose some milli-voltage. Mitochondria can be recycled & the milli-voltage drop causes that cell to use ATP to power over failing mitochondria to processing vacuoles inside the same cell. When the protein degrading enzymes (protease) cleave loose the peptides from an old mitochondrial protein amino acid(s )the cell can use them & their nitrogen again. This is quite likely what is happening with an instar: the mitochondria are starting to "fail" from all the high activity & the larvae needs to form lots more of those functional energy plants....When this programmed cell re-cycling (a type of autophagy) shifts into gear it is because the mitochondrial (oxidative) respiration chain is stumbling & this means less super oxide generated. That in turn allows more nitric oxide to hang around &, as mentioned nitric acid holds down H2O2 levels; while the plunge in H2O2 stymies fat body build up (Akt not getting H2O2 signal that there's plenty to store up)....If the larvae did not enter an instar they would suffer more & more mitochondria getting fried. The point of no return for recycling a failing mitochondria is when not only has it's nitric oxide levels risen, but the super oxide that is still being spun off can't phase away it's reactive oxygen as H2O2. Once the super oxide has no outlet as H2O2 molecule(s) another ROS , called peroxy-nitrate, starts to hang around inside the mitochondria; & unfortunately sustained elevated levels of peroxynitrate can cause the mitochondrial proteins too severe damage for them go on to recycle peptides (nitrogen). Peroxynitrate is an adverse reactive molecule caused when super oxide & nitric oxide interact with one another in an attempt to resolve one of them out of the way of the other, so to speak. If there is not enough of either super oxide or nitric oxide to establish a definitive reduction of peroxynitrate then it acts to "nitrate" (nitrosylate) mitochondrial proteins....An instar is like a time out. They stop moving because movement muscles had begun to operate undersuch high levels of nitric oxide that the nitric oxide inhibited an actor (cytochrome co-oxidase "C") of the ATP production sequence & the muscle output of ATP for moving went way down. Muscle cells use oxygen & it is cytochrome C (if nitric oxide kept in check) that lets more of the oxygen already in the cell become available for the muscle cell....Some larvae from the same egg clutch do not survive all the way to pupate. These suffered from a simultaneous excessive nitric oxide at the same time as excessive super oxide & by extension inadequate H2O2; which means those larvae had problems sustaining ATP. Once the ATP became too low they didn't even have enough ATP to power enough metabolism of their own fat store for a net gain in energy....Pupae at too low relative humidity can suffer mortality; since not feeding they are not spinning off super oxide that would otherwise knock out nitric oxide & the nitric oxide level rises in the pupae. Remember, nitric oxide inhibits enzyme cytochrome C & one of the functions of this enzyme is to get oxygen O2 inside the cell to form up into water inside the cell; cytochrome C has a neat role because it make one O2 molecule into 2 water H2O molecules. When the relative humidity falls way below a recommended % the failure of some pupae is likely due to exasperating the falling lower internal moisture from the nitric oxide predominance during the pupal period.... Female insect pupae tend to eclose (emergence of adult) later than males & this is likely programmed by estrogen using ROS signaling. As estrogen levels build up the estrogen receptor up-regulates, which activates the enzyme involved in making nitric oxide & thus downstream the female's nitric oxide levels increase. The male pupal nitric oxide level will be lower than in female pupae.... When conditions are right for pupal metamorphosis, if it is to lead to a successful adult's emergence, there's a steroid needed to push along key events (not detailed here). The insect steroid is called ecdy-steroid & is also a player in insect molts....This ecdysteroid bio-synthesis is regulated by an enzyme (cytochrome P450) & this enzyme uses oxygen to perform it's function in a part of a cell called a microsome. The enzyme needs to pass off a lot of that oxygen & the pathway leads to super oxide production right inside the microsome; down the line conversion into H2O2 is how that reactive oxygen gets away from the microsome. In contrast extra nitric oxide will tag directly onto the P450 enzyme & this keeps the rate of enzymatic oxygen dispersal down(slower); meaning that the female can take longer to get the same ecdysteroid steroid boost.

  • @gringojay - my main takeaway from this is that too low a relative humidity can result in failed molts and pupation, but are there also dietary consideration that can influence (positively or negatively) this mitochondrial cycle? like ensuring sufficient levels of phosophorus, nitrogen, etc.. in the feed?

  • Humidity & larvae mitochondria: in layman's terms?....Larvae bring their air into trachea (like our throats, & instead of mouth/nose insect's have pore structures called spiracles). The trachea network of tubes step down in gauge to branch into tiny (~ 1 micro-meter diameter channels) insect tracheoles that deliver oxygen inward & take back out CO2 (carbon dioxide) to go out; they don't have a central gas exchange site, like lungs...Insects don't have diaphragms, oxygen comes into the trachea under the force of what is called oxygens' partial pressure. When the oxygen reaches a tracheole it presses against an actual "plug" of water inside that tracheole....In simple language oxygen first meets a natural control feature that stops it from diffusing farther before it actually reaches any of the insects' inside cells. The design is arranged so that the cell(s) fed by that tracheole move some of the cells' soluble ions that deal with osmosis & the osmotic change physically pulls the tracheole's water "plug" inward; then the pressure of the oxygen moves the oxygen to get right to a cell (&/or circulatory system) .... Obviously, the tracheole water "plug" must open to let CO2 into it for that gas to go outward & leave the insect. The CO2 exerts it's own type of force, which in the insect hybrid circulatory system (like blood & lymph systems, but all in one) is called haemolymph CO2 partial pressure..BUT, there is another factor that keeps the water "plug" out of the way & the tracheole/trachea airway wide open. Low metabolic rate itself causes the airway to be open ended & then the insect can lose it's sparse internal water....Mitochondria drive the metabolic rate; whether from age decline or lots being taken off line for re-cycling mitochondria can't always run the insect at a high metabolic rate. It is precisely the move to improve mitochondrial capability for the next instar installment that leaves the front door open to danger, so to speak.... Humidity becomes important at this point & different species range limits of humidity do vary. The main point is if humidity is too LOW for them they will keep losing water through their airway when it is open during the time it's metabolic rate is low....This is because dry air has higher pressure than moist air & it will force it's way in deeper faster. Also drier air, that is not stalled because a tracheole water "plug" left a direct tube channel to inner cells, can "rob" some cellular water....It is counter-intuitive, but humid air weighs less than dry air weighs; it is less dense. Thus moisture in the air acts to lower it's pressure - even though the % of oxygen (~21%) & % of nitrogen (~78%) stays the same regardless of humidity . Water vapor, which we call humidity, is lighter than both the molecules of nitrogen & oxygen)....Larval instar/molting (which requires mitochondria re-cycling) require oxygen to stay inside the haemolymph for optimal ion regulatory functions. At these vulnerable phases of change it is important to also change the ratio of oxygen partial pressure (external factor) to (internal factor) insect's content of oxygen.... In other words, during those times when mitochondria can't ratchet up the metabolic rate & the airway is uncharacteristically open humid air will press in less than dry air. Haemolymph can hold some fall back oxygen for a limited time bound to a molecular component called haemocyanin; so there is no need to press in more oxygen via drier air....Now, the problem with too humidity is that internal cells lose water & what is in those cells become more concentrated in the remaining fluid. Haemolymph dehydrates & nitrogen based molecules build up....Here is the problem: "ammonia", which is tricky to explain because we have to distinguish between a form with no ionic feature, NH3 (called ammonia) & the ionic form NH4+ (called ammonium). These "ammonia" comes mostly from enzymes breaking down(catabolism) of proteins/amino acids (a smaller amount are byproducts of purine/pyrimidie metabolism). Point to understand is that too much "ammonia" can be toxic to a cell & too low humidity makes it more likely to get to toxic levels....First, for those who do not know it, ionic regulatory activity is both the main cellular activity & the largest consumer of ATP energy. One way ions work is to "pump" one ion across some cell membrane & exchange it with a cooperating ion; then when appropriate "pump" them back the other way & so on, back & forth....Probably the most strategic for our larvae's situation is the "pump" that uses sodium(Na) in conjunction with potassium(K). And since a "pump" uses ATP energy to boost it into action you'll see this called "Na+/K+ATPase"....Well guess what: K+ ion & ammonium NH4+ ion both orbit their + ion at the same radius. Which means NH4+ can jump into place instead of K+ & then usurp an otherwise benign natural exchange of positions with sodium....In simple terms a normal ion process lets the wrong ion in & the potentially toxic ammonia in the form of NH4+ (ammonium)gets into a cell when it shouldn't. Not only that but more NH4+ can come along & hop the same way into that same cell & build up undesirably inside that cell....When the humidity is too low the haemolymph's thicker ammonia concentration has to be off-loaded to cells. So this usurping of the Na/K pump is meant to be a transitory safety valve; it is impossible for NH4+ to diffuse either in or out of any membranes, it needs an electro-chemical gradient to move across membranes....The stashing of NH4+ ammonium is meant to be temporary & not a fatally toxic accumulation. First NH4+ has to shed a H+ & become just NH3....Here a problem arises when there are mitochondria off-line/re-cycling; the H+ must use ATP to "pump" it out of the cell & ATP to pump in Na in exchange for the safer NH3. Except there is not much ATP hanging around when the mitochondria are getting sorted out.... But if there is not enough ATP those promisingly split up NH4+ components of H+ & NH3 go to a part (vesicle) of that cell with a low pH & the pH recombines that NH3 with any loose H+ back into the more toxic NH4+....In summary, the mitochondria use reactive oxygen (detailed previously) to signal they are ready to be recycled, the larvae do this so they can go on to their next development phase with adequate energy (ATP), their metabolic rate slows until mitochondria can ratchet it back up, the airway temporarily doesn't re-install it's water "plug", outside access to inner cell water is open, if the humidity too low for too long the oxygen pressure leads to quicker cell water loss, relative concentration of "ammonia" in haemolymph goes up, ammonia hijacks sparse ATP to get pumped into cells, until mitochondria back on line with enough ATP the ammonia can't be pumped out of the cells & finally it is ammonia as NH4+ molecule that is toxic at high levels.... Larvae can often survive this, although if similar low humidity stresses at enough low ATP instar phases repeatedly occur the number of instars that larvae goes through until pupation will be more than usual. It's the mitochondria signalling with reactive oxygen they need another "tune up" again & then can get back to work.... Some larvae may survive a few instars by bringing mitochondria back on line before recuperating all the mitochondria it ideally should make for meeting the pending instar energy needs. Then each instar that follows will have less re-cycled mitochondria & the larvae is progressively more vulnerable. An inopportune low humidity, that wasn't too low before but with mitochondria wanting in capacity, could eventually indirectly cause irreparable ammonia toxicity & some larvae suddenly die when everything seemed to be going fine for a long while.

  • Hi Andrew, The trace minerals involved in modulating reactive oxygen are copper, zinc & manganese. They form up enzymes called super oxide reductase (reducers)& then the step finishing the neutralizing into oxygen & hydrogen peroxide (H2O2) takes another cooperating enzyme (glutathione peroxidase). One reducer enzyme (abbreviated as SOD; for super oxide dismutase) works in the mitochondria, another in the general interior of the cell (cytosol) & another outside a cell. The cell can make super oxide in the mitochondria & also make it in cytosol's part called a microsome.... The reducer in the mitochondria uses manganese (written in short hand as MnSOD). It is under the control of genes in the nucleus & not mitochondrial genes; MnSOD is imported into the mitochondria.The amount of manganese needed is considered a trace amount so apparently wheat bran naturally has enough. If not feeding wheat bran I would look up the manganese content of wheat bran & then see if what intend to use as an alternate feed has at least that content of manganese.... In the mitochondria if superoxide gets acted on by nitric oxide an MnSOD is not used & the 2 reactive oxygen species (ROS) segue a molecule of per-oxy-nitrite (ONO2-), this is instead of super oxide going into hydrogen peroxide. The problem arises when per-oxy-nitrite builds up & lingering too long acts to "nitrate" proteins in the mitochondria. Nitrated tyrosine binds up MnSOD & not only does this inhibit MnSOD from doing it's clearing out intervention (ie: making super oxide into hydrogen peroxide), but the nitrated tyrosine can inhibit it's captive MnSOD molecule until that mitochondria gets recycled. Not all nitrated tyrosine is potentially insidious; sometimes it is serves a role in temporarily dampening down what goes on at the first 2 mitochondrial "complex" when oxygen is too low; which could be a condition the instar must transition through. But once oxygen level raises the nitrated tyrosine can be shucked from those complex, unlike how it binds to MnSOD...As the mitochondria gets on in it's life it starts to falter in productivity due to super oxide & nitric oxide interactions losing equilibrium. Then a cycle of compensation leads to more per-oxy-nitrite lasting long enough to nitrate proteins & even more MnSOD gets irrevocably tied up. That cell is not able to make ATP like it should & larvae signaled to take a time out & re-make mitochondria for the next instar....When a mitochondria gets taken for re-cycling it's nitrated proteins are taken apart & the amino acids assembled into new proteins that are no longer nitrated. In a sense the larvae don't need to do their next instar until their mitochondrial proteins are again nitrated to the degree they impair function & likewise, if the last instar got stressed (low humidity, excessive heat) had to cut short it's reformulation of the total mitochondria it is designed to have then the sooner it will nitrite it's mitochondrial proteins & need to seek another instar for refreshing it's energy plants... If the larvae are exposed to foreign micro-organisms their defense is an immunological ramping up. This can happen when the natural larval symbiotic micro-organisms that live on the larvae are reduced &/or displaced by "invading" micro-organisms. Probably the most frequent hostile takedown is from micro-organisms living as symbionts on other insects that get into the mealworm substrate; ants & cockroaches come to mind....Immune activity entails both super oxide & nitric oxide levels rising in a coping situation. This provokes a lot of per-oxy-nitrite formation & as detailed leads to a drop in MnSOD availability. In turn, with MnSOD off line. Meanwhile the mitochondrial keeps spinning off super oxide; but it can't use the relief pathway of hydrogen peroxide so even more per-oxy-nitrite appears & all that nitrating of mitochondrial proteins impairs that mitochondria's functional apparatus. The ATP output falters, the mitochondria must use the sparse ATP just to keep it's own membrane milli-voltage up & cell(s) suffer an energy deficiency; if not resolved in time that mitochondria will die....The other reactive oxygen reducer in the cell body uses 2 trace minerals as co-factors; these are copper (Cu) & zinc (Zn), so outside the mitochondria the cytosol's reactive oxygen reducer is a hybrid (written in short hand as Cu/ZnSOD). For this latter reducer zinc's role can be performed by substituting the zinc with cobalt, mercury or cadmium - but you'd not want to add any extra of the latter 3. There is no other trace mineral that can replace copper in the hybrid enzyme. Again I'd peg the trace amounts of both copper & zinc to what wheat bran contains....Revisiting the concept that reactive oxygen species are crucial signaling molecules & the interplay of super oxide & nitric oxide. It is the molecule per-oxy-nitrite whose action on zinc found in other enzymes (xanthine oxidase &/or nitric oxide synthase) that lead to situations where super oxide predominates over nitric oxide. Nitric oxide has it's own role regulating trace metals because it works on the transcription factor (brings gene online) group called zinc finger....Nitric oxide has an electron in it's orbit that is missing a pair; it can hook up with iron, copper & other metals. This feature is important because by influencing metals it (nitric oxide) stops the more damaging reactive oxygen called a hydroxyl (OH-) being formed. If the cell is not putting limits (via nitric oxide) on the amount of super oxide, & there's too much to shunt away as hydrogen peroxide, then the super oxide acts on the iron/sulphur components of the mitochondrial complex I & complex II. The result is iron disassociates to become a free agent in both the mitochondria & cytosol. Excess iron on the loose leads to more hydroxyls & any loose hydroxyls are what can sear the first molecule they come in contact with, even genetic....As for protein having any benefit as a protective supplement for an instar it is apparently not always ideal. Experiments on Drosophila found that viability goes down when more protein than usual is given, but only if there is a problem with oxygen level in the insect. A relative hypoxia (low oxygen) can occur when the new instar animates in a high concentration of CO2, such as might exist in aging feed.... If look back into the mitochondria it's cytochrome C uses oxygen & if the nitric oxide set-point is too low there's no feedback loop to stop that mitochondria from using even more oxygen. What sets the base line nitric oxide level low is if there were too few fully functional mitochondria put back online in the instar. Then there are fewer mitochondria to match growing larval needs of ATP being cranked out; so those fewer mitochondria then increase the milli-voltage of the mitochondrial inner membrane for speedier delivery of the sparks (protons' force) borrowed for making ATP. That impulsivity, in turn, results in even more super oxide than normal & then, since super oxide decreases nitric oxide this adaptative measure compounds an already low nitric oxide operating state if the larvae/instar has insufficient numbers of robust mitochondria.... Whereas when amino acids are low & oxygen also low there are changes leading to more of key gene (TOR gene) expression in the fat body. This specific gene upregulation in the fat body leads in turn to changes in insulin signalling out & about in other cells - they do better. But, if all of a sudden we make the protein amino acids in feed substrate higher than before & there is some relative hypoxia the intermediary adaptation (upregulated TOR) in the fat body doesn't happen. (For larval TOR & insulin: see 2012 "Nutrient/Tor Dependent Regulation ... Controls Tissue & Organismal Growth..."; free full text = http://emboj.embopress.org/content/31/8/1916

  • PostScript: Where discuss the mitochondrial signalling response of the immune system add the stress challenge from "mold". A too moist vegetable isn't going to drown larvae, but a mold can damage the symbiotic micro-organism(s) living on the larvae & evoke an immune response. It may not kill the larvae outright by simple contact, but be the cause of mitochondrial insufficiency which can lead to death.

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