Cellular Responses, Age-Related Changes, Gut Microbiota and Beyond
Insects lack an “adaptive” immune system. They don’t have the long-lived B- & T- cells that target specific pathogens and remember infections for future encounters. Their primary approaches involve encapsulation of bacteria and parasites, along with an antimicrobial peptide-driven response. Paternal genetics, hormones, nutrition, microbiota and age indirectly influence this process, either enhancing or hindering it depending on the creature’s condition.
The individual honey bee’s immune system shares many aspects with other insects, and this article can be thought of an overview to insect immunity from a honey bee lens. Part Two will cover immunity at the colony level, and we see our individual bee placed as part of a greater whole who provides immunity to the “superorganism”.
Cellular Level Responses are Consistent in the Order Insecta
Insect immune systems are similar across the insect families in terms of their pathogen recognition, immune signalling and responses against said pathogens. Much of what we know about Apis mellifera was deduced by Dipteran studies on the fruit flies Drosophila & the mosquitoes Anopheles. We start with a description of their innate immune system1:
Innate Immunity is the First Line of Defence: Physical Barriers, Cells and Chemicals
Innate immunity attempts to stop pathogens from entering using physical barriers like an insect’s cuticle or gut mucous membranes. Should the invader breach those barriers, they encounter the second line of defence, those cells and chemicals that neutralize and degrade foreign material. Insects have a limited arsenal for the second line; mostly consisting of phagocytes and haemocytes (the former being a functional class of the latter).
Humoral responses (“humoral” as in “relating to the body’s humours” [a historical belief]) involve antimicrobial peptides, linkage proteins, melansiation & coagulation proteins. What molecules their immune system deems foreign is a result of germline cell programming (sperm & ova) – the offspring inherit the immune system of their parents.
In honey bees, inherited immunity does not occur through learning or memory, rather via selection and maternal immune priming. When queens are exposed to pathogens, components of those pathogens (as bacterial cell wall fragments, toxins etc..) can be transferred to developing eggs via the yolk protein vitellogenin (Salmela et al., 2015). This exposure does not encode specific memories instead “biasing” the offspring’s’ immune systems against these material.
At the same time, queens differ genetically and epigenetically in how effectively their immune systems detect and control microbes that escape the gut. Queens with traits that better regulate immune responses are more likely to survive and reproduce. Over many generations, natural selection favours these traits, leading to colonies whose workers are collectively more efficient at recognising and managing opportunistic infections.
Cellular Immunity
The cells in the bees’ haemolymph (“blood”) act like our white blood cells’ phagocytes: they encapsulate and phagocytose (“consume”) foreign material, with an added step of melanisation. Small foreign agents are phagocytized for removals. Larger ones, or an aggregation of smaller agents, trigger encapsulation, which is the removal of foreign material through cooperation by several haemocytes. Melanisation may take place here, with the goal of eliminating large numbers of foreign matter.
Prophenoloxidase is a haemolymph protein that mediates melansiation and is activated upon detection of pathogen associated molecular patterns (PAMPs) by the pathogen-recognition receptors on haemocytes. Once activated, they surround the invading agent and “encapsulate it” (in the literal, rather than immunological sense), releasing prophenoloxiadase along with melanin and other proteins to eliminate the foreign material (for a review of melansiation in Drosophila, see Tang, 2009).
Humoural Immunity
The second line of defence involves antimicrobial peptides (AMPs), which are synthesised by the insect haemocytes, salivary glands and fat body cells (adipocytes)2 in response to infections. Honey bees have reduced humoral activity relative to the fruit fly/mosquito models, and I wager that that this reflects their social lifestyle – less individual energy invested in AMPs as the colony also provides protection. A. mellifera’s AMPs belong to four families (reviewed in Rahnamaeian et al., 2015):
- Abaecin, acts via DnaK inhibition to target affects Gram-negative bacterial membranes, disrupting protein folding and ribosome formation
- Apidaecin, similar to abaecin, targeting Gram-negative bacteria, disrupting ribosome binding
- Hymenoptaecin also targets Gram-negative bacteria, destabilises their cell membrane
- Defensin, two genes (Defesin-1 & Defensin-2) that target Gram-negative bacteria’s lipopolysachride-containing outer membranes.
Bumble bee studies showed these AMPs synergise to provide greater antimicrobial effects, and deploying multiple AMPs may be more energy efficient than a single family on account of this non-linear synergy (e.g. Marxer et al., 2016 or Duong et al., 2021).
Humoural immunity Targets Gram-Negative Bacteria Because they’re Commonly Encountered by Bees
That most of the immune genes target Gram-negative bacteria is a reflection of the selective pressures that honey bees encountered over evolutionary time. You can see this in their gut microbiota as their core community is largely Gram-negative. When the gut barriers are disrupted due to stress or pathogens, their microflora escapes the gut and into the bloodstream; triggers the bee version of sepsis (“systemic inflammation”).
Further, many flower families harbour Gram-negative bacteria, naturally. Here it becomes cyclical: Bees evolved defences against Gram negative bacteria because they’re the most common type of bacteria in the environment, and since Gram-negative bacteria is they pose the greater risk of systemic infection, bees have evolved defences against them.
What of Gram-positive pathogens? Consider Paenibacillus larvae (causative of AFB) & Melissococcus plutonius (causative of EFB) – both infect the brood, rather than adult bees. The pathogens’ specialisation means they can survive the larval antimicrobial peptides, osmotic pressure, acidic gut conditions etc… it’s a different selective pressure. While adult bees do encounter Gram-positive bacteria in the natural environment, they rarely cause systemic infections in adults and thus exert weaker selective pressure on haemolymph immunity.
The Insect Immune Response is Triggered Upon Pathogen Recognition
The insect immune system is switched “off” until it detects a foreign agent. The event sequence is generalised as so: recognition, activation of signalling pathways and deploying pathogen-eliminating mechanisms. Pathogen recognition starts with detecting PAMPs, whether it’s lipopolysaccharides in gram negative bacteria, or double-stranded RNA from viruses. Once detected, signalling events activate other aspects of the immune system, and recruit haemocytes to phagocytose the foreign matter.
Activation of signalling pathways is technical, and I summarise as so: the Toll signalling pathway is conserved (“common”) in many insects, and it triggers antimicrobial peptides & lysozymes. The Immunodeficient (Imd) pathway is involved in antimicrobial peptide synthesis & further downstream signalling via JAK/STAT. Speaking of JAK/STAT, in bees, these receptors mainly induce the phagocytosis response by haemocytes and also coordinate antiviral responses. Last one is the RNAi (RNA interference) pathway3, which is used against viral infections by silencing its replication cycle. The pathway produces small single-stranded RNA that binds to the virus’ messenger RNA and silencing it, preventing subsequent viral protein production.
How the Honey Bees’ Immune System Differs from Other Insects
A. mellifera has about one third as much immune genes as Drosophila & Anopheles. This low number highlights the importance of “social immunity” in protecting bees; workers have more genes dedicated to odour recognition and foraging than phagocytosis or AMP production.
Two proposals: this shows the importance of social defences (i.e. based on social behaviour) and/or honey bees’ tendency to be attacked by a limited set of pathogens. For example, A. mellifera has a single prophenoloxidase gene, whereas Drosophila have three and Anopheles have nine (reviewed in Larsen et al., 2019). Activation of prophenoloxidase leads to series of reaction that aim at destroying the foreign agent (melanisation, along with reactive oxygen species etc…).
Early genomic work by Evans and colleagues, followed on from the results of the Honey Bee Genome Sequencing Project and found that these reductions in immune genes were observed in all of the bees’ life stages, from recognition and signalling to the actual immune effectors. (Evans et al. 2006). The honey bee immune system favours combatting Gram-negative bacteria, as they are the most abundant in their internal & external natural environment, and it is to the gut bacteria that we turn to next.
A Bee’s Native Gut Microbiota Prevents Pathogens from Establishing Themselves
The Gram-negative bacteria’s peptidoglycan triggers the Imd pathway in bees, resulting in upregulated antimicrobial peptides. Work from Horak & colleagues found that gut bacteria Snodgrassella alvi reduces the activation of this pathway and drives the immune system to favour the Toll pathway (Horak et al., 2020). Perhaps it is a case of cross-activation for a more effective immune response (e.g. reported by Alejandro et al., 2022 in kissing bugs)?
As in ourselves, the gut microbiota has multiple functions with immunity being one of them. The microbiota serves as a “barrier” that stops foreign pathogens from colonising the gut (Steele et al., 2021). Bees with a disturbed gut microbiota had reduced survival after encounters with Serratia marcescens (a model bacteria for pathogen studies), whereas those with the native gut population better resisted pathogenic colonisation.
The microbiota’s metabolic activity alters bee gut chemistry, changing pH, oxygen concentrations, and/or reactive species concentration. The two primary S. marcescen antagonists were proposed to be S. alvi and Gilliamella apicola, on account of having a shared bacterial secretion system (“T6SSs”).
The native gut bacteria can trigger body temperature increases and induce a “fever” as way to “selectively favour the growth of symbionts and their inhibition of parasites, such that the presence of symbionts effectively reduces parasite heat tolerance” (Palmer-Young et al., 2023 with American spelling correction by Yours Truly). Beneficial gut bacteria were more tolerant of higher bodily temperatures than parasites, here the trypanosomatids had optimal growth at 25-28°C whereas honey bee gut bacteria 35-37°C, some being able to grow above 40°C. Note that parasites still grew at those 35°C, however the native bacteria had greater growth at this temperature and outcompeted the invaders.
Host Lactobacillus strains produce acids as metabolic byproducts, lowering the environmental pH and contributing to chemical inhibition of the pathogens. This acidification occurs in varying degrees throughout the digestive tract with the aim of making a bee’s internal environment harsher to invaders.
Nutrition as a Form of Immunity & Its Metabolic Costs
That starvation (“caloric restriction”) impairs immunity is well known, and this goes beyond bees; maintaining an immune system is metabolically costly. Pollen provides proteins, and these proteins may be used in the formation of antimicrobial peptides, whereas nectar and honey provide the carbohydrates for powering the cells of the innate immune system. Worth mentioning are the secondary plant metabolites (as in black locust or sunflower honey) whose digestion provides some antimicrobial properties (Erler et al., 2014).
Unsurprisingly, bees fed a varied diet of natural forage had better overall health outcomes, with one recent study from Mexico comparing avocado, maize, and polyfloral pollen diets on the immune system (Quesada-Bejar et al., 2022). The authors measured the protein content (via AMPs) in the haemolymph of bees fed these varied diets and found that AMPs were highest in the avocado and polyfloral pollen groups. They also noted that when total haemolymph protein was high, prophenoloxidase activity was lower, suggesting an inverse regulation.
That’s not to say that bees cannot utilise both AMP & prophenoloxidase systems at once, it just comes at a cost of ~25% increased metabolic rate (Ardia et al., 2012, based on studies in house crickets, yellow mealworm beetles, American cockroaches and green June beetles). The overall picture shows that a high-quality diet produces a high-quality immune system.
The Antimicrobial Properties of Hive Products
I’ll mention this here and expand on it when discussing colony immunity. In short:
- Honey’s antimicrobial components are mainly in hydrogen peroxide (Manuka honey has something else), which damages the microorganism’s DNA and cell membrane(s)
- Certain flowers carry phenolic compounds like flavonoids and phenolic acids which can inhibit the growth of Gram-negative bacteria
- Propolis’ antimicrobial activity varies with its foraged source. Of note are flavonoids, phenolic acids, and terpenoids, known for their antimicrobial, antioxidant, and anti-inflammatory properties; go read the propolis article
- Royal jelly has various bioactive molecules, there is 10-HDA, the major royal jelly protein families and the yelleines – more on this in the royal jelly article
- Wax comb itself holds antimicrobial effects. The antimicrobial activity likely stems from the hydrocarbons, fatty acids, esters and other substances that make up the wax molecule, as well as adsorption of antimicrobial compounds such as propolis-derived phenolics from the environment (see Fratini et al., 2016 for a mini review).
Bees Reared at Lower Temperatures Have a Reduced Immune Response
One of the more unsurprising findings from literature showed that larvae reared at lower temperatures had a reduced immune response (Butolo et al., 2021). Researchers tested 8, 16, 24, and 32°C (as opposed to the standard range of 34-36°C) and found that colder temperatures reduced the number of haemocytes in all development stages. Low haemocyte larva lead to low haemocyte adults. No surprise, considering that cold temperatures impact every other system, from digestion to flight capacity to learning and memory formation. Likewise with elevated temperatures beyond the optimal, these individual bees also emerge with stunted immune systems.
Why am I unimpressed? Because real-world colonies are programmed to control their brood box temperature tightly. A colony that cannot maintain a constant 34-36°C is no colony at all, odds are it will collapse long before those larvae become adults. Still, such research is important to confirm what we suspect and gives other authors a reason to cite papers.
“Immune Memory” is the Insect Version of B-& T-Cells
Insects don’t have an adaptive immunity like higher vertebrates and while RNAi is similar, it is not the same thing. Instead, they have “immune memory”, whereby an individual’s immune system “remembers” a previous infection and can better tolerate it upon subsequent exposure.
Burciaga & colleagues tested groups of nurse bees exposed to pathogens at a sub-lethal and lethal dose, and found that the groups who were exposed to a sublethal dose first followed by the lethal dose had better survival than a control group who received the lethal dose first (Burciaga et al., 2023). The keyword is “same pathogen”, as the enhanced immune memory was pathogen-specific- bees who survived a sublethal dose of the bacteria S. aureus or M. lysodeikticus did not show better survival outcomes when a lethal dose of E. coli was administered.
To determine the mechanisms behind this, the authors investigated haemolymph protein content and found no significant differences between the treatment groups, suggesting that a wider array of testing is required (i.e. measuring antimicrobial peptide & lysozyme levels), as their suggestions that it was due to lytic activity and phenoloxidase function were inconclusive. No matter, their results are a step further towards protecting bees from natural infections by developing “bee-vaccines” against bacteria (or viral infections using particles of inactive virus).
Knowledge of Immune Memory is Not New; Further Research Required
The 2014 study from Hernandez Lopez & colleagues showed that queens stimulated with the bacteria that causes AFB (P. larvae) produced offspring that were more resistant to AFB than untreated brood (“Trans-generational immune priming”; Hernandez Lopez et al., 2014). Alas, the study only followed one generation of workers- would it still hold for offspring 10 generations later?
I say no, since it’s been eleven years since that study and nowhere do we see adverts for “AFB-resistant queens!”, so it is a question of how long does this immune memory last?
Age-Based Immunity: Foragers have a Weaker Immune System than Nurses
Worker bee immunity declines with age and is the least efficient in the foraging caste. Odd, considering that foragers are the ones encountering the most pathogens when visiting flowers4. Lourenco & colleagues exposed foragers and nurse bees from the same colony to S. marcescens to determine the survival rates between the two groups, both of which were forced to perform “age uncoupled tasks” (Lourenco et al., 2019)5.
They found that foragers could still activate their immune system like the nurse bees do (as determined by levels of Defesin-1). They suggest that high juvenile hormone levels cause reduced immune activity, citing a previous paper whereby topical application of JH resulted in haemocyte shrinkage. Younger bees have a low JH titre (and higher vitellogenin; the two are inversely related) and it is this low JH-high Vg balance that is favours a stronger immune response, (foragers have a high JH-low Vg balance; Lourenco et al., 2019 [these results are also supported by similar research from Byhro et al., 2019]).
Differences Between Immunosenescence & Task-Based Immune Reallocation
Some definitions: Immunosenescence is irreversible biological aging, whereas task allocation & reallocations are hormonally-mediated shifts in immune investment and as you’ve seen from the work above, they are fluid and reversible. Failing to separate these concepts leads to incorrect conclusions like “task roles and aging are the same process” or “foragers have weaker immunity because they are old” or “immunity always declines with age”. Honey bees and colonies are flexible, they will shift their physiology to meet whatever demands placed on them by the hive or environment.
One Step Further: Colony Level Immunity
Some of the mechanisms described above can be scaled up to the colony level. Other aspects require a shift in perception to one where the honey bee worker is no longer viewed as an individual unit, rather one that is a part of a superorganism. In other words, the colony approach to immunity is not a continuation of what you’ve read above – there is more to consider when the organism is “scaled up”.
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Footnotes
- Higher vertebrates (fish, birds, mammals etc…) have adaptive immune responses; the B- & T-cells that are specific against repeat foreign invaders and whose activation results in a stronger immune reaction. Insects only have an innate system, as an adaptive immunity is costly, requiring long-lived cells, permanent tissues, stable blood volumes, large genomes and several other aspects. Metamorphosis from larva to adult is a nail in the metaphorical coffin – how are you supposed to have long-lived immune structure if you undergo a complete replacement of your internal anatomy in order to enter the adult stage of life? ↩︎
- There was a shift from calling them “adipocytes” to “trophocytes” in literature, as adipocytes implies a single function: fat storage, whereas “trophocytes” suggests that it’s a multifunctional cell providing nourishment (troph- being Greek or “nourish” or “feed”, and -cyte meaning cell; nourishing cell). Don’t take my word for it? Here an excerpt: “The trophocytes are also called adipocytes, an improper denomination since these cells are not simple lipids deposits, but have a much complex metabolism and storage and synthesis of proteins and carbohydrates” Roma et al., 2010 ↩︎
- RNAi can be thought of being “adaptive-immunity-like”, as the pathway targets specific viral sequences for destruction, and can persist in the insect body for weeks, just like “immune memory”. This method is exclusive to viral invaders and occurs at the molecular level rather than the cellular. For an overview of the process, see Brutscher & Flenniken, 2015 ↩︎
- It becomes less odd when viewed from a metabolic perspective, as mounting and maintaining an immune response is energetically costly. Diverting energy away from haemocyte production or antimicrobial peptide synthesis is one reason why foragers can handle the extreme demands of foraging. ↩︎
- “Age uncoupling” is a scientific way of stating that bees were forced to move up in the division of labour. In this case, the nurse bees were forced to do foraging because they were placed in an artificial hive.
From the authors: “This colony was composed of ca. 4000 1-day-old worker bees, a frame with honey, pollen and brood, and a naturally mated queen…Because this colony initially contained no old bees, some colony members initiated foraging earlier than usual, enabling us to sample precocious foragers and age-right nurses, all being 6–8 days old… the development of over-aged nurses [i.e. older bees who reverted to nursing] was achieved by replacing the frame of the single-cohort colony, at this moment containing bees near to the emergence, by a new one, containing younger brood. Then, we collected over-aged nurses and age-right foragers, all 21–23 days old.” (Lourenco et al., 2019) ↩︎
May I use some of your material for a PowerPoint presentation to my beekeeper friends?
Certainly can, help yourself