In humans, aspartame is completely hydrolyzed by digestive enzymes into phenylalanine (50%), aspartate (40%), and methanol (10%) [3].

Intact aspartame does not enter the blood, nor the large intestine.

Aspartame therefore is never present in the placenta, amniotic fluid, or breast milk [4].

The authors imply the transplacental passage of aspartame in humans based on a recent study [5] that explicitly stated they did not measure aspartame “due to its rapid degradation . . .

upon ingestion”.

The authors discuss the mechanisms of the metabolites of aspartame implicated in the development of autism spectrum disorder, based primarily on high-dose animal studies.

However, for even the highest human consumers of aspartame, these mechanisms are not biologically plausible.

The contribution of amino acids from aspartame is trivial in comparison to dietary proteins and results in no change in human blood levels nor any change in brain amino acid uptake [3].

Using dietary surveys, when all sources of methanol are considered (including methanol present in or produced during digestion of fruits, vegetables, juices, coffee, alcoholic beverages, and other food additives, plus the endogenous methanol production), the methanol from aspartame in humans ranged from 1 to 10% of total methanol [6].


Another significant limitation of this study is the high risk of reverse causality, where high cardiometabolic risk—encompassing obesity, type 2 diabetes, and cardiovascular disease—prompts the increased intake of low-calorie sweeteners as a risk mitigation strategy.

Reverse causality is well documented in the literature evaluating low-calorie sweeteners [7,8].

The authors failed to account for or adjust for key cardiometabolic risk factors, such as maternal obesity and diabetes, which are established risk factors for autism spectrum disorder.

Although the authors recognize these limitations, such shortcomings substantially undermine the study’s validity.


In summary, the association between diet soda/beverage and aspartame exposure and autism spectrum disorder reported by Fowler et al. is unreliable, not biologically plausible and is confounded by reverse causality.

Caution should be exercised when suggesting that a mothers’ dietary habits play a possible role during pregnancy based on unreliable associations.

Therefore, these findings should be interpreted with significant caution, and we urge for higher standards of quality in nutritional epidemiological studies.


Thank you for the opportunity to respond to the concerns raised by Ayoub-Charette et al. [ 1].

As they disclosed, the senior authors of this Comment have longstanding relationships with industry associations that promote the manufacture and sale of non-nutritive sweeteners (NNS), diet sodas, and other diet products.

Four of the five authors have disclosed conflicts of interest directly related to the promotion and/or manufacture and sale of NNS and products containing them.

The intermingling of industrial and academic priorities in this group is concerning; we therefore appreciate the opportunity to respond to their comments.


Overarching issues:


Before addressing specific concerns raised by the authors of the Comment, we would repeat and emphasize a key statement from the conclusions section of our paper [2]: These associations do not prove causality.

Our data were collected through a case–control study, the limitations of which were previously described in our paper and are reiterated below in response to specific comments by Ayoub-Charette et al. Given these limitations, our results—which are consistent with earlier prospective cohort studies that found increased health risks among offspring gestationally exposed to these substances—raise a warning flag about maternal consumption of these products during pregnancy and breastfeeding but cannot prove causality.


Nonetheless, taking a historical perspective, it should be noted that case–control studies, in general, are often used as a first step in the epidemiologic search for the causes of rare diseases.

Case–control studies, for example, provided early warnings of the link between cigarette smoking and lung cancer before more definitive data on these associations became available through prospective cohort studies.

( Please see White’s 1990 discussion of the relative roles of case–control and prospective cohort studies in identifying cigarette smoking as a major causative agent of lung cancer [3]).


As White described, in studying the association between cigarette smoking and lung cancer, the epidemiologic study designs that were employed progressed from small case–control studies to larger, meticulously designed case–control studies and then on to large-scale prospective cohort studies [3].

Using this historical sequence by way of context, our study would reside within the category of small case–control studies, with the limitations we already described.

In the earlier investigations into cigarette smoking and lung cancer, small case–control studies identified the association between smoking and lung cancer as early as 1939, but it was not until two larger case–control studies replicating these findings were published in 1950 that the associations were taken more seriously.


As White described, associations between cigarette smoking and lung cancer—identified through case–control studies—were next studied through prospective cohort studies, the results of which were critical in moving medical and public health opinion to accept “. .

.the disturbing finding that a pleasant and popular habit was, in the long term, potentially lethal.

Conviction that this was so did not come easily” [3].

Thus, these investigators followed a classic progression: from studying associations using case–control studies to studying them within prospective cohort studies, results from which were less influenced by several limitations of case–control studies.


Beyond prospective cohort studies, the ideal, gold-standard epidemiologic study design for the evaluation of the relationship between an exposure and an outcome is a clinical intervention, the classic example of which is a randomized controlled trial (RCT).

In the classic RCT, the exposure is hypothesized to be a beneficial one, which can prevent or reduce the health burden from the condition under study.

There are two main arguments against the use of an RCT in evaluating the associations reported from our study, however: the first is ethical, and the second is logistical.


In the first category, based on results from both animal studies and case–control and cohort studies in humans, it is our assessment that it would be unethical to conduct clinical interventions in which soon-to-be pregnant women were randomized to a study arm in which they were instructed to consume diet beverages, NNS in general, or aspartame specifically, daily, in dosages comparable to those found in our study.

Indeed, based upon results from earlier studies in children, Sylvetsky et al. recently wrote, “We propose that the FDA restrict NSSs [non-sugar-sweeteners] in products marketed to children until definitive evidence of benefit and lack of harm is available” [4].

We agree with the concerns expressed by these investigators and feel that these concerns are equally valid regarding exposing an embryo or fetus in utero to these products and their metabolites.


As to the second issue—logistical difficulties—it would be enormously challenging and expensive to conduct an RCT to study these associations, and also, by our calculations, it might take up to seven years or more to conclude such a study and finish data analyses.

Thus, for both reasons, conducting an RCT to study this question would be problematic.


This, therefore, raises the question of which next steps could be pursued to provide more definitive answers to these questions more rapidly.

Because data are already available from large-scale prospective cohort studies, we would recommend analyses of these data, with adjustment for additional potential confounders, to further explore these associations without the limitations of our own study.

Meta-analyses of results from multiple cohort studies could be used to further evaluate these associations.

Meanwhile, analyses of existing data from individual prospective cohort studies would be an important next step in evaluating the associations that emerged from our study.


Response to specific concerns raised by Ayoub-Charette et al.:


With regard to the limitations in our study data noted by Ayoub-Charette et al., it is worth restating two that we had previously described in our paper.

First, we could not include maternal body mass index, diabetes status, and other maternal cardiometabolic risk factors as covariates in our analyses and thus cannot exclude the possibility that our results may have been confounded by reverse causality: maternal overweight/obesity and diabetes themselves increase the risk of autism and could increase a woman’s motivation to use diet products.

In our sensitivity analyses, the daily consumption of non-nutritive sweeteners was no higher within key sociodemographic strata previously associated with an increased risk of overweight/obesity and diabetes, but adjustments for individual cardiometabolic risk factors will be critical for future analyses of prospectively collected data.

Second, all data were recorded retrospectively, years after the diagnosis of autism or autism spectrum disorder (ASD).

Future analyses of prospectively collected dietary data are warranted to evaluate these associations without these limitations.


We thank Ayoub-Charette et al. for the opportunity to affirm that aspartame is indeed rapidly metabolized within the small intestine into aspartic acid (40%), phenylalanine (50%), and methanol (10%) [5], which, in turn, is converted into its highly reactive first-phase metabolite, formaldehyde, a known neurotoxin [6].

Formaldehyde can be further metabolized into formic acid.

For this reason, unlike other NNS detected in human amniotic fluid [7], cord blood [7], and breast milk [8], aspartame itself is not detectable in these fluids.

Instead, within and beyond the small intestine, it is the aspartame metabolites and the molecules, bacteria, and tissues with which they interact that would affect the cecal and gut microbiota.

Along with aspartame metabolites themselves, other metabolites produced by the gut microbiota [9–11] also enter the bloodstream and, thence, the tissues, organs, and fluids beyond.


Ayoub-Charette et al. expressed concerns about the lack of plausible mechanisms at biologically relevant exposure levels underlying our findings.

From multiple studies in both animals and humans, however, plausible biological mechanisms for our findings emerged from two overarching patterns in aspartame-fed animals and their offspring: adverse impacts on intracellular metabolic processes and adverse impacts on the gut microbiota.

Because of the questions raised by Ayoub-Charette et al., these two categories of mechanisms are described below.

Unless otherwise noted, references were provided in our paper [2].


1. Disruption of One-Carbon Metabolism (OCM); Lowered Availability of Reduced Glutathione (Glutamine Sulfhydryl: GSH); Decreased Antioxidant, Detoxification, and Methylation Capacity; and Increased Oxidative Stress in Aspartame-Fed Animals


A pre-eminent impact of aspartame consumption in animals was a decreased availability of GSH, a major antioxidant critical for a wide range of functions within neurons and throughout the body [2].

GSH protects cells from oxidative stress, provides detoxification protection from both endogenous and exogenous toxins, and supports methylation processes [2].

GSH availability is maintained by one-carbon metabolism (OCM), three interrelated metabolic pathways that support the supply of multiple molecules necessary for defense against toxins and oxidative stress, methylation, and other critical cellular processes [12].

Animals fed aspartame at dosages within normal human consumption ranges [13] demonstrated the following: strikingly decreased levels of GSH, with significantly reduced antioxidant and detoxification capacities [2,13–16]; significantly increased oxidative stress, including elevated levels of lipid peroxidation and mitochondrial dysfunction [2]; and disruptions of processes within OCM [15], which could explain the GSH depletion and each of the findings above.


These findings are strongly congruent with metabolic patterns observed among individuals with autism [12]: decreased GSH availability and total antioxidant capacity; reduced detoxification capacity; and increased oxidative stress, including increased mitochondrial dysfunction [17–20].

Autism typically emerges through the overlaying of toxic or other adverse exposures upon individual genetic susceptibility – or, as Heilbrun et al. wrote, “‘dose plus host’ determines toxicity” [21].

Through its deleterious impacts on OCM—thereby simultaneously reducing antioxidant and detoxification capacities while increasing toxicant load—aspartame could contribute to the perfect storm which underlies the etiology of autism.

Importantly, it could leave exposed individuals more vulnerable to environmental toxins known to increase the risk of autism.


Ayoub-Charette et al. stated, however, that, at normal consumption dosages, the metabolites of aspartame would not enter the maternal gut or bloodstream in sufficient concentrations to cause harm to gestationally exposed offspring.

To examine this question, it is useful to consider the results from several early studies in both animals and humans.

These evaluated the impact of aspartame consumption on circulating levels of two of its metabolites—methanol and phenylalanine—using a dose of 34 mg of aspartame/kg body weight (mg/kg), a dosage representing the upper limit (99th percentile) of normal consumption in the general population.

For comparison, U.S. and European acceptable daily intake (ADI) levels are 40 and 50 mg/kg, respectively.


When this dosage was fed in a single serving to one-year-old infants [22] and to adults in two studies [23,24], their plasma phenylalanine concentrations increased within 30 to 45 min by 90% [22], 54% [23], and 96% [24], respectively, and remained significantly elevated until 2 h after the dose [24].

Increases in serum methanol concentrations were even more dramatic.

In rats [25] and adult humans [23] fed the same dosage, serum methanol concentrations increased by over 130% [25] and by 170% [23], respectively, within 30 to 60 min and remained significantly elevated for three hours [25].


In another study, adult males were fed a lower dose of aspartame: 500 mg total, the FDA’s estimated average daily intake of aspartame in humans—equivalent to about 2.8 cans of diet cola—which represented a dosage of approximately 6 to 8.7 mg/kg in the male volunteers in this study [25].

Following this lower dose of aspartame, serum methanol concentrations rose more than 50% above basal levels within 45 min and remained significantly elevated three hours following the dosage [25].

These dosages, given in a single serving, would result in higher individual spikes of circulating phenylalanine and methanol than the average consumer would experience following each of multiple servings throughout the day.


Nonetheless, these results suggest a greater impact of aspartame consumption on circulating levels of methanol than the 1% to 10% range reported by Ayoub-Charette et al. In addition, the magnitude of each temporary elevation of circulating metabolites might provide a better indicator of their potential metabolic impacts, compared with the mean overall percentage of their contribution, averaged over 24 h. (Please note variations in phenylalanine concentrations reported by Stegink et al. in 1988, for example [26]).


A different approach to evaluating the impact of aspartame consumption, however, is to examine its actual impact on tissues.

Our paper already described the adverse metabolic impacts of aspartame consumption in a number of studies [2].

In addition, however, a 2023 study [27] published after our paper reported that pregnant mice treated with aspartame by oral gavage at doses as low as 3.5 mg/kg (comparable to 1.4 cans of diet cola consumed by a 160 lb woman) experienced impaired placental growth and function, with significantly reduced placental and fetal weight [27].

The investigators also demonstrated a significantly increased production of reactive oxygen species, downregulation of expression of the antioxidant MnSOD, increased intracellular oxidative stress, and diminished cellular proliferation in placental trophoblasts exposed to phenylalanine in vitro in concentrations comparable to 10 mg/kg of aspartame [27].

Thus, using biologically relevant doses, this recent study further demonstrated adverse metabolic impacts from aspartame consumption or exposure – in this case, on the placenta itself [2,13].


As Choudhary and Pretorius wrote, “The existing animal studies and the limited human studies suggest that aspartame and its metabolites, whether consumed in quantities significantly higher than the recommended safe dosage or within recommended safe levels, may disrupt the oxidant/antioxidant balance, induce oxidative stress, and damage cell membrane integrity, potentially affecting a variety of cells and tissues and causing a deregulation of cellular function, ultimately leading to systemic inflammation” [13].


2. Adverse Impacts on the Gut Microbiota


As many as 90% of children with ASD have been estimated to experience gastrointestinal disorders [28], which may exacerbate other symptoms of autism [29].

Animal studies demonstrated the following impacts of aspartame on the gut microbiota, including several which were found in individuals with autism [30]: increases in total bacteria and the abundance of Enterobacteriaceae and Clostridium leptum [10], significantly decreased lactose fermentation and increased abundance of Clostridium cluster IV [9], and reduced diversity within the microbiota [31].

Disturbances in biodiversity within the microbiome have been reported to cause increased permeability of the gut [28], which can increase inflammation systemically, including within the central nervous system [28].

Aspartame and two other leading NNSs—sucralose and saccharin— have also increased oxidative stress and apoptosis in intestinal epithelial cells and increased epithelial barrier permeability [32], which would further exacerbate inflammation systemically.

Inflammation itself has been reported to increase autism risk [33].


Investigators have also observed alterations in the production of gut metabolites, including increased production of the cecal short-chain fatty acids (SCFA) propionate, butyrate, and isobutyrate in aspartame-fed Sprague–Dawley dams, as well as changes in the cecal microbiome of their offspring, resulting in an increased abundance of propionate-producing strains in the offspring gut microbiome [34].

Aspartame-fed rat dams exhibited a doubling of serum concentrations of propionate [10], which the investigators hypothesized resulted from increased production of propionate by colon microbiota [10].

Propionate and other SCFAs have been hypothesized to trigger ASD [35].

As we noted earlier [2], elevated propionate concentrations have been associated with increased permeability of both the gut and the blood–brain barrier [35,36].

This combination would allow increased access of toxins from the gut into the bloodstream and, thence, into the brain, as well as throughout the body.

Not surprisingly, therefore, elevated serum propionate levels have been associated with decreased GSH levels and increased oxidative stress, excitotoxicity, and neuroinflammation [28,37,38] and with an increased risk of autism in several studies [10,35–39].


Aspartame-fed animals have also exhibited significantly increased serum concentrations of kynurenine [11], another metabolite produced by the gut microbiota which is elevated in individuals with autism [28,33,35,40].

Elevated kynurenine has been hypothesized to cause increased excitotoxicity and neuroinflammation [33] and to increase the risk of neuroanatomical problems characteristic of ASD, which arise during fetal brain development [41].


Animal studies have demonstrated that maternal aspartame consumption can adversely impact not only the gut microbiota but also weight gain and glucose tolerance in both the dams themselves and their gestationally exposed offspring who did not themselves consume aspartame [9].

Germ-free mice that received fecal microbiota transplants (FMT) from these aspartame-exposed offspring subsequently developed the same cardiometabolic problems the offspring had experienced [9].

That these adverse impacts can be replicated through interspecies FMT from offspring offers strong evidence that maternal aspartame consumption provokes deleterious changes in the offspring’s microbiota.


Aspartame consumption has also had adverse impacts on gut microbiota in humans and their offspring.

Among healthy volunteers fed aspartame for two weeks, significant microbiome composition and functional changes, and heightened glycemic responses were noted in some volunteers—termed ‘responders’—and changes were observed in their plasma metabolome [11].

Germ-free mice that received FMTs from these ‘responders’ developed similarly elevated glycemic responses [11].

In another study, three-month-old human infants whose mothers consumed diet beverages daily during pregnancy exhibited increased concentrations of spermidine and succinate—urine metabolites produced in the gut by the metabolism of putrescine—compared with the offspring of mothers who rarely or never consumed these beverages [42].

Urinary succinate concentrations were associated with increased body mass index in this study [42].

These studies in both animals and humans demonstrate adverse changes in the gut microbiota resulting from consumption of, or gestational exposure to, aspartame and other NNS.


Some patterns found in the gut microbiota and metabolites in aspartame- and NNS-exposed animals are similar to those observed in individuals with ASD.

As noted earlier, an increased abundance of Clostridium species, for example [43,44], and a lower percentage of Akkermansia [29] have been found in individuals with ASD, as well as among aspartame- and NNS-exposed animals.


Because our primary, dichotomized exposure variable was daily early-life exposure to ≥1 diet soda/day, and aspartame, Ace K, and sucralose are the three leading NNS used in diet sodas [45], it should be noted that sucralose [11,46] and Ace K [47] have both also been found in multiple studies to have adverse impacts on the gut microbiota [11,46–48].

One study found that the consumption of low doses of sucralose and Ace K by pregnant and lactating mice resulted in significant changes in the gut microbiota and associated processes in their pups [49].

These included significant increases in Firmicutes and decreases in Akkermansia muciniphila in the gut microbiota, changes in concentrations of metabolites produced by the gut microbiota, and diminished hepatic detoxification processes [49].

The authors noted that similar impacts on the gut microbiota in humans had been associated with increased cardiometabolic risk and stated that “NNS consumption during pregnancy and lactation may adversely affect infant metabolism” [49].


Adverse impacts on the maternal gut microbiota similar to those seen in animal studies would thus result in increased toxicant load systemically, as noted earlier [2], with increased toxicant exposures and oxidative stress for both the mother and the fetus.

Correspondingly, a pro-oxidant environment could contribute to the dysbiosis of the gut microbiota.

In the context of decreased GSH availability and increased vulnerability to oxidative stress and toxins, the overall result would be diminished neuroprotection.

Assertions by Ayoub-Charette et al. that aspartame and its metabolites either do not reach critical organs or reach them in insufficient concentrations to affect either the mother or the child are refuted by the results from these earlier studies.


In our paper, we reported earlier findings of the presence of NNS in 65% of human milk samples [8], 77% of amniotic fluid samples [7], and 100% of cord blood samples studied [7], in order to highlight the extent to which these products are being consumed by pregnant women and to underscore their pervasive presence in the fluids to which both unborn children and infants are exposed.

Even though intact aspartame cannot be measured in these fluids, the detection of the other two leading NNSs used in diet sodas and other diet beverages in all infant cord blood samples tested and in the large majority of amniotic fluid and milk samples underscores the striking degree to which offspring are now being exposed to these products during periods of profound neurodevelopmental vulnerability.

Although aspartame does not persist intact in the body long enough to be detected in maternal blood or in these fluids, animal studies have repeatedly demonstrated that, through its metabolites, aspartame has pervasive influences on the health of both the consuming individual and offspring exposed in early life: either gestationally, through nursing, or both.


Our study is not the first to find associations between gestational/early-life exposures to aspartame or diet beverages and subsequent health risks in human offspring.

Instead, our results follow and are congruent with those from an earlier case–control study [50], which found that maternal dietary methanol intake during pregnancy, calculated mainly from the consumption of aspartame-sweetened beverages and other products, was more than twice as high among the mothers of children with autism compared with the mothers of neurotypically developing children [50].

Our findings are also consistent with reports from large-scale prospective studies in finding significantly increased developmental risks among human offspring exposed in utero to diet sodas and other diet beverages [2].

These earlier cohort studies employed a prospective collection of dietary recall data and included covariates for several maternal cardiometabolic and dietary risk factors in their analyses.

Among offspring gestationally exposed daily to diet sodas or to diet beverages in general, these earlier studies showed a 67% increased risk of early preterm delivery [51], a 57% increase in the risk of infants being large for gestational age at birth [52], an approximate doubling of the risk of being overweight or obese at the age of one year [53] and at the age of seven years [52], and a 30% increased risk of being diagnosed with asthma [54].

We hope that results from future analyses of data from large-scale prospective studies, with adjustments for additional risk factors, will soon become available to shed further light on the question of whether the daily maternal intake of diet sodas and/or aspartame, specifically, is associated with increased autism risk in exposed offspring.

In the meantime, we would reiterate that while our results do not prove causation, they also do not stand alone: they are the latest in a long series of findings from both animal and human studies, suggesting that caution may be advisable for women considering the use of these products during pregnancy and breastfeeding.


This study is also not the first to sound a warning about the potentially increased risk of developmental problems in offspring exposed to diet sodas, other NNS, and/or aspartame specifically.

In 2018, the following warning appeared in a Science Advisory from the American Heart Association, entitled ‘Low-Calorie Sweetened Beverages and Cardiometabolic Health [55]: “On the basis of the available evidence, the writing group concluded that, at this time, it is prudent to advise against prolonged consumption of LCS [low-calorie-sweetened] beverages by children” [55].

In 2021, Laforest-Lapointe et al. wrote, “Our study suggests that infants exposed to ASB [artificially sweetened beverages] through their mothers may be at higher risk of shifts in microbial community structure related to early-life predisposition to metabolic diseases” [42].

In 2020, Palatnik et al. wrote, “Emerging evidence from animal studies warns against NNS consumption [in pregnant women] and present specific effects that harm the metabolism in offspring. .

.As evidence accumulates and with about 30% of pregnant women self-reporting NNS consumption and possibly more with unintentional exposure, it is critical to question the safety of NNS consumption during pregnancy” [56].


Given the combined results from studies in both animals and humans in finding an increased risk of adverse health outcomes among offspring exposed gestationally to diet beverages, NNS, and/or aspartame specifically, we have chosen to follow the precautionary principle and add our voices to those of earlier investigators in encouraging pregnant women to carefully weigh the possible risks to their unborn children when considering the use of these products.


Author Contributions: Conceptualization, S.P.F., L.P.H. and R.F.P.; investigation, R.F.P., L.P.H. and S.P.F.; data curation, L.P.H., R.F.P. and S.P.F.; writing—original draft preparation, S.P.F.; writing—review and editing, R.F.P., D.G.R.d.

P., P.S.G., L.P.H., M.D.S. and S.P.F.; project administration, R.F.P. and L.P.H.; funding acquisition, R.F.P. All authors have read and agreed to the published version of the manuscript.


Conflicts of Interest: The authors declare no conflict of interest.


Venomous snakebites are common in South and Southeast Asia, Africa, as well as in Central and South America [1].

The significant morbidity and mortality resulting from these injuries are mostly in rural areas that are poorly equipped to handle such medical emergencies [1-4].

Snakebite envenomation (SBE) causes around 150,000 deaths and 500,000 disabilities worldwide every year [1].


In India alone, around 58,000 deaths occur annually and most of these are attributed to the "Big Four" snake species which include Toxins 2022, 14, 805.

https://doi.org/10.3390/toxins14110805 https://www.mdpi.com/journal/toxins Russell's viper (Daboia russelii), Indian cobra (Naja naja), saw-scaled viper (Echis carinatus), and common krait (Bungarus caeruleus) [5].


SBE victims are often agricultural workers who accidentally encounter snakes while performing their regular activities.

The victims mostly lack basic protection such as suitable footwear and gloves which might prevent bites and envenomation [3,6].

The envenomation effects depend on several factors but the species responsible and the amount of venom injected are the key determinants.

The most common envenomation effects are neurotoxic, myotoxic, cytotoxic, nephrotoxic, and coagulopathic complications [7].


However, SBE may cause several rare complications such as priapism [8], spleen rupture [9], Wunderlich syndrome [10], pseudoaneurysm [11], and sialolithiasis [12].


Optic neuritis is a frequently observed clinical condition that occurs due to the inflam- mation of optic nerves [13].

This condition is mostly associated with multiple sclerosis; however, there are several factors such as autoimmune disorders and infections which can also induce optic neuritis [13].

The delay in diagnosing and treating this condition may result in permanent loss of vision.


SBE-induced ocular complications are frequently reported, although their clinical pathology, underlying molecular mechanisms, appropriate diagnosis, and treatment methods are not well understood [14].

While most of the reported ocular complications are caused by venoms sprayed directly into the eyes from spitting cobras, the non-spitting snakes (including vipers) are known to induce ocular complications following injection of venom in other body parts such as lower and upper limbs [14].

For example, central retinal artery occlusion (CRAO), acute angle-closure glaucoma (AACG), cataract, corneal oedema, macular infarction, and subconjunctival haemorrhage have been rarely reported in victims following envenomation by vipers or other non-spitting snake species [14-17].


To the best of our knowledge, common krait-induced optic neuritis has not been reported previously.

Hence, we report the clinical presentation, diagnosis, and treat- ment of optic neuritis that developed in a patient following envenomation by a common krait and compare their features with the one developed in a victim following a cobra bite.

Despite antivenom treatment, the ocular manifestations developed in both patients several days after the envenomation.

This article provides awareness and approaches for the diagnosis and treatment of optic neuritis induced by common krait envenomation (similar to cobra bites) which will be beneficial for SBE management, especially in rural areas.


Patient 1, a healthy 32-year-old female homemaker, was bitten by an Indian cobra on her right foot.

She developed bilateral ptosis and respiratory depression within 30 min following the bite.

After evaluation in a local hospital, she was treated with an intravenous infusion of 10 vials (100 mL) of polyvalent antivenom raised against the Indian 'Big Four' snakes as well as neostigmine and atropine.

Although she improved, on the fifth day after the bite, she complained of decreased vision in both eyes and therefore was referred to the emergency department of our hospital.


The patient arrived at the emergency department without any neurological deficits.

However, an ophthalmic examination of both eyes revealed sluggish pupillary reactions to light, papilledema (Figure 1A), and the best- corrected vision was finger counting close to the face in both eyes.

Anterior slit lamp examination, intraocular pressure and extraocular motility were all normal.

Based on these findings, benign intracranial hypertension was suspected, and therefore, brain computed tomography (CT) imaging with contrast was performed but it showed normal results.

Lumbar puncture showed normal opening pressure of cerebrospinal fluid (CSF).

Visually evoked responses showed increased latency and decreased amplitude indicative of bilateral optic neuritis.


Finally, an MRI of the brain revealed effacement of the perioptic-optic CSF space and a diffuse increase in T2 signal involving both optic nerves on T2-weighted images (Figure 1B).

MRI also revealed a hyperintense thickening of the perioptic nerve sheath surrounded by CSF giving a 'polo mint' appearance on fat-suppressed T2-weighted images (Figure 1C) confirming the diagnosis of optic neuritis.

Based on the clinical features, the patient received three doses of intravenous methylprednisolone (1 g of methylprednisolone over 40 min for each dose) over three days to treat optic neuritis.

Following this treatment, the pain subsided rapidly, and the patient was able to see shapes on the following day and her symptoms gradually returned to the baseline vision at 45 days.

The patient was monitored regularly for up to six months and no other residual effects were observed in their vision or eyes.

22, 14, x FOR PEER REVIEW 3 of 9 sheath surrounded by CSF giving a 'polo mint' appearance on fat-suppressed T2- weighted images (Figure 1C) confirming the diagnosis of optic neuritis.

Based on the clin- ical features, the patient received three doses of intravenous methylprednisolone (1 g of methylprednisolone over 40 min for each dose) over three days to treat optic neuritis.

Fol- lowing this treatment, the pain subsided rapidly, and the patient was able to see shapes on the following day and her symptoms gradually returned to the baseline vision at 45 days.


Patient 2, a 30-year-old male farmer, was bitten by a common krait while working on his farmland.

He was transported to the emergency department within one hour follow- ing the bite.

He was unconscious and in respiratory distress upon admission.

He was im- mediately intubated and started on mechanical ventilation.

He was intravenously admin- istered 20 vials (200 mL) of polyvalent antivenom (Bharat Serum and Vaccines Limited, Mumbai, India) raised against the Indian 'Big Four' snakes.

His sensorium, muscle


He was transported to the emergency department within one hour following the bite.

He was immediately intubated and started on mechanical ventilation.

He was intravenously administered 20 vials (200 mL) of polyvalent antivenom (Bharat Serum and Vaccines Limited, Mumbai, India) raised against the Indian 'Big Four' snakes.

His sensorium, muscle strength and oxygen saturation improved and were extubated two days after admission/bite.


On the fourth day after the bite, he complained of blurred vision and diminished visual acuity similar to patient 1.

So, he underwent a thorough neurological examination.

His motor and sensory system examinations were normal without any signs of cerebellar dysfunction or meningeal irritation.

His ophthalmic examination was notable for a visual acuity that was limited to the perception of light and round pupils but sluggishly reactive to light.

His intraocular pressure was within normal limits and ocular motility and anterior segments were unremarkable.

Fundoscopic examination revealed bilateral papilledema.

The visual evoked potentials were indicative of maculopathy and bilateral axonopathic optic neuropathy consistent with optic neuritis.

MRI of the brain revealed high-intensity changes in both optic nerves on T2-weighted images (Figure 2A) and enlarged optic nerves surrounded by CSF giving a 'polo mint' appearance on fat- suppressed T2-weighted images (Figure 2B) supporting the diagnosis of optic neuritis.


Like patient 1, this patient also received three doses of intravenous methylprednisolone over three days.

Following this treatment, the pain reduced rapidly, and the patient was able to perceive hand movements from the second day of treatment.

His vision gradually improved to bilateral visual acuity of 6/60 on the sixth day and 6/6 in both eyes within 30 days.

Regular monitoring for up to six months did not reveal any further residual effects in their vision or eyes.


strength and oxygen saturation improved and were extubated two days after admis- sion/bite.


His intraocular pressure was within normal limits and ocular motility and ante- rior segments were unremarkable.


MRI of the brain revealed high-intensity changes in both optic nerves on T2-weighted images (Figure 2A) and en- larged optic nerves surrounded by CSF giving a 'polo mint' appearance on fat-suppressed T2-weighted images (Figure 2B) supporting the diagnosis of optic neuritis.


Anatomically, greater than one million axons of each optic nerve connect the rods and cones of each eye to the optic chiasm in the brain [18].

The optic disc serves to connect the axons of the retinal ganglion cells with the optic nerve.

The optic nerves are considered an extension of the central nervous system and are divided into four segments which include the intraocular, intraorbital, intracanalicular, and intracranial [18].

The retina and choroid are rich in blood vessels, but the cornea and lens lack vasculature.

Optic neuritis commonly refers to a demyelinating condition and is often described in patients with multiple sclerosis [19].

However, this term is also used more broadly to describe other optic neuropathic complications which result from inflammation of the optic nerves [13].

Autoimmune disorders such as systemic lupus erythematosus and Sjogren's disease, microbial infections, and other local or systemic inflammatory processes are known to induce optic neuritis.

The early symptoms of optic neuritis include blurred or diminished vision, but the delay in seeking appropriate treatment may result in permanent loss of vision.


Although SBE has been known to induce a plethora of local and systemic complica- tions, ocular manifestations are rarely encountered following envenomations, specifically by non-spitting snake species.

SBE-induced ocular complications include subconjunctival haemorrhage, AACG, CRAO, acute anterior uveitis, external ophthalmoplegia, exuda- tive retinal detachment, photophobia, macular infarction, and optic neuritis [14].

Several species of spitting cobras including Naja atra, Naja nigricollis, Naja pallida, and Hemachatus haemachatus from various geographical locations are known to induce ocular complications including optic neuritis.

In many cases, optic neuritis developed a few or several days after envenomation.

In some patients, simple water irrigation of the eyes was used to manage ocular damage, while in others, a range of treatment methods was used [14].

While spitting cobras directly spray their venom into the eyes and cause ocular complications, the non-spitting snake species induce complications through the injection of venom in other body parts that may induce direct or indirect effects on the eyes based on their pharmacokinetic behaviour [20].

The impacts of various toxins present in elapid and viper venoms on the ocular system vary based on their molecular mechanisms.

Therefore, it is important to thoroughly investigate the ocular complications induced by SBE specifically from non-spitting species to underpin their clinical pathology and develop appropriate diagnostic and therapeutic strategies.


In this study, we report a unique case of optic neuritis that developed in a patient following envenomation from a common krait and compare their clinical features with optic neuritis developed in another patient who was bitten by a cobra, both non-spitting elapid species.

In both cases, optic neuritis developed a few days after the bite despite antivenom treatment.

The subjective complaints of blurred vision in both scenarios prompted thorough neurological and ophthalmological examinations followed by CT and MRI scans which revealed the development of optic neuritis.

Despite envenomations by two different snakes in these patients, the clinical features developed in both were very similar.

Moreover, the use of MRI was beneficial in diagnosing optic neuritis and identifying the unique 'polo-mint' appearance of enlarged optic nerves in both patients.


The envenomations from non-spitting cobras, including the Indian cobra, were previously reported to induce optic neuritis in a few cases.

For example, an Indian cobra bite in a 50-year-old male caused diminished visual acuity on the sixth day after SBE and improved over the next few days [21].

This patient displayed sluggish pupils and diminished visually evoked responses although their fundus was normal.

The patient was treated with an intravenous infusion of dexamethasone for three days and a tapered oral prednisolone regimen over three weeks with gradual improvement in his vision.

Similarly, another case of Indian cobra bite developed optic neuritis six days after the bite despite receiving the antivenom treatment [22].

This patient was treated with intravenous methylprednisolone for seven days followed by a prednisolone taper regimen, which improved their condition.

However, a case of optic neuritis developed in a 7-year-old child following suspected envenomation by a Cape cobra resulting in a permanent disability [23].

While there are limited reports available for optic neuritis following non-spitting cobra bites, we were unable to find any reports of optic neuritis following krait envenomation, although they predominately induce neuromuscular effects [24].

Hence, this study reports the first case of optic neuritis developed following krait envenomation, and its clinical features are comparable to cobra- induced optic neuritis.


In addition to elapid snake species, some vipers such as Crotalus atrox (which is known to spit venom in some cases) [25,26], Daboia russelii [27], and Macrovipera lebetinus [28] as well as specific colubrid snakes were reported to induce optic neuritis [14].

In a Russell's viper bite victim, bilateral blindness developed within 30 min following the bite and improved upon treatment with methylprednisolone [27].

In contrast, Macrovipera lebetinus induced blurred vision and optic neuritis in a patient six days after the bite [28].

In two cases of viper bites, the venom injection occurred directly in the eyes of the patients resulting in severe necrosis and subconjunctival haemorrhage [29,30].

While in some cases, ophthalmological examinations revealed optic neuritis, in others, CT and MRI scans were required to ascertain the development of this condition.

As used in this study, we strongly recommend the use of these sophisticated methods to ascertain the precise nature of damage induced by optic neuritis.

Moreover, most of the reported optic neuritis cases were treated with intravenous and/or oral administration of corticosteroid drugs.

In patients with mild ocular effects, water irrigation, topical prednisolone, and/or specific antibiotics were used to successfully manage this condition.


There are several potential causes reported for the development of optic neuritis following SBE [14].

For example, haemorrhage (including those distant from the ocular structures), allergic reactions to antivenoms, and capillary or other vascular injuries are likely to induce inflammation and subsequent ocular damage.

Snake venom toxins are well known to induce systemic inflammatory responses with leucocytosis and release various inflammatory mediators such as IL-4, IL-6, IL-8, and TNFα [31].

Therefore, this may play a critical role in the development of optic neuritis along with vascular damage and haemorrhage.


Venom metalloproteases induce the degradation of extracellular matrix and basement membrane in capillaries which subsequently results in haemorrhage [32].

They are known to induce systemic haemorrhage in addition to their effects on local tissues around the bite site.

Hence, their effects on the ocular system, which is rich in blood capil- laries (retina and choroid), are largely attributed to ocular haemorrhage and subsequent secondary inflammatory responses [14].

In addition, phospholipase A 2 (PLA 2 ) has been shown to induce anti-coagulant effects resulting in haemorrhage.

Venom metalloproteases, PLA 2 and L-amino acid oxidases can induce inflammatory responses [31].

Therefore, these activities may collectively induce ocular complications including optic neuritis.


Cytotoxic three-finger toxins, often abundant in elapid venoms, can damage cell membranes [33] and may affect the integrity of the optic nerve.

Similarly, PLA 2 present in both elapid and viper venoms [34] are likely to directly affect the nerve membranes in the ocular system in addition to their wide range of neurotoxic effects.

The membrane damage may lead to inflammatory responses which in turn affect the optic nerve.

However, the optic nerve contains both myelinated sections and unmyelinated areas within the eye that fuse with the retinal cells.

Hence, further research is required to underpin exactly how different venom toxins might directly interact with these types of neurons and lead to inflammatory responses resulting in optic neuritis.


Neurotoxins present in elapid venoms can induce ocular muscle paralysis.

The movement of four of the six eye muscles (ocular muscles) is controlled by the oculomotor nerve (also known as Cranial Nerve III).

We note that both patients displayed attenuated pupil reaction to light which could be explained through the involvement of the sensory nerve (optic), or the motor response mediated by the motor nerve (oculomotor).

There is much debate about the structure of the human ocular nerve with some suggesting that it is unmyelinated, whereas others show that the degree of myelination is low and uneven during early years but thereafter increases significantly with age [35].

Cadaver tissue profiling of the oculomotor nerve from people of the approximate age of our two patients has a clear sign of myelination.

Hence, the abnormal reaction to light could in part be attributed to the demyelination of the oculomotor nerve as well as the optic nerve.


Generally, SBE-induced posterior segment complications are attributed to direct actions of venom toxins mostly on blood capillaries, while anterior segment com- plications may arise because of secondary inflammation.

While vascular complications are likely to occur following envenomation by vipers, due to the low abundance of pro- teolytic enzymes in elapid venoms, they are unlikely to induce such complications with high severity.

Therefore, the underlying mechanisms in the development of optic neuritis following envenomations by krait and cobras should be further scrutinized to underpin the molecular relationship between their venom toxins and components of the ocular system.


It is apparent that envenoming by any venomous snake species including elapids, vipers, and colubrids can induce optic neuritis.

Hence, this condition, as well as other ocular manifestations, are not only attributed to typical envenomations of spitting cobras.

The clinicians who treat SBE should be aware of this complication arising from non-spitting snake species specifically several days after the bite despite antivenom administration.

Fundoscopic examination as well as CT and/or MRI scans together with thorough ophthal- mological and neurological examinations can be used to identify optic neuritis.

As reported in most cases, intravenous administration of corticosteroids may be effective in treating this inflammatory condition.

Although optic neuritis upon envenomation by non-spitting cobras was reported previously, the development of this condition in a patient with krait envenomation emphasises the need for further research in this area.

Moreover, the molecu- lar interactions of various venom toxins with the optic nerves and other components of the eyes should be studied in detail to establish the underlying molecular mechanisms.

Finally, the impact of antivenom components in the development of this condition should be investigated as this may be another potential cause which could be resolved through further refinements to antivenoms.


Both patients provided written informed consent to collect and publish the data presented in this article.

The patients and their relatives were informed about the purpose of this study and its wider use in scientific and clinical communities to improve the clinical management of SBE.

The identity of the offending snake species was confirmed by an experienced herpetologist by examining the dead specimens brought by the patients.

The patients were diagnosed and treated as normal using standard protocols from the World Health Organisation and local health authorities.

They received polyvalent antivenom raised against the Indian 'Big Four' snakes from Bharat Serum and Vaccines Limited, India.

The diagnosis of optic neuritis was performed using standard ophthalmic and neurological examinations as well as using CT and MRI scanning equipment available within the hospital.


The authors declare no conflict of interest.


It is with interest that I read the case report by Senthilkumaran et al. [ 1], in which the authors described two cases of severe optic neuropathy following envenomation by an Indian cobra (Naja naja) and a common krait (Bungarus caeruleus).

Both patients were treated with antivenom due to systemic envenomation, but developed bilateral optic neuropathy four and five days after the bite regardless.

There was severe visual loss and papilledema with hemorrhages; the diagnosis of optic neuropathy (or optic neuritis) was established using MRI.

Both patients regained full visual acuity after a high dose of intravenous methylprednisolone.

The authors present various possible mechanisms for the cause, including “allergic reactions to antivenoms” amongst other causes.

I would like to take the opportunity to present a few arguments from other studies that suggest antivenom as a cause for the reported symptoms.


In our review of the online available literature [2], ten other case reports of optic neuropathies after a snakebite can be found [3–12].

Nine describe a similar syndrome of severe bilateral vision loss and papilledema with hemorrhages, several days (mostly six) after initial envenomation.


Symptoms of neurotoxicity after snakebite envenomation usually develop more quickly (several minutes up to nineteen hours [13]).

A conflicting temporal association between the bite and the onset of ocular symptoms would make a direct effect of the venom less likely.

Various snakes are described in the literature; three patients were envenomated by elapids [7,8,11] and four by vipers [3,4,9,10].

While elapid venom is mostly neurotoxic, viper venom is predominantly hemotoxic.

Due to species-specific differences in venoms, venom as a common cause for optic neuropathy is therefore questionable.


Antivenom, however, is a common cause; the reported patients received intravenous antivenom as part of the initial treatment, before the development of ocular symptoms [3–11].

Interestingly, in one of the reported cases, the offending snake was discovered to be non-venomous after the antivenom had already been administered [5].

This patient, too, developed visual deterioration with papilledema six days after antivenom was given.

The same syndrome has even been described in a case of scorpion envenomation, in which a (similar) antivenom was given to a patient without signs of systemic involvement [14].

She developed bilateral optic neuropathy after five days as well.


Though there are a few cases of optic neuropathy after a snakebite, antivenom could be one of the probable causes.

However, it is hard to draw definitive conclusions, and other causes should not be overlooked.

Inflammation caused by a snakebite, triggering an aberrant immune response, could be another cause explaining the above cases.

Since antivenom is given in a majority of venomous snakebites, this could be a probable cause of bias in this regard.

Also, in most reports, there is no clear description of the used antivenom.

Authors are encouraged to specify the type (e.g., serum immunoglobulins or immunoglobulin fragments), dose, and producer of antivenom in future reports.

This information can contribute to the development of a new generation of antivenoms.


We thank the author for showing interest in our article [1] and proposing antivenom as a potential cause for the snakebite-induced bilateral optic neuritis that we reported in two snakebite victims [2].

Our primary aim of this article was to report the “polo mint” appearance that we observed using MRI in those patients and to emphasise the necessity for early diagnosis using sophisticated techniques such as MRI and treatment with corticosteroids to promptly tackle this condition.

Moreover, to the best of our knowledge, krait bite-induced optic neuritis has not been reported previously, and thus, we reported the first case of krait bite developing this condition.

Through this article, we also wanted to caution clinicians that they should continue to monitor patients for any delayed complications such as optic neuritis including in krait envenomation.

They should consider using techniques such as MRI to identify the precise damage and use corticosteroids promptly to manage this condition.


This condition can be caused by various issues including bacterial and viral infections [3,4].

Optic neuritis and similar manifestations have been previously reported in some viper [5–7] and elapid [8] snakebite victims.

However, the underlying molecular mechanisms that induce optic neuritis in snakebite victims were not completely understood.

We highlighted a list of potential causes for snakebite-induced optic neuritis in our article including snakebite-induced haemorrhage and inflammation, venom, and antivenom.

As the precise mechanisms for optic neuritis following snakebite envenomation are largely unknown, further research is essential to establish the molecular relationships between snakebites/venoms and optic neuritis.

Hence, we agree with the author that antivenom could be a potential cause along with several other factors for optic neuritis following envenoming.

Further scientific research could establish the causes and underlying mechanisms for developing optic neuritis in snakebite victims.


The role of epigenetic mechanisms in the regulation and evolution of behavior is becoming increasingly recognized.

Changes in the epigenetic status of DNA provide mecha- nisms for the control of gene expression, with processes such as DNA methylation being associated with the up- regulation or downregulation of genes that influence be- havior.

Some of the clearest examples to date come from the growing body of evidence that epigenetic state is asso- ciated with differences in behavior among members of so- cial insect colonies (e.g., Kucharski et al. 2008; Lyko et al. 2010; Smith et al. 2012; Amarasinghe et al. 2014).

How- ever, epigenetic processes may also be associated with ge- nomic imprinting (Haig 2002; Patten et al. 2014).

Genomic imprinting is the epigenetic phenomenon by which alleles in offspring vary in their pattern of expression as a result of the parent from which those alleles are inherited.

This means that a maternally inherited allele (inherited from the mother and called a matrigene by Patten et al. 2014) has a different pattern of expression from a paternally in- herited allele (inherited from the father; a patrigene), lead- ing to parent-of-origin specific gene expression (POGE).


Much of the interest in terms of the evolution of geno- mic imprinting has revolved around intragenomic conflict, whereby different actors-such as males and females or parents and offspring-are under opposing forces of selec- tion to control gene expression in focal individuals, which might include themselves (e.g., Spencer 2000; Haig 2002; Úbeda and Haig 2004; Rice 2013; Holman and Kokko 2014; Patten et al. 2014).

Under the kinship theory of ge- nomic imprinting, the conflicts arise due to differences among individuals in their patterns of relatedness to each other; for instance, females are always related to all of their offspring, but males may be related to only some of the off- spring of a female he has mated with.

This means that males would be favored by selection if they could manip- ulate their offspring to obtain as many resources as possi- ble from their mother.

This can bring maternally and pa- ternally inherited alleles within an individual into conflict, for instance, over maternal investment, and POGE is ex- pected to evolve (Haig 2002).

Epigenetic mechanisms then provide the opportunity to influence the expression of par- ticular alleles.

DNA methylation is a key molecular mecha- nism of imprinting, whereby differing "imprints" are laid down in eggs and sperm, and the subsequent inheritance of these marks leads to differential gene expression at im- printed loci in the offspring (Reik and Walter 2001).


Here, we investigate the role of DNA methylation in facultative sex allocation behavior in the parasitoid wasp Nasonia vitripennis and what this role could mean for * Corresponding author; e-mail: nc52@st-andrews.ac.uk.

All use subject to JSTOR Terms and Conditions intragenomic conflict over sex allocation.


Female Nasonia allocate sex broadly in line with Hamilton's theory of local mate competition (LMC; Hamilton 1967, 1979; Burton- Chellew et al. 2008; West 2009).

As with all Hymenoptera, N. vitripennis is haplodiploid, with males developing from unfertilized (haploid) eggs, and females developing from fertilized (diploid) eggs.

This means that females are in putative control of sex allocation, either releasing sperm stored in their spermatheca to fertilize an egg prior to ovi- position, hence producing a daughter, or not releasing sperm and instead laying an unfertilized egg that will develop into a son.


Females laying eggs alone on their blowfly pupae hosts produce very female-biased sex ratios (considered through- out as proportion male).

As mating takes place when adults emerge from the remains of the host (males are brachypter- ous in this species, with adult females dispersing away after mating), this female bias reduces competition among sons for mates and maximizes the number of mates for those sons (Taylor 1981).

However, females produce less-biased, or even male-biased, sex ratios, depending on the number of other females (foundresses) ovipositing together on a host and the relative clutch sizes produced by those females (Hamilton 1967; Werren 1980; West 2009).


While we have a detailed understanding of the information used by fe- male Nasonia during sex allocation (e.g., Werren 1980, 1983; King et al. 1995; Shuker and West 2004; Shuker et al. 2005, 2006a; Burton-Chellew et al. 2008), we have only a rudimentary understanding of the genetic basis of sex al- location (Pannebakker et al. 2008, 2011, 2013).


Recent theory has shown that maternally and paternally inherited alleles have different patterns of optimal sex al- location under LMC (Wild and West 2009).

In haplo- diploids such as Nasonia, maternally inherited alleles in a female are more closely related to female offspring than paternally inherited alleles.

This means that maternally inherited alleles are selected to produce more female- biased sex ratios than their paternally inherited counter- parts (fig. 1A), although the predicted differences in spe- cies with female control of sex allocation (as in Nasonia; but see Shuker et al. 2006b) are small.

Crucially, parent-of- origin information must be associated with each allele, for instance, via epigenetic marks (see "Discussion" for further information).


Recent work has confirmed genome-wide DNA meth- ylation in Nasonia, with approximately 30% of genes hav- ing methylated CpG sequences, mostly in exons, with methylation typically associated with increased gene ex- pression (Wang et al. 2013; see also Beeler et al. 2014).

Here we use the demethylating agent 5-aza-2 0 -deoxycytidine (5-aza-dC) to manipulate the genome-wide DNA methyla- tion status of female N. vitripennis.

There are a number of possible outcomes of this manipulation.

First, the sex allo- cation machinery, from the perception of LMC cues (e.g., Shuker and West 2004) to the control of fertilization, could be independent of any influence of DNA methyl- ation.

We have recently shown that facultative sex alloca- tion in N. vitripennis is not associated with changes in gene expression, even though oviposition itself is (Pannebakker et al. 2013; N. Cook, U. Trivedi, B. Pannebakker, M. Blaxter, M. Ritchie, E. Tauber, T. Sneddon, and D. Shuker, unpub- lished manuscript), so there may be limited scope for DNA methylation to influence short-term gene expression of sex ratio-related genes.


On the other hand, disrupting DNA methylation could destroy facultative sex allocation, with All use subject to JSTOR Terms and Conditions females producing either (i) effectively random sex ratios (mean of 0.5, high variance), (ii) pathologically low sex ra- tios (all or nearly all daughters), or (iii) pathologically high sex ratios (all or nearly all sons).

In the case of (ii), this would suggest that females might be expected to produce sex ratios similar to that of single foundresses (in the range of 0.1-0.2) regardless of the numbers of foundresses on a patch and the level of LMC that male offspring will experience.

This might suggest that disrupting DNA methylation is disrupting infor- mation gathering and/or processing.

In the case of (iii), this might suggest that disrupted methylation leads to disruption of control over fertilization, with females no longer able to fertilize eggs (thereby producing only sons).

The remaining alternative is that facultative sex allocation is not destroyed, and females still shift sex ratios with increasing foundress numbers but the shape of the LMC curve is altered (Wild and West 2009; fig. 1A).

We do not have an a priori prediction in this instance, as the shift in the LMC curve will depend on whether what we usually see (and in our control females) is closer to a maternally inherited allele optimum, a pater- nally inherited allele optimum, or a form of evolutionary com- promise between the two.

However, if the genes of interest follow the pattern of being upregulated when methylated (Wang et al. 2013), then an overall increase in sex ratio across the LMC reaction norm following our demethyla- tion manipulation would imply the silencing of genes asso- ciated with producing a lower sex ratio.


Nasonia vitripennis are gregarious parasitoids of dipteran pupae (Whiting 1967).

The focal females used in this exper- iment were from the wild-type AsymC strain, the genome of which was recently sequenced (Werren et al. 2010).

Wasps are maintained on Calliphora spp.

hosts at 257C with a 16L∶8D photoperiod.

Cofoundresses were drawn from the red-eye mutant STDR strain, allowing us to track the offspring of a focal AsymC female using eye color.


We used 5-aza-dC (Sigma Aldrich, Gillingham, UK) at a nonlethal concentration to reduce methylation across the genome in adult female N. vitripennis.

Widely used in epigenetic anticancer treatments (Jones and Taylor 1980; Seidel et al. 2012; Yang et al. 2014), 5-aza-dC is a hypo- methylating agent that works by inhibiting DNA methyl- transferase activity (Creusot et al. 1982; Christman 2002).

During continued exposure, 5-aza-dC molecules are incor- porated into DNA during replication in the place of cyto- sine.

This results in a passive loss of methylation across the genome as DNA methyltransferases become irrevers- ibly bound to the 5-aza-dC residues in DNA, thereby in- hibiting their function.


Mated AsymC females were collected from stock popula- tions and individually allowed to parasitize three Calliphora vicina hosts.

These hosts were incubated, and emerging brothers and sisters were allowed to mate for 48 h. We then took one female per sibship and individually pretreated each female with access to a fresh host for 24 h (which al- lows host feeding and egg development).

We then allocated experimental females to one of two treatments: (1) control females, which were fed a 20% sucrose solution from a microcentrifuge tube lid for 48 h; and (2) treated females, which were fed a 20% sucrose solution, supplemented with 10 mM 5-aza-dC, also for 48 h. Fresh solution was provided after 24 h, and all solutions were colored with green food col- oring.

Subsequent dissection of 20 females from each treat- ment showed that all females had traces of green in the gut and, as such, were confirmed to have fed.


Treated and control females were then individually placed in one of three foundress conditions (alone, 5 foun- dresses, 10 foundresses) in a standard LMC manipula- tion (e.g., Werren 1983; Shuker and West 2004).

The 5 and 10 foundress treatments consisted of a focal female and 4 or 9 STDR cofoundresses, respectively.

In all treat- ment combinations, three hosts were provided.

After 60 min, one-way escape tubes were fitted to the vials to allow females to disperse away from patches following oviposition if they chose to.

After 24 h, we removed all females and incubated the hosts.

Throughout, all wasps and parasitized hosts were kept at 257C with a 16L∶8D photoperiod.

All emerging offspring were sexed, geno- typed by eye color (black or red), and counted to determine the sex ratio produced by each focal female.

Any clutches composed entirely of male offspring were removed from further analysis, as these are likely to have arisen from virgin females.


Given the small effect sizes predicted (Wild and West 2009) and observed (see below), the entire experiment was performed twice (with experimental block included in the analysis).

A total of 520 sex ratios across two ex- perimental blocks, with N p 64-107 for each treatment combination, were analyzed (raw data are deposited in the Dryad Digital Repository: http://dx.doi.org/10.5061/dryad .15nj0 [Cook et al. 2015]).

As is standard for sex ratio anal- yses (Wilson and Hardy 2002), we used a generalized linear modeling approach implemented in SPSS 21 (IBM, Ar- monk, NY), analyzing sex ratios with a binomial error struc- ture and a logit link function.

We used F tests to test for significance to account for slight overdispersion.


As expected, untreated females produced the classic LMC response, with sex ratio increasing with foundress num- ber (fig. 1B).

Treated females also displayed facultative sex allocation, again producing less female-biased sex ra- tios with increasing foundress number (foundress number:


All use subject to JSTOR Terms and Conditions F 1, 515 p 94.18, P < .001; foundress number 2 : F 1, 515 p 36.49, P < .001; fig. 1B).

However, treated females produced slightly less female-biased sex ratios than control females (F 1, 515 p 5.28, P p .022), a pattern that was similar across foundresses (interactions: P > .05).

Importantly, clutch size varied with foundress number as expected, with focal fe- males in multifoundress groups producing fewer offspring than single foundresses (F 1, 516 p 467.28, P < .0001), but there was no difference in the clutch sizes produced by treated versus untreated females (F 1, 516 p 0.47, P p .50).

This suggests that the chemical treatment of mothers did not lead to differential mortality of male or female offspring.


Experimental manipulation of genome-wide DNA meth- ylation using a common demethylating agent leads to sub- tle changes in sex allocation in Nasonia vitripennis.

Im- portantly, the general shape of facultative sex allocation is not altered.

Females still respond to increasing numbers of cofoundresses, as predicted by classical LMC theory (Ham- ilton 1967; fig. 1).

This suggests that the presence or ab- sence of epigenetic marks on genes associated with sex allo- cation does not influence whether facultative sex allocation occurs.

However, the curve is subtly shifted up following treatment with the demethylating agent 5-aza-dC. Clutch size did not vary with 5-aza-dC treatment.

Therefore, there is no evidence that embryonic lethality as a result of reduced DNA methylation in focal females (Zwier et al. 2012) has in- fluenced the sex ratios observed.


Our data are consistent with predictions of genomic conflict over sex allocation under LMC (Wild and West 2009), whereby disrupting DNA methylation has shifted the balance between maternally and paternally inherited alleles within ovipositing female wasps.


In their 2009 pub- lication, Wild and West comprehensively examined the ex- tent to which conflict between maternally and paternally inherited alleles could arise over the allocation of resources to male and female offspring.

They explored conflict in a range of situations encompassing competitive and cooper- ative interactions between relatives, ploidy, and whether sex allocation is controlled by the mother or the offspring.

In a haplodiploid species with maternal control over the sex ratio, it is predicted that paternally inherited alleles will favor a more even sex ratio, whereas maternally inherited alleles will favor a more female-biased sex ratio (fig. 1A).


To understand the differing perspectives, it is necessary to consider three generations.

Briefly, consider a grandmater- nal allele: this allele can find its way into granddaughters via both sons and daughters.

The grandpaternal allele can achieve the same only via daughters.

This means that the maternally inherited allele is more closely related to daugh- ters than the paternally inherited allele.

Therefore, the ma- ternally inherited allele places a greater relative genetic value on daughters than does the paternally inherited allele, lead- ing to a more female-biased sex ratio.


The sex ratios pro- duced by untreated females in our experiment would ap- pear to be closer to a maternally inherited allele optimum, with the treatment shifting sex ratios toward greater male production (as predicted for paternally inherited alleles).


Taking into consideration that methylation is typically associated with increased gene expression in N. vitripennis (Wang et al. 2013), we hypothesize that maternally in- herited alleles might be "winning" the conflict, with meth- ylation perhaps associated with upregulation of the mater- nal allele(s).

This is the first (albeit indirect) experimental evidence for such a genomic conflict over sex allocation, using recently developed theory to guide our interpretation of the phenotypic changes.

Of course, a more complete test requires proof of genomic imprinting, such that DNA meth- ylation "imprints" carry parent-of-origin information across generations, for instance, by manipulating the DNA meth- ylation status of males and females across generations using the techniques described here and observing changes in sex ratio.


Given that the N. vitripennis methylome has recently been sequenced (Wang et al. 2013; Beeler et al. 2014), future research will determine which methylated DNA sequences are involved in sex allocation.

As we have recently shown that there is (so far) no detectable differential gene expres- sion associated with facultative sex allocation in Nasonia (N. Cook, U. Trivedi, B. Pannebakker, M. Blaxter, M. Ritchie, E. Tauber, T. Sneddon, and D. Shuker, unpublished man- uscript), it would appear that disrupting DNA methylation is not having its effect by changing patterns of gene expres- sion during oviposition, but rather the effect must be up- stream, perhaps changing the sex allocation machinery prior to the female actually having to allocate sex.

We are still some way off of uncovering what this machinery is.

More generally, our data show how elucidating the mechanism of familiar behaviors can enrich our understanding of them, in this case, showing that we may need to consider genomic conflict in our models of sex allocation under LMC.

Fur- thermore, our data confirm that new theoretical and em- pirical insights are still there to be discovered, even in well- studied systems such as sex allocation in parasitoid wasps.


The use of DNA demethylating agents has been popular in epigenetic studies.

Recently, Cook and colleagues, in a 2015 American Naturalist article, claimed an effect of 5-aza-20-deoxycytidine (5-aza-dC) on the sex ratio of a parasitoid wasp without verifying its effect on DNA methylation.

We repeated the 5-aza-dC feeding treatment to test its effectiveness.

We used bisulfite amplicon sequencing of 10 genes that either were heavily methylated, previously showed a response to 5-aza-dC, or were suggested to regulate fatty acid synthesis epigenetically, and we demonstrate that wasps fed 5-aza-dC did not show reduced DNA methylation at these loci.

Therefore, the conclusion that demethylation shifts sex ratios upward needs reconsideration.


Recently, Cook et al. ( 2015) applied 5-aza-dC to test for an effect of DNA demethylation on the sex ratio response of the parasitoid wasp Nasonia vitripennis, which is known to adjust the sex ratio of its clutch based on the number of founding females in a host brood [13][14].

In this experiment, wasps that were fed 5-aza-dC in a 20% sucrose solution showed a slight but consistent shift in sex ratio in the direction predicted by theory.

However, no validation of the actual demethylating effect of 5-aza-dC was carried out.

In fact, demethylation efficacy should be verified for each organism and each concentration at which 5-aza-dC is applied because 5-aza-dC is known to have a wide range of antimetabolic activities that could confound its demethylating effect [6].

Therefore, the lack of such evidence in the study by Cook and colleagues precludes the observed change in sex ratio in N. vitripennis to be attributed to a decrease in DNA methylation.


Several other studies followed Cook et al. ( 2015) and administered 5-aza-dC in an identical manner to the same or other parasitoid species, with no [15] or very limited [16] quantification of its effect on DNA methylation.

In the latter study, only four CpG sites were tested, each one in a different gene.

Although subtle changes were seen in methylation status for three out of the four loci, loci were not tested separately for significant changes, and the number of CpG sites tested was too low to obtain a representative estimate of the effect of 5-aza-dC on the epigenome [16].

Here we want to emphasize the importance of validating the demethylating effect of 5-aza-dC when testing hypotheses about the relationship between DNA methylation and phenotypic expression.

In addition to sex allocation [13] and induction of diapause [16], several other phenotypes have been implied in parasitoid wasps to be regulated epigenetically, including caste determination [17] and fatty acid composition [15].

For a more reliable interpretation of all experiments with orally administered 5-aza-dC to parasitoids, it will be necessary to have a better idea of the extent and pervasiveness of demethylating effect of this method.


Therefore, we applied oral 5-aza-dC treatment on N. vitripennis in the same dose as previous studies [13][16] and tested its effect on DNA methylation of 155 CpG sites distributed over 10 different genes.

We included (i) genes that were heavily methylated based on several published genome methylation scans in this species [18][19][16], (ii) genes that were previously shown by quantitative polymerase chain reaction (PCR) to change their CpG methylation status in response to 5-aza-dC [16], and (iii) genes that are implied to be regulated epigenetically based on the lack of sequence variation despite different expression levels and phenotypes [20][21][22].

For the latter category, we chose genes that are involved in the fatty acid synthesis pathway, as N. vitripennis lacks the ability to convert dietary sugars into fatty acids [23][20], similar to most other parasitoid wasps [24][25][26].

No apparent gene loss or pseudogenization has been observed in pathways relevant to lipogenesis [27], and DNA methylation has been suggested as a regulator of fatty acid synthesis in Drosophila melanogaster [21].

Therefore, we hypothesize that N. vitripennis may regain lipid production after demethylation.


Methylation status of the genes was assessed using high-throughput bisulfite amplicon sequencing (BSAS) on an Illumina platform [28].

With this method it is possible to accurately estimate the percentage of methylated CpGs per site at very high coverage (on average 12,000 times per sample in our experiment).

We tested the hypothesis that 5-aza-dC treatment following established methods in N. vitripennis [13] results in significant DNA demethylation.

In addition, we measured lipid reserves of wasps in control and 5-aza-dC treatments over a 9-day period.

These phenotypic measurements served a twofold purpose: first, if 5-aza-dC treatments result in significant changes in DNA methylation, we can test whether there is a concomitant change in lipid levels.

Second, if no effect of 5-aza-dC treatments on DNA methylation is found, the phenotypic measurements allow us to ascertain the general effectiveness of our 5-aza-dC treatment, as the antimetabolic activity of the demethylation agent is expected to cause a decrease in physical condition of the wasps, for which lipid reserves are a general and sensitive measure [24][29].


The fully homozygous wild-type strain AsymCX of Nasonia vitripennis of which the genome sequence is available [27] was chosen for our experiment.

These wasps were reared on Calliphora vomitoria pupae at 25°C, 16L:8D photoperiod, and 75% relative humidity.

To test the effect of 5-aza-dC on N. vitripennis, newly emerged females (<4 h old) were randomly assigned to either of two treatments: 5-aza-dC and control.

Each treatment consisted of four biological replicates, with each replicate consisting of 10 pooled females.

Wasps were kept in a plastic tube (height×diameter = 75.0 mm×23.5 mm) with a sponge stopper.

Wasps in the control treatment were fed a 20% sucrose solution, while the wasps in the 5-aza-dC treatment were fed a 20% sucrose solution to which 10 mM of 5-aza-dC was added, following the methods applied by previous studies [13][16].

In both treatments, wasps were kept without food for several hours before giving them access to the sucrose solution to ensure a high motivation to feed.

Wasps were observed to start feeding immediately when food was offered.

This is important because 5-aza-dC has low solution stability and a half-life of about 25 min in human cytoplasma due to high activity of cytidine deaminase [6], an enzyme for which the gene is also present in N. vitripennis (LOC107980937).

After 2 days, wasps were put in 70% ethanol and stored at -20°C for further analysis.

No individuals were removed or excluded from the experiment because all survived our treatments.


As a positive control of 5-aza-dC effectiveness, another set of wasps was given the same feeding treatments as above in parallel.

After feeding for 2 days on the same solutions as above, these wasps were given clean sucrose solution for another week to test for an extended effect of 5-aza-dC incorporation.

Randomly chosen samples of wasps from both treatments were frozen on days 0 (emergence), 2, and 9.

We measured their per capita lipid levels following standard methods [30][24][26].

All data on lipid levels have been deposited in the Dryad Digital Repository: https://dx.doi.org/10.5061/dryad.d305qm1 [31].


All four biological replicates (each consisting of 10 wasps) were washed with 70% ethanol, vacuum dried, and crushed in 100 µL of phosphate-buffered saline.

For DNA isolation, we followed a modified protocol of Promega.

In short, we added 100 µL of Nuclei Lysis Solution (Promega) and 2 µL of Proteinase K (20 mg/mL; Roche) and incubated the lysate for 60 min at 60°C.

After incubation, we added 170 µL of Lysis Buffer (Promega), vortexed shortly, and centrifuged the mixture for 10 min at 14,000 rpm.

We used spin columns and DNA wash buffer (Promega) to isolate the DNA from the supernatant.

For each sample, an aliquot of DNA was converted with bisulfite following the protocol of the EZ DNA Methylation-Gold kit (Zymo).

We selected 10 different gene fragments for measuring the DNA methylation level.

Genes aa and rnapol were chosen based on their consistently heavy methylation pattern in both Wang et al. ( 2013) and Beeler et al. ( 2014).

Genes perq and wdr36 contain highly methylated sites in the study by Pegoraro et al. ( 2016) and had reduced DNA methylation in their dmnt3-knockdown experiments.

Genes acc, fabd, fasn1, fasn2, fasn3, and mcd are part of the fatty acid metabolic pathway, which has been suggested to be epigenetically regulated [20][21].


For every fragment, we developed primers on the bisulfite-converted sequence.

Furthermore, we added nonconverted PCR products of aa and rnapol to counterbalance the low CG content in the sequencing library due to bisulfite conversion of C → T. Primers and PCR conditions are listed in table 2.

For PCR, we used the GoTaq Flexi DNA Polymerase protocol (Promega) according to the manufacturer’s specifications.

PCR products were loaded on a 1.5% agarose gel, and bands of the correct size were cut out.

PCR fragments were purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel).

DNA concentrations of all 96 (two treatments, 12 amplicons, four replicates per amplicon) purified PCR fragments were measured with Qubit DNA-HS technology (ThermoFisher Scientific).

Amplicons were diluted to a concentration of 0.2 ng/mL. We prepared dual index libraries following the Nextera XT DNA Library prep kit and the Nextera Index kit (Illumina).

Ninety out of 96 samples passed our quality check on an Agilent 2100 Bioanalyzer system, and these were normalized and pooled to a final pool of 3 nM. The pooled amplicon library was sequenced at Macrogen using Illumina MiSeq 150 bp paired-end sequencing technology.

No reads mapping to fasn2 were recovered, indicating that the library preparation failed for this amplicon.

The raw short-read sequence data are available in the National Center for Biotechnology Information Short Read Archive under BioProject number PRJNA52613.


Differences in lipid levels of wasps were tested using ANCOVA with feeding treatment and time as independent variables and fat-free dry mass as the covariate.

ANCOVAs were used for every day separately as post hoc tests (with a p < 0.05/3) to verify on which day the lipid levels diverged between treatments.

Raw sequence data were checked for quality with FastQC version 0.11.3 [32].

Illumina primers were trimmed with Trimmomatic version 0.35 [33] and demultiplexed with a Python script.

Reads were mapped to target gene sequences using Bowtie 2 version 2.2.6 [34], and single-nucleotide polymorphisms (SNPs) were called using the mpileup function in SAMtools version 0.1.19 [35] and summarized using BCFtools.

Because we used an entirely homozygous strain (AsymCX), SNP calling of reads against the reference genome gives only sites with varying methylation level plus a small number of sequencing errors.

Efficiency of the bisulfite conversion was measured on the proportion of C → T SNPs in non-CpG sites.

SNPs in non-CpG sites were removed from this data set.

Only CpG sites with >100 times the coverage were retained.

We calculated the proportion of methylated cytosines per site and plotted the results in R version 3.2.5 [36].

Using this pipeline, we obtained a data set of per-site methylation proportions for all amplicons and treatments.

Differences in methylation level per site were tested separately for each amplicon using a two-way ANOVA with treatment and site as factors, including interactions.

It is important to emphasize that site was included as a fixed effect in the model with each site assigned a unique code, so we did not test for a nonzero slope.


After quality filtering and trimming, the DNA sequence data set consisted of 1,513,412 paired-end reads in total.

Of these, 90.8% mapped to the reference gene sequences.

We obtained 2,101 SNPs in total, of which 1,357 had a coverage of >100 times per sample.

Bisulfite conversion was highly effective at a rate of 98.86% ± 0.39% (mean ± SE).

No C → T SNPs were observed in the non-bisulﬁte-converted amplicons.

Figure 2 shows a histogram of the coverage per site for all amplicons combined.

Of the 155 CpG sites of our target amplicons, 148 were SNPs in our data set.

Some CpG sites had a lower proportion of cytosine than predicted by the bisulfite conversion efficiency (i.e., these sites are probably never methylated in situ).

The proportion of methylated cytosines is plotted per CpG site per amplicon in figure 3.


For a substantial proportion of the 155 individual CpG sites, methylation level increased or decreased in the 5-aza-dC relative to the control, but we tested changes in methylation status at the whole amplicon level (table 3).

In all amplicons we found significant differences in methylation level per site over the length of the amplicon (all P < 0.05).

There were no significant differences in methylation levels between 5-aza-dC and control treatments (all P > 0.1) and no significant interactions between treatment and site on the amplicon (all P > 0.05).


We expected 5-aza-dC-fed wasps to have a lower proportion of methylated CpG sites compared to the controls.

However, no significant differences between 5-aza-dC-fed and control treatments were found in any of the genes we examined at the 10-mM doses provided, the dose commonly used in other studies in this model system [13][16].

Although for a substantial proportion of the 155 individual CpG sites we noted a decrease or increase in methylation status, our results demonstrate that 5-aza-dC does not consistently reduce DNA methylation in the nine amplicons that we tested.

This result is corroborated by a recent genome-wide methylation study in Nasonia vitripennis, also showing a lack of uniform demethylating effect of 5-aza-dC treatment across sites [37].

We observed the same result for each of the three categories of genes we tested: there was no reduction in DNA methylation in highly methylated genes, nor in genes previously shown to have varying methylation levels, nor in genes involved in lipogenesis, a metabolic process that has been suggested to be epigenetically regulated.


We found a negative effect of 5-aza-dC treatment on lipid levels of N. vitripennis, most likely due to the detrimental effects of antimetabolic activity of 5-aza-dC [6].

Therefore, previous studies that attributed phenotypic changes in 5-aza-dC-fed wasps to a decrease in DNA methylation should be reconsidered in the light of the current results.

The high proportion (98.86%) of C → T SNPs in non-CpG sites proved that the bisulfite conversion efficiency in our study was high, also compared to other methods [38].

In CpG sites, the average methylation level was 0.09%–80.73% per site.

These two observations imply that the lack of a demethylating effect of 5-aza-dC was not due to inefficient bisulfite conversion or improper detection of DNA methylation.


Two CpG sites were tested in both our BSAS study and the MethylQuant-based study by Pegoraro et al. ( 2016).

The MethylQuant-tested sites correspond in our study to sites 113 and 220 for perq and wdr36, respectively.

Site 113 for wdr36 is the only site in this amplicon with a detectable methylation level, with considerable variation between samples.

Site 220 for perq seems to have a higher methylation level in 5-aza-dC-treated wasps.

However, most other sites in this amplicon show no difference, similar to the rest of the data set.

These differences could be life stage specific, a general pattern also found by Wang et al. ( 2013).


In plants and fungi, genome-wide DNA methylation levels have repeatedly been shown to decrease after 5-aza-dC treatment, often with associated changes in phenotypes such as growth, flowering phenology, or secondary metabolite production [39][1][7][40].

The evidence for a demethylating effect of 5-aza-dC in arthropods is much less strong.

This is partly because the major insect genetic model Drosophila lacks a DNA methylation system [41].

In other arthropod model systems, DNA methylation is present.

In Daphnia, for instance, 5-aza-dC had a clear demethylating effect on four out of the five tested genes, which differed among CpG sites [10].

However, few insect studies have shown a consistent effect of 5-aza-dC. In bumble bees, 5-aza-dC has an effect on overall DNA methylation, but its effect is erratic, as hyper- and hypomethylation were both observed after 5-aza-dC treatment [9].

A similar variable effect of 5-aza-dC treatment on methylation status of individual CpG sites was also found in a genome-wide study of methylation in N. vitripennis [37].

Our study adds to this growing body of literature by showing a lack of consistent demethylating effect of 5-aza-dC in N. vitripennis.


There may be several causes for this lack of a demethylating effect of 5-aza-dC in N. vitripennis and in insects in general.

The 5-aza-dC is usually administered via the food, and one explanation may be that the animals did not feed enough.

In our study, however, we observed all wasps to feed within minutes after the food was offered, ensuring that 5-aza-dC was ingested by all wasps.

Also, the phenotypic effect of 5-aza-dC treatment on lipid reserves indicates that the 5-aza-dC solution was effective.

An alternative explanation for the lack of demethylating effect of 5-aza-dC could be that there is a paucity of cell division in adult insects [42].

Incorporation of 5-aza-dC into the DNA happens only during cell division and is necessary to inactivate the DNA methyltransferase 1 enzyme [6].

In adult insects, only a few tissues contain regenerative cells, such as cells involved in neurogenesis in the brain of the house cricket Acheta domesticus [43], intestinal stem cells in the midgut of Drosophila melanogaster [44], and germ cells continuously dividing in ovaria of N. vitripennis [45].

Compared to organisms with continuous growth, the amount of cell division in adult insects is low, yet an effect of 5-aza-dC on DNA methylation would have been possible.


We showed a small but significant negative effect of 5-aza-dC on lipid levels in N. vitripennis.

Lipid levels decreased over time in wasps in both treatments, but the rate of decrease was higher in 5-aza-dC-treated wasps.

However, this is probably not biologically significant but due mainly to detrimental effects of 5-aza-dC on wasp health.

The five genes involved in the fatty acid synthesis contain many CpG sites, but methylation levels varied substantially among genes.

In FASN1, FASN3, and ACC, methylation levels at CpG sites were on average less than 1%, whereas MCD and FabD showed high methylation levels at CpG sites on various locations in the amplicon.

However, 5-aza-dC did not influence the DNA methylation levels in any of these genes.

Our results suggest that phenotypic differences observed in adult wasps after administering 5-aza-dC arise through a mechanism independent of DNA methylation—for instance, an effect on other metabolic processes.

Even though we did not quantify sex ratio in our study, our results warrant further investigation of the previously reported relationship between DNA methylation and sex allocation in N. vitripennis [13].

Due to the inconsistent effect of 5-aza-dC for DNA demethylation, it would not be advisable for future studies on the link between CpG methylation and phenotypes in N. vitripennis.

Pupal knockdown of DNA methyltransferases could be a good alternative, although this will also remove methylation of sites that could be essential for successful metamorphosis and thus lead to premature mortality.

A second alternative is the use of the nonspecific DMNT1 inhibitor RG108 [46], but as far as we know this method remains untested in N. vitripennis.

In all scenarios, the study should always include evidence showing consistent changes in DNA methylation.


We previously demonstrated that treatment with the demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) alters the offspring sex ratios produced by females of the parasitoid wasp Nasonia vitripennis [1].

Females allocate offspring sex ratio in line with Local Mate Competition theory, producing more or less female-biased sex ratios as the number of other females laying eggs on a patch varies, thereby reducing competition amongst their sons for mates.

Interestingly, treatment with 5-aza-dC did not ablate the facultative sex allocation response.

Instead, sex ratios became less female-biased, a shift in the direction of the optimum sex ratio for paternally-inherited alleles according to genomic conflict theory.

This was the first (albeit indirect) experimental evidence for genomic conflict over sex allocation.

Ellers et al. ( 2019) have since assayed the effects of 5-aza-dC on DNA methylation in ten Nasonia genes, finding no evidence of demethylation in these 10 genes, from which they conclude that 5-aza-dC has no demethylating capability in Nasonia vitripennis.

Quantifying the efficacy of 5-aza-dC in terms of demethylation is indeed crucial to in-depth interpretation of studies using 5-aza-dC to link phenotypes to epigenetic regulation.

Here, we outline the mode of action of 5-aza-dC and demonstrate that determining the efficacy of 5-aza-dC in insect systems requires a whole-genome approach.


DNA methylation, sex allocation and 5-aza-2′-deoxycytidine In a previous study, we demonstrated that treatment with the demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) alters the sex allocation response of Nasonia vitripennis females [1].

As with all Hymenoptera, N. vitripennis is a haplodiploid species, with males arising from unfertilized, haploid eggs and females developing from fertilized, diploid eggs.

Females are in putative control of sex allocation, either laying unfertilized eggs that will become sons or releasing sperm to fertilize eggs that will become daughters.

Female Nasonia allocate sex broadly in line with Local Mate Competition (LMC) theory [2][3][4]; females laying eggs alone will produce very female-biased sex ratios to minimize competition amongst their sons for mates.

As the number of laying females (foundresses) on a patch increases though, the level of LMC consequently declines and less female-biased sex ratios are produced.

Recent theory, however, has demonstrated that maternally- and paternally-inherited alleles should have differing patterns of optimal sex allocation under LMC, with maternally-inherited alleles selected to produce more female-biased sex ratios [5].

Importantly, parent-of-origin information must be associated with each allele, for instance via epigenetic marks such as DNA methylation.

We demonstrated that disrupting DNA methylation with 5-aza-dC did not ablate the facultative sex allocation response, but instead found that sex ratios were shifted in the direction of the paternal optimum (sex ratios became more less female-biased; [1]).

This result was consistent with predictions from genomic conflict over sex allocation theory [5], suggesting that unmanipulated sex ratios are closer to the optimum for maternally-inherited alleles (Figure 1).

Ellers et al. ( 2019), in their valuable comment on this study, raised the valid point that the efficacy of 5-aza-dC in insect systems should be tested, and have suggested from their own experimentation that 5-aza-dC does not disrupt methylation in Nasonia vitripennis.

To fully address their comments, we will begin by briefly reviewing the mechanism by which 5-aza-dC brings about the demethylation of DNA, before showing that assessing the role of 5-az-dC requires a whole genome approach.