Cardiovascular disease (CVD) is the leading cause of death in Type 1 Diabetes (T1D). The molecular basis for atherosclerosis in T1D is heavily influenced by hyperglycaemia and its atherogenic effects on LDL. Ongoing research into the distinct pathophysiology of atherosclerosis in T1D offers exciting opportunities for novel approaches to calculate CVD risk in patients with T1D and to manage this risk appropriately. Currently, despite the increased risk of CVD in the T1D population, there are few tools available for estimating the risk of CVD in younger patients. This poses significant challenges for clinicians in selecting which patients might benefit from lipid-lowering therapies over the long term. The current best practice guidance for the management of dyslipidaemia in T1D is generally based on evidence from patients with T2D and the opinion of experts in the field. In this review article, we explore the unique pathophysiology of atherosclerosis in T1D, with a specific focus on hyperglycaemia-induced damage and atherogenic LDL modifications. We also discuss the current clinical situation of managing these patients across paediatric and adult populations, focusing on the difficulties posed by a lack of strong evidence and various barriers to treatment. CVD is the most common cause of death in Type 1 Diabetes (T1D) . HoweverThe majority of cases of T1D are diagnosed in the paediatric population and the incidence of the condition appears to be increasing . This isThe high morbidity and mortality associated with CVD in Type 1 Diabetes suggests a need for early identification of risk factors and evidence-based measures to attenuate that risk. Much of the current guidance on the management of dyslipidaemia in T1D is based on studies composed primarily of patients with T2D despite the differing underlying pathophysiology. There are few clinical trials that have investigated the approach to dyslipidaemia in T1D, in either paediatric or adult cohorts. The aim of this review article is to describe the pathophysiology of dyslipidaemia in T1D and to examine the current evidence for the screening and management of dyslipidaemia in patients with T1D across both paediatric and adult populations.There is evolving evidence that differences exist in the pathophysiology of atherosclerosis in individuals with T1D versus individuals who do not have diabetes. Examination of atherosclerotic plaques in patients with diabetes has shown that they are more lipid-rich and have an increased amount of macrophages and thrombus than those in non-diabetic patients . The patEndothelial cells line the inner surface of all blood vessels and are responsible for controlling the vessel structure and function through production of vascular mediators including nitric oxide, angiotensin 2, endothelin 1, and prostacyclin. Nitric oxide (NO) is a key regulator of many processes including haemostasis, vascular smooth muscle relaxation, inflammation, and thrombosis . OverallIn addition to reduced nitric oxide bioavailability, diabetes is associated with increased production of vasoconstrictors, the most important of which is endothelin-1. Increased activity of endothelin-1 may be mediated by insulin dependent gene expression, increased gene transcription caused by oxidised LDL or stimulation of the receptor for AGEPs ,26. EndoThe pathways above also result in an increased production of the transcription factors NFKB and activator protein 1 which regulate the expression of many atherosclerotic mediators. This leads to increased expression of leucocyte adhesion molecules, monocyte attracting chemokines, and pro-inflammatory mediators found in atheroma such as TNF alpha and leukotriene-1 . Overall, these hyperglycaemia-induced changes lead to significant endothelial damage, resulting in decreased production of nitric oxide and increased endothelin 1/angiotensin 2 with an end result of vasoconstriction and activation of inflammatory mediators. This creates an environment that favours the adhesion of circulating macrophages and invasion into the arterial intima where they are activated and take up lipoproteins, leading to the formation of foam cells and the fatty streak, which are the hallmarks of early atherosclerosis ,22. Vascular smooth muscle cells (VSMCs) are key to the development of atherosclerosis. Once fatty streaks are formed, VSMCs migrate to the intima where they lay down a complex extracellular matrix. Hyperglycaemia has the effect of activating protein kinase C, NFkB, and receptors for AGEs in VSMCs as it does in endothelial cells, leading to additional oxidative damage. Enhanced migration of VSMCs has been observed in patients with diabetes. The presence of VSMCs in an atheroma provide collagen and stability, making it less likely to rupture. Plaques which have ruptured and resulted in thrombosis tend to have fewer VSMCs present . HoweverThe concentration of glucose in platelets is equivalent to the extracellular concentration as glucose uptake in platelets is not mediated by insulin . HyperglIt has been shown that not all LDL particles are atherogenic and that native LDL does not necessarily cause prominent accumulation of lipids in the arterial wall. It is the modification of native LDL that leads to its atherogenic properties. The relationship between small, dense LDL (sdLDL), and dyslipidaemia in diabetes has been extensively researched. The process of LDL modification is thought to be initiated by desialylation of LDL. Sialic acid is an important component of native LDL and it has been proposed that desialylation and multiple subsequent modifications lead to increased atherogenicity . CholestAGEs play a role in multiple steps of atherosclerosis. Glycosylation of LDL particles is a key atherogenic change in the context of chronic hyperglycaemia. Glycosylation occurs on the ApoB and phospholipid portions of LDL and leads to a significant decrease in the recognition of specific LDL by the LDL receptor. This results in decreased LDL-receptor mediated uptake and degradation of glycated-LDL as compared to native LDL. Furthermore, glycated LDL is more readily recognized by macrophages, leading to increased uptake and formation of foam cells via non-specific scavenger receptors on macrophages rather than via the specific LDL-receptor . GlycatiIn general, inflammation is considered central to the pathophysiology of atherosclerosis and inflammation appears to be more prominent in individuals with diabetes compared with non-diabetic controls. Individual with diabetes have higher levels of inflammatory markers such as interleukin-2, CD-40 ligand, and C-reactive protein ,38. The As discussed, modification of lipids promotes inflammation through increased expression of adhesion molecules and increased release of chemokines and pro-inflammatory cytokines in both macrophages and vascular cells . Above, p < 0.03), a finding previously demonstrated in adult studies [In general, individuals with well-controlled Type 1 Diabetes appear to have similar lipid profiles when compared with the general population . However studies . Studies of lipoprotein subclasses in adults with Type 1 Diabetes have shown more conflicting evidence. More advanced analysis of lipoprotein subclasses in individuals with T1D have been published with inconsistent results. Nuclear magnetic resonance spectroscopy is a method which can quantify the number, size, and composition of lipoproteins and can reveal more subtle dyslipoproteinaemia . FurtherInterestingly, a larger, more recent study of adults with T1D from a Mediterranean population did not replicate the results, showing a more atherogenic profile in T1D. The mean HDL-P size was larger in the T1D group versus healthy controls and the triglyceride content was not found to be increased . PossiblThey did, however, note a potentially more atherogenic NMR profile in women. The gender differences seen in the LDL parameters of non-diabetic individuals were not seen between males and females with T1D   .Screening for dyslipidaemia should commence in all children diagnosed with Type 1 Diabetes soon after diagnosis (once diabetes is stabilised) from the age of 11 years.If there is a family history of early CVD, hypercholesterolaemia, or if the family history is unknown, screening should begin as early as 2 years of age.If the initial screen is normal, this guideline recommends repeat screening every 5 years.Fasting lipid profiles are not necessary as the initial screening test. However, if fasting LDL or triglycerides are elevated, fasting studies are then required.Screening and Ongoing Surveillance:High LDL cholesterol is defined as >2.6 mmol/L (100 mg/dL).High LDL should be managed initially with lifestyle interventions, including diet and exercise.If lifestyle interventions do not lower LDL to ≤3.4 mmol/L, initiation of statin therapy should be considered in children aged >10 years.The ideal treatment target is <2.6 mmol/L.Management:The American Diabetes Association released new guidelines in 2021 entitled Standards of Medical Care in Diabetes. Chapter 13 addresses guidelines for children and adolescents . Their rRecommend initial lipid screening from 2 years of age.If initial screening shows an LDL <2.6 mmol/L, repeat screening at age 9–11 years.Repeat screening is recommended every three years if LDL is normal (versus the 5 years recommended by ISPAD).Screening and Ongoing Surveillance:If lipids are abnormal, they recommend initial conservative management with optimisation of blood glucose and medical nutritional therapy to limit the amount of calories from fat to 25–30%, saturated fat to <7%, cholesterol <200 mg/day, avoidance of trans fats, and an aim of approximately 10% of calories from monounsaturated fats.Consideration of statin therapy in children over the age of 10 who have persistently raised LDL despite adequate lifestyle interventions and medical nutrition therapy.Statins are to be considered with an LDL >4.1 mmol/L or an LDL of >3.4 with one or more additional cardiovascular risk factors.The target LDL was the same at <2.6 mmol/L.Management:The American Diabetes Association  closely In adults not taking statins or other lipid-lowering therapy, it is reasonable to obtain a lipid profile at the time of diabetes diagnosis, at an initial medical evaluation, and every 5 years thereafter if under the age of 40 years, or more frequently if indicated.Screening and Ongoing Surveillance:Lifestyle interventions focusing on weight loss (if indicated); application of a Mediterranean style or Dietary Approaches to Stop Hypertension (DASH) eating pattern; reduction of saturated fat and trans fat; increase in dietary n-3 fatty acids, viscous fibre, and plant stanols/sterols intake; and increased physical activity should be recommended to improve the lipid profile and reduce the risk of developing atherosclerotic cardiovascular disease in patients with diabetes.Recommend a lipid profile at initiation of statins or other lipid-lowering therapy, 4–12 weeks after initiation or a change in dose, and annually thereafter as it may help to monitor the response to therapy and inform medication adherence.Management: 40–75 years: Moderate intensity statin therapy for all patients with diabetes without ASCVD. Additionally, if there are multiple ASCVD risk factors present in a patient between 50–70 years, it is reasonable to use high-intensity statin therapy.In adults with a 10-year ASCVD risk >20%, it may be reasonable to add ezetimibe to maximally tolerated statin therapy with an aim to reduce LDL-C >50%.The recommendations for patients with diabetes less than 40 years of age and/or T1D with other CVD risk factors is not entirely clear as the evidence in this group is lacking.They state that similar statin treatment approaches should be considered for patients with Type 1 and Type 2 diabetes, particularly in the setting of additional risk factors. They recommend that the health care provider has a discussion regarding the relative risks and benefits and to consider the use of moderate intensity statin therapy.Pharmacotherapy for Primary Prevention:All patients with diabetes and atherosclerotic cardiovascular disease should be commenced on high-intensity statin therapy in addition to lifestyle interventions.In patients with very-high risk ASCVD with an LDL >1.8, consider the addition of a non-statin cholesterol-lowering medication such as a PCSK9 inhibitor or ezetimibe.Pharmacotherapy for Secondary Prevention:European Guidelines:Statins are recommended in all patients with T1D who are in the “high risk or very high-risk category”.High Risk: Patients with DM without target organ damage, with DM duration >10 years, or another additional risk factor.Very High Risk: DM with target organ damage, or at least three major risk factors, or early onset of T1D of long duration (>20 years).Statin therapy may be considered in both T1D and T2D patients aged <30 years with evidence of end organ damage and/or an LDL-C >2.5 mmol/L, as long as pregnancy is not being planned.EAS/ESC Statins Recommend offering atorvastatin 20 mg as primary prevention of CVD to people with T1D who are >40 years old, have had diabetes for more than 10 years, have established nephropathy, or have other CVD risk factors.NICE guidelines on lipid modification from 2014 RecommendAll current guidelines for the management of dyslipidaemia in general recommend the calculation of total cardiovascular risk. There are many CVD risk calculators in use—the ESC/EAS guidelines recommend using risk charts based on country-based cohort data where possible. The SCORE (Systematic Coronary Risk Estimation) can be recalibrated for use in different populations and is the calculator recommended by the ESC . The ADAScreening for dyslipidaemia is currently carried out clinically through assessing the standard lipid profile. There is ongoing research into the use of novel cardiac biomarkers to build individual risk profiles for patients. These biomarkers could potentially be incorporated into T1D-specific risk calculators to increase their accuracy. Candidate biomarkers that have been shown to correlate with CVD include ApoB, lipoprotein A, modified LDL  and LDL-immune complexes, receptors for AGEs, metalloproteinases, and highly sensitive C-reactive protein . CurrentLastly, there is potential for the incorporation of imaging modalities into risk calculators in T1D. A recent prospective study of the use of echocardiography for the assessment of CVD risk in individuals with T1D without cardiovascular disease showed that measures of myocardial dysfunction are significantly associated with major adverse cardiovascular events over a 7-year follow-up. These echocardiographic parameters were shown to identify at-risk individuals at an improved rate compared with conventional risk factors alone . CarotidOverall, patient selection criteria for the treatment of dyslipidaemia in T1D in younger patients is unclear due to the lack of evidence. Development of accurate risk calculators for this population would be of great benefit going forward.The initial management of dyslipidaemia in children as recommended by best practice guidelines revolves around lifestyle interventions including diet and exercise. A 6-month-long Italian trial showed that a dietician-led programme prioritising a Mediterranean-style diet improved the quality of nutrient intake and subsequently showed a decrease in LDL-C and non-HDL-C . AnotherIn addition to these lifestyle interventions, improved glycaemic control has been shown to improve lipid profiles, but may not be sufficient to fully normalise dyslipidaemia alone . IntensiSimilar to paediatric guidelines, adult diabetes guidelines recommend lifestyle interventions for the management of dyslipidaemia. The Mediterranean diet when supplemented with extra virgin olive oil or nuts was associated with a lower rate of major adverse cardiovascular events . This diCurrent recommendations on management of dyslipidaemia in paediatrics are based on adult studies or on evidence of preclinical atherosclerosis, as there are no trials in paediatrics showing a relationship between treatment cut-offs and a decrease in CVD events in the future. Effective lifestyle interventions to change behaviour can be exceedingly difficult to achieve and may be insufficient on their own for optimal cardiovascular health ,84. FurtThere is evidence that LDL-C is among the most important risk factors for myocardial infarction  in T1D with each 1 mmol/L increase in LDL-C associated with a 35–50% increased risk of ASCVD events. The study showed that LDL-C has an almost linear relationship with the risk of outcomes, especially death and myocardial infarction. Additionally, there is evidence to suggest that the optimum levels of LDL-C for reducing risk of cardiovascular disease may be lower than currently recommended by guidelines [The strongest evidence for statin use in diabetes in general exists in the 40–75-year-old group, with several randomised controlled trials of moderate intensity statin use leading to reductions in ASCVD  ,95,96. HThe evidence for lipid-lowering medications in T1D in patients <40 years is lacking and the age at which statin therapy should be commenced is largely unclear. While the short-term risk of ASCVD in this age group is generally low, there is evidence that the risk increases with time, especially in those with a long duration of disease (>20 years) . ChildreDespite the well-documented increased risk of CVD in T1D and the established role dyslipidaemia plays in atherosclerosis, a significant amount of uncertainty still exists regarding optimal management strategies for these patients. As discussed, there is strong evidence that atherosclerosis begins in childhood and that children with early onset diabetes have high rates of complications in early adulthood. Many of the recommendations made in paediatric guidelines are based on data from adult trials  and rely on expert consensus or opinion. Nevertheless, these guidelines do give clear indications for statin use and clinical targets. In practice, however, we have seen that management of this patient group is not reflective of published guidelines. The barriers to treatment in paediatrics include persistent concerns for the teratogenic potential of statins and consequently their use in adolescent females. Additionally, young people and their parents may be reluctant to commence further medications when already contending with the heavy burden of multiple insulin injections. Parental education is made more difficult by the fact that there is no long-term safety or efficacy evidence to point to in the discussion for commencing statins. What are the main areas that need to be addressed to enhance treatment in this group? Further research into the lipoprotein subclasses in youths with T1D and their association with the development of atherosclerosis could be useful in the assessment of risk and selection of patients for treatment. More information on the teratogenicity of statins would be significant. Most importantly, RCTs examining the efficacy and safety of statin use exclusively in younger people with T1D that includes long-term follow-up would mark a significant advance in clinical practice.At a basic level, ongoing research into the differing pathophysiology of atherosclerosis in T1D vs. the general population and even T2D could yield promising treatment targets. More research is required to evaluate the role of lipoprotein subclasses in risk stratification of patients with T1D, as the results so far have been conflicting. There were also discrepancies seen between results in adult patients versus those in children and adolescents. Possible explanations for these discrepancies are the effects of pubertal hormones on insulin resistance, which could affect lipoprotein subclasses. The higher insulin requirements seen in adolescence could also play a role. Additionally, use of lipid-modifying drugs could account for some of the differences seen, with much lower frequency of statin use reported in the paediatric studies than those in adults. The issues regarding selecting ideal candidates for statin therapy and the optimal age at which to commence treatment is also present in adult practice. There is a similar lack of randomised controlled trial evidence for the use of statins in T1D, especially under the age of 40 years. Key questions that need to be answered include what is the significance of CVD risk factors in adolescents and young adults and what are the indications to intervene? When is the optimal time to commence statin therapy, and in whom? Are statins safe and efficacious over the long term in this population?Tools for risk-assessment and therefore correct patient selection for treatment will be essential going forward. The further validation of the existing CVD risk calculators for T1D and the development of novel calculators could play a key role in patient selection. Further research into novel biomarkers of CVD in T1D is another exciting research opportunity for risk stratifying younger people with T1D. A promising area is the evaluation of the role of modified LDL , which are immunogenic and result in the production of autoantibodies. These antibodies then form circulating immune complexes with modified LDL and these LDL-immune complexes (LDL-ICs) have been shown to be pro-inflammatory and pro-atherosclerotic. There is accumulating evidence that quantifying modified LDL and associated immune complexes may have a better predictive value than conventional lipid measurements . MeasureIn summary, the risk of CVD is well documented in this group. The difficulty remains in the selection of the appropriate younger patients who may benefit from lipid-lowering drugs, the treatment of whom will be long-term by virtue of patient age. The main advances that need to occur to overcome this issue are RCT evidence for the longer-term safety and efficacy of statins in T1D specifically. Additionally, new approaches to individually assess risk in T1D patients would significantly improve and standardise management of risk factors in T1D. As discussed, keys to this include further evaluation and optimisation of risk calculators and the discovery and development of novel biomarkers and imaging methodologies in order to create individualised risk profiles going forward. This will represent a personalized medicine approach to management.Cardiovascular disease remains the leading cause of death in T1D despite advances in glycaemic control and risk factor management. Ongoing research into the pathophysiology of atherosclerosis and the lipoprotein subclasses in T1D is essential to our understanding of the disease and may offer new targets for therapy. What remains clear is that there is a significant gap in evidence from randomised controlled trials for the management of dyslipidaemia in both paediatric and adult individuals with T1D. Significant advances have been made in the management of this condition, but more clinical research in this area is required to guide clinicians and to inform evidence-based practice.


Only 5 dogs, compared to 14 pre-treatment, received any oral NSAID or other analgesic for the duration of the 6-month study period. The TPI, and change in TPI from baseline, were not statistically significantly different between the two treatments at any time point. These data suggest clinical efficacy of both APS and HAT, but fail to show superiority of one treatment vs. the other. The inability to detect a statistically significant difference between the two treatments could be attributable to a true lack of a difference, or a type II statistical error.Twenty-three dogs with bilateral hip osteoarthritis were used to compare the efficacy of intra-articular injections of autologous protein solution (APS) to hyaluronic acid plus triamcinolone (HAT). Prior to treatment, owner assessments of pain and mobility were obtained using the canine brief pain inventory (CBPI) and Liverpool Osteoarthritis for Dogs (LOAD) questionnaires. Owners were also asked to list all medications used to control signs of pain associated with hip osteoarthritis (OA). In addition, objective kinetic data using a pressure sensitive walkway was used to quantify the relative weight bearing of each of the limbs . One hip was then selected using a random number generator for injection with HAT and the contralateral hip was injected with APS under the same sedation event. At 1-, 3-, and 6 months following injection, medication usage was recorded and dogs were re-assessed using the CBPI and LOAD questionnaires and using objective gait analysis to determine the TPI. Twenty dogs completed all aspects of the study and statistically significant ( Autologous protein solution (APS) is an autologous blood product involving preparation of leukocyte-rich platelet-rich plasma (L-PRP) from a single centrifugation cycle followed by desiccation with polyacrylamide beads. In horses and people, this preparation process results in a bioactive milieu with a favorable concentration of anti-inflammatory proteins, such as interleukin-1 receptor antagonist protein . No simiTwo of the unanswered questions with regard to APS use in dogs pertain to the duration of benefit, and whether APS provides a greater improvement than alternative injectable treatments such as hyaluronic acid and/or corticosteroid. This latter question is relevant because hyaluronic acid and corticosteroid are available “off-the-shelf” and are typically less expensive than APS. As for duration of benefit, the previous studies in dogs have followed patients just 1- and 3- months post injection, and so whether APS provides a longer benefit in dogs is unclear , 7. Two The purposes of the current study were to compare APS to hyaluronic acid combined with triamcinolone in dogs with naturally-occurring, bilateral hip osteoarthritis. Further, we sought to assess outcomes with a longer follow-up time than has been used previously when assessing dogs treated with APS. Based on aforementioned data in people, horses, and dogs, we hypothesized that there would not be a significant difference between the two treatments at first follow-up (1 month), but that the benefit of APS would be superior to that of HAT at subsequent follow-ups including up to 6 months post treatment.With approval by the Animal Care and Use Committee of Colorado Canine Orthopedics and Rehab, and based upon a previous pilot study in which we compared APS to saline in dogs with hip OA using the same study methodology , we soug®, CIR Systems, Franklin, NJ) to collect objective gait data. We sought to obtain a minimum of 5 valid trials. A trial was valid if the leash was slack and not influencing the dog's gait, the dog was trotting in a straight line while looking straight ahead without turning its head, and in which the dog maintained a relatively consistent pace. Dogs were typically trotted at least 20 times across the walkway in order to ensure obtaining five or more acceptable trials. All trials were video-recorded for subsequent review.Following screening, dogs that met the inclusion criteria were enrolled and all owners provided written, informed consent for inclusion in the study. Prior to treatment owners then completed the Canine Brief Pain Inventory (CBPI) and the Liverpool Osteoarthritis in Dogs (LOAD) questionnaires. Dogs were also trotted across a pressure sensitive walkway  and 0.2 mg/kg of Butorphanol  administered intravenously. An area over the jugular vein was then clipped and aseptically prepared and an 18-gauge 2″ IV catheter was then placed in the jugular vein. A 60 ml syringe that was pre-loaded with 5 ml of ACD-A  was then attached to the catheter and filled with blood to its full 60 ml volume. The syringe was then gently inverted multiple times to mix the blood and anti-coagulant. A small volume (0.5–1 ml) of such blood was placed in a small purple top tube with EDTA for performing a whole blood complete blood count (CBC) on an in-house blood analyzer . It is worth noting that no validation of methods for counting cellular components, including platelets, in canine autologous protein solution specifically, has been performed.® APS device, Owl Manor, Warsaw, IN). Following APS preparation 1 ml of APS was collected for administration to the patient and ~0.5–1 ml was taken for a CBC on the APS.The remaining blood was then used to prepare APS according to manufacturer instructions . The patient was then rolled into the opposite recumbency, the contralateral hip was aseptically prepared, joint fluid aspirated, and 1 ml of APS was injected. Following treatment dogs were reversed from their sedation using Atipamezole  and discharged to the owners. Owners were provided 2 days of a non-steroidal anti-inflammatory drug  to use in the first 2 days following injection as experience has shown that many owners complain of patient discomfort in the first 2 days. Owners were also provided a daily log into which they recorded any adverse events or use of any medications or supplements. Owners were informed that they were allowed to use medications or supplements if necessary but were requested to contact the principal investigator prior to initiating using of any medications or supplements. All dogs were allowed to resume activity without limitation.One hip was then selected for injection with HAT using a random number generator. The injection site was aseptically prepared, joint fluid was aspirated to confirm intra-articular needle placement and then the joint was injected with Triamcinolone  combined with 1 ml of a medium molecular weight hyaluronic acid . The percentage of all 4 limbs adds up to 100% and a normal distribution is typically about 30% on each forelimb and 20% on each hindlimb. Only those trials in which there was a minimum of 2 full gait cycles and <10% variation in the dog's velocity were included.Once the gait data were quantified the TPI of the two pelvic limbs were exported for assessment. The TPI and the change in TPI from baseline were selected as the outcome variable of interest before the study was conducted or the data analyzed. These data (TPI and change in TPI from baseline) were evaluated using a linear mixed model (LMM). The LMM included fixed factors for treatment and the time of the assessment  and a treatment by time interaction effect. The LMM also included random intercepts for each dog and each limb to account for within dog and within limb correlations. Satterthwaite degrees of freedom method was used. F-tests of simple treatment effects at each time point were performed. Model residuals were examined to evaluate the assumption of normality. These analyses were performed using SAS V 9.4 .The CBPI is a validated owner questionnaire for assessing canine pain and consists of 4 questions in which the owner assesses the dog's pain and six questions that evaluate the dog's function. For each of these questions, owners can provide an answer from 0 to 10 with 0 being consistent with a normal dog and 10 being consistent with either more pain or decreased function. The total scores for the first 4 questions  were summed for each dog both prior to and at each time point  following treatment as has been done in previous study  with higher scores consistent with decreased mobility, as done in previous study . As for post-hoc between the change in TPI (from baseline to 3 months) in the APS-treated limb and both the leukocyte concentration and the platelet concentration in the APS using a Pearson product moment correlation.Correlation was assessed T-test .The mean TPI in the APS-treated limb at 3 months post treatment was compared between dogs that did or did not receive any carprofen following treatment (excluding the first 48 h following injection), using a T-tests .The CBPI and LOAD scores at 3 months for pain, function, overall function, general mobility, and mobility at exercise were all compared between dogs that did or did not receive any carprofen following treatment using post-hoc assessments because as shown below , month 3 was when the APS treated limbs had shown their greatest improvement (based on change in TPI) and were at their best (greatest TPI).The 3-month time frame was chosen for these Twenty-three dogs met the inclusion criteria and were enrolled. Twenty dogs completed the study and 3 were removed from the study. The 20 dogs that completed the study included 4 German shepherd dogs, 3 each of border collies, Labrador, and Golden retrievers, and one Australian shepherd, Doberman, Irish setter, Poodle mix, Rottweiler, Siberian husky, and St. Bernard mix. Ten dogs were spayed females, nine were castrated males, and there was one intact female. The mean weight was 33.62 kg (stdev 6.8 kg). The mean age was 4.8 years (2.2).Both hips were classified as having mild hip dysplasia in 1 dog, moderate hip dysplasia in 2 dogs, and severe hip dysplasia in 15 dogs. In two dogs one hip was graded as severe and the contralateral was graded as moderate hip dysplasia. In one of these two dogs the randomization resulted in the hip with moderate dysplasia being treated with APS and in the other dog the hip with moderate dysplasia was treated with HAT based on the random number generator.The 3 dogs that were removed from the study included a 2-year-old Husky that was attacked by another dog <4 weeks after treatment and had wounds to the left forelimb and associated left forelimb lameness at the 4-week recheck, thus precluding accurate objective assessment of the relative weight bearing on each of the hindlimbs. A 2-year-old German Shepherd dog developed myasthenia gravis 2 months following treatment, hampering his ability to walk and thus precluding assessment of gait. An 8-year-old castrated male German Shepherd dog developed a septic joint and polymyositis within 48 h following treatment. All data from these three dogs were omitted from all analyses.Prior to study enrollment 5 dogs were not receiving any oral medications or supplements to help control pain or lameness attributable to hip osteoarthritis. One dog was treated with cannabidiol oil only. Fourteen of 20 dogs were receiving carprofen prior to study enrollment. Of these 14 patients receiving carprofen, eight were also receiving a glucosamine chondroitin supplement, four were being treated with Gabapentin, three were receiving fish oil supplementation, one was receiving Tramadol, and one was receiving Grapiprant.All these medications and supplements were discontinued a minimum of 1 week prior to treatment and all owners were provided with 2 days of carprofen to use in the 2 days immediately following treatment. After this 2-day period of carprofen use, five dogs received carprofen for 23 cumulative days (mean 4.6 days/dog) over the subsequent 6 months. One of those dogs accounted for more than half (12/23 days) of the carprofen usage with the remaining 4 dogs averaging fewer than 3 days of carprofen use over the 6 months. One of these dogs also received 3 days of Gabapentin concurrent with the carprofen. One additional dog didn't receive any caprofen but the owner re-started glucosamine and chondroitin 3 months following treatment.Six owners reported perceived discomfort following injections that resolved by 48 h post injection in all cases.Escherichia coli. The patient was treated with two rounds of hypodermic needle lavage of the affected coxofemoral joint, ultrasound-guided drainage of intramuscular pockets of fluid in the vastus lateralis, and oral and intravenous antibiotics and the infection resolved.One aforementioned patient developed a septic arthritis and septic polymyositis following APS injection. Bacterial culture of fluid obtained from the affected joint and the vastus lateralis both confirmed infection with The results of the CBCs showed a mean increase of 11.1 times (stdev 2.1) the leukocytes in the APS in comparison to the baseline whole blood. There was a mean increase of 1.4 times (1.1) the platelets in the APS when compared to the whole blood.p < 0.01) at all time points for assessment of pain, function, and overall score as assessed using the CBPI. These data are shown in There was a statistically significant improvement (p < 0.05) at all time points for assessment of general mobility and mobility at exercise as assessed using the LOAD questionnaire. These data are shown in There was a statistically significant improvement (p = 0.62), at 1 month (p = 0.7), 3 months (p = 0.22), or 6 months (p = 0.44); p = 0.36), 3 months (p = 0.16), or at 6 months  between the TPI at 3 months in APS treated limbs, or the change in TPI between 0 and 3 months in APS-treated limbs, and either the leukocyte concentration or the platelet concentration in the APS.There was no statistically significant correlation (p = 0.59) in the TPI at 3 months in APS treated limbs for dogs that did or did not receive any carprofen. Similarly, there were no statistically significant differences (p > 0.05) in CBPI or LOAD scores at 3 months post injection between dogs that did or did not receive carprofen in the 6 months following treatment.There was no statistically significant difference  and the TPI of one limb is affected by the weight bearing of the contralateral limb. This phenomenon is easily visualized in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Colorado Canine Orthopedics and Rehab Animal Care and Use Committee. Written informed consent was obtained from the owners for the participation of their animals in this study.SF designed the study, executed data collection, and wrote the manuscript. AF assisted with study coordination, data collection, patient treatment, and manuscript revision. Both authors contributed to the article and approved the submitted version.SF and AF are owner/operators/employees of Franklin Research and Innovation.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Bacillus thuringiensis (Bt) crops have been widely planted and the effects of Bt-crops on populations of the target and non-target insect pests have been well studied. However, the effects of Bt-crops exposure on microorganisms that interact with crop pests have not previously been quantified. Here, we use laboratory and field data to show that infection of Helicoverpa armigera with a densovirus (HaDV2) is associated with its enhanced growth and tolerance to Bt-cotton. Moreover, field monitoring showed a much higher incidence of cotton bollworm infection with HaDV2 in regions cultivated with Bt-cotton than in regions without it, with the rate of densovirus infection increasing with increasing use of Bt-cotton. RNA-seq suggested tolerance to both baculovirus and Cry1Ac were enhanced via the immune-related pathways. These findings suggest that exposure to Bt-crops has selected for beneficial interactions between the target pest and a mutualistic microorganism that enhances its performance on Bt-crops under field conditions. Bacillus thuringiensis bacteria, known as Bt-crops, have become important tools for the management of insect crop pests , was associated with significantly enhanced tolerance of cotton bollworm, H. armigera, to a baculovirus    , and thearmigera . HaDV2 wnd 2012) . The dennd HaDV2 .H. armigera, HaDV2, and Bt-cotton, to establish its relevance to field populations and to test the hypothesis that the widespread adoption of Bt-cotton in China has selected for cotton bollworm carrying the densovirus. Laboratory experiments show that HaDV2 infection in both Cry1Ac-resistant and Cry1Ac-susceptible strains of cotton bollworm enhances larval tolerance to Bt and overall fitness. Field experiments indicate that H. armigera populations infected with HaDV2 also have a higher tolerance to Bt-cotton relative to those not infected with HaDV2. Moreover, field monitoring over a 10-year period indicates that the frequency of HaDV2-infected cotton bollworm is significantly higher in regions planted with Bt-cotton than in those areas where Bt-cotton is not grown, and that in Bt-cotton-growing areas, the prevalence of HaDV2 infection increases with time since Bt-cotton adoption and with the proportion of cotton that is grown which is transgenic. Further, we found that increased Bt tolerance is associated with activated immune pathways to HaDV2 infection. These results indicate that increased HaDV2 infection in cotton bollworm is correlated with the wide-scale adoption of Bt-cotton in China. Our data are consistent with the notion that exposure to Bt-crops selects for beneficial interactions between the target pest and a microorganism that enhances their fitness in response to Bt exposure under field conditions.Here, we further explore the interaction between H. armigera was infected with HaDV2, it increased its tolerance to the Cry1Ac toxin (H. armigera populations that differ in their susceptibility to Cry1Ac due to different mechanisms of resistance. Two susceptible H. armigera strains infected with HaDV2 (96S and LF) both showed 1.5 times greater tolerance to Cry1Ac toxin, relative to their corresponding non-infected controls : χ21=848.16, p<0.0001; Strain*log10(Cry1Ac toxin concentration): χ28=97.30, p<0.0001; HaDV2-status: χ21=9.53, p=0.0020). However, there was no evidence that the benefits of hosting HaDV2 are affected by the tolerance level of the H. armigera strain, as reflected in the LC50 of the non-infected insects . At 8 days post-hatching, H. armigera larvae were significantly lighter  and had lower HaDV2 viral loads in individuals feeding on diet containing Bt than the ones without Bt , suggesting that Bt decreased the replication rate of HaDV2 by suppressing the growth of H. armigera larvae  and were infected or not infected with HaDV2. Enhanced Bt-resistance in H. armigera LF, LF5, LF60, and LF240 strains was associated with lower larval survival rates, prolonged larval and pupal development (in both sexes), reduced pupal weight, lower adult emergence rate, and reduced fecundity and egg hatch rate; in contrast, sex ratio at emergence and the longevity of adults of both sexes were not influenced by the capacity to resist Cry1Ac toxin .To determine whether infection with HaDV2 reduces the costs associated with evolving resistance to Bt, a range of fitness traits  were meaAc toxin . When trains, R0 , this reains, R0 ; strainsH. armigera strains showing a significant increase in R0 relative to their non-infected counterparts, averaging around 38% higher  were comparable to that of the non-infected susceptible (LF) strain  strain .H. armigera larvae was also analyzed on Bt-cotton plant leaves. Larval weight after 9 days growth was significantly affected by the cotton variety (Bt or non-Bt) and by the HaDV2 infection-status  and Anci (Hebei Province) in northern China, two locations where Bt-cotton has been widely planted over the last decade   . Associa<0.0001) . Moreove<0.0001) .H. armigera densovirus infection levels and the adoption of Bt-cotton, we monitored HaDV2 infection rates at 36 locations across 16 provinces during the period 2014–2016, including locations planted with transgenic Bt-cotton (29 monitoring points across 12 provinces) and locations where Bt-cotton has not been planted (9 monitoring points across 4 provinces) (H. armigera were significantly higher at locations where Bt-cotton was planted (mean=82%) than in those where it was not (15%) . There was also a significant year-by-crop interaction  due to HaDV2 infection levels being uniformly high across the 3 years at sites growing Bt-cotton (81–90%), whereas infection levels gradually increased from 2014 to 2016 at non-Bt sites .To further test the association between ovinces) . Across ot (15%)    . Adding environmental variables to this minimal model, either singly or in combination, did not significantly improve the model fit . These results are consistent with the notion that the benefits of HaDV2 infection are greatest for H. armigera exposed to Cry1Ac-producing cotton and that selection favoring HaDV2 infection increases the longer the insects are exposed to the Bt-cotton.Moreover, across the provinces where Bt-cotton is grown, the mean prevalence of HaDV2 in <0.0001)  and alsop=0.021) . HaDV2 pTo try to understand better the mechanisms increasing the Cry1Ac tolerance levels and enhanced fitness of HaDV2-infected insects, we conducted an RNA sequencing experiment .The principal component analysis of the transcriptome with differentially expressed genes (DEGs) data clearly distinguished HaDV2-positive from HaDV2-negative individuals at three different time points: 24, 48, and less so at 72 hr after HaDV2 inoculation . Taken tWe performed pathway enrichment analysis on the DEGs, focusing particular attention on pathways related to the development and immune systems . Genes iHaDV2-negative insects after exposure to Cry1Ac (673 upregulated and 900 downregulated). We focused on DEGs and pathways related to Bt tolerance and immune systems. Seven ABC transporter genes, which are related to immunity, were differentially expressed (four upregulated and three downregulated)  ; eight tgulated) ; two cargulated) . Genes igulated) .HaDV2-positive insects after exposure to Cry1Ac (165 upregulated and 84 downregulated). One trypsin gene was downregulated; genes in the ascorbate and aldarate metabolism pathway, which is related to carbohydrate metabolism were upregulated; genes in drug metabolism – cytochrome P450 pathway, metabolism of xenobiotics by cytochrome P450 pathway and drug metabolism – and other enzymes pathways, which are related to detoxification, were also upregulated  when this exposure has been for prolonged periods of time. Intensive monitoring in two provinces where Bt-cotton has been grown over a number of years showed a strong temporal increase in HaDV2 infection levels, from around 40% in 2007 to nearly 100% in 2016, with high levels of HaDV2 infection being associated with higher larval growth rates on Bt-cotton in the field. Indeed, at locations where Bt-cotton is not grown, H. armigera shows much lower rates of HaDV2 infection (12–44% vs. 81–90%), consistent with weaker selection for HaDV2-infected H. armigera in the absence of Bt-crops. Moreover, the prevalence of HaDV2 infection increased with the number of years since Bt-cotton was first introduced to a province and with the proportion of cotton grown that is transgenic, consistent with Bt-cotton being a key selection pressure. There were no significant relationships between HaDV2 prevalence and the proportional cover of any other main crops grown locally  or with any of the environmental variables tested . Thus, our data suggest that the Cry1Ac-toxin selected for HaDV2-infected H. armigera following exposure to Bt-cotton and our RNA-Seq analysis suggests that the increased tolerance of HaDV2-infected insects is caused, in part at least, by the activation of a series of immune pathways and pathways that enhance development.Commercialization of transgenic Bt-crops has brought some significant benefits to farmers . These BRickettsia sp. nr. bellii, swept through a population of invasive sweet potato whitefly, Bemisia tabaci, in just 6 years   . Consistth model  suggeststh model .H. armigera in China, it is possible that the association between the HaDV2 and its host is more ancient, but that the increase in HaDV2 prevalence in recent years is because it has only recently evolved from a parasitic or commensal relationship into one that is more mutualistic , which is located in Bohai Sea between Bt- and non-Bt-cotton areas and where there were no resident populations of H. armigera  following the wide-scale adoption of Bt-cotton in China and the subsequent reduction in pesticide application rate on crops   ; howeverH. armigera, despite the failure to identify the key genetic locus conferring resistance in these populations  . In a prulations . The relulations  suggestselopment . Lepidopelopment . HaDV2-id larger . Therefod larger , and thed larger , so immuH. armigera larvae. Due to the observed effects of HaDV2 on its host, we focused on molecular pathways related to immunity and development  would be determined by the interaction between HaDV2 infected-status and cotton plant Bt-status. A similar experiment to this was conducted by Rickettsia, who found that over five generations, the prevalence of Rickettsia increased from 14% in the parental generation to 40–70% on three different host plant species. Repeating such experiments with H. armigera is feasible but logistically much more difficult due to their much larger size. In summary, here, we report the rapid increase in the prevalence of a mutualistic virus in a major crop pest and show that its spread is greatest for field populations exposed to Bt-cotton for prolonged periods. Our findings support a novel mechanism by which insects may cope with changes in engineered host plant defenses, namely via infection with a mutualistic virus that enhances their tolerance to the toxins expressed by Bt-cotton crops. Our study has significant implications for understanding the co-evolution of host-pathogen-symbiont interactions. Although we show that HaDV2 increases the tolerance of its host to Bt, it is not feasible to manipulate HaDV2 infection status in the field in order to enhance the capacity of Bt to manage H. armigera because of the lack of suitable antivirals (against HaDV2) and because of the efficient vertical and horizontal transmission of this virius in the field.Whilst data from the field are inevitably correlational, when combined with the laboratory experiments, we feel that the most parsimonious explanation for the observations reported here is that HaDV2 directly or indirectly enhances tolerance of Cry1Ac was obtained as a gift from Dow AgroSciences  in product formulation MVPII (20%). To avoid degradation, Cry1Ac protoxin was stored at −80°C. To exclude the possibility of a decline in protoxin potency over time, the susceptible strains (LF) were used as the internal control in different years .H. armigera strains were used: two susceptible strains (LF and 96S) and eight Cry1Ac-resistant strains , with different resistance ratios due to different resistant mechanisms , and in Anci County, Hebei Province (with a multiple-crop farming system that includes cotton). The history of Bt-cotton deployment in the two locations during 2007–2013 has been reported previously . In the reported .GGATTGGCCTGGGAAATGAC and HaDV-R: CGTTGTTTTTATATCCGAGG. The remaining debris of HaDV2-positive individuals was transferred to 1 mL phosphate-buffered saline buffer on ice. The homogenate was centrifuged and the liquid supernatant was subsequently filtered with 0.22 µm membrane filter (Sigma-Aldrich). The HaDV2-containing filtered liquid (200 µL per tube) was collected and stored immediately at −80°C. Quantification of the virus was performed using the qPCR method  in 24-well plates. The plates were covered with a plastic lid to prevent escape and 72 larvae were tested per Cry1Ac concentration. After 7 days, the mortality was recorded and body mass of individuals that were still alive was measured. Both dead larvae and those with a body mass of less than 5 mg were recorded as dead. For each strain, the median lethal concentration (LC50) value was determined using a Probit analysis  and the resistance level to Cry1Ac indicated by the resistance ratio was calculated with LC50 of the tested strains divided by the LC50 of the susceptible strain.reported . In brieR0) was calculated for each H. armigera strain  were obtained with life-table techniques . The coma strain  as R0=NtTo analyze the fitness cost associated with Cry1Ac-resistance, the larval weight of the different strains was analyzed on Bt/non-Bt-cotton. Newly hatched larvae were first orally inoculated with either filtered-liquid containing HaDV2 or filtered-liquid from non-infected individuals. After that, they were placed in each treatment Petri dish for 2 days to ensure that larvae ingested the treated diet. They were then transferred individually onto Bt-cotton (Zhongmian 29) or non-Bt-cotton (Zhongmian 24) leaf in plastic wells in 24-well plates. After 9 days, the weight of individuals was measured. Each treatment had 48 larvae with three repeats.To obtain F1 generation offspring of each of the laboratory strains, one virgin adult male and one virgin adult female were paired in a 250 mL clear plastic cup. Adult females collected from the field were placed individually into 250 mL clear plastic cups and covered with gauze to provide a substrate for egg laying. Eggs were collected on a daily basis. At larval hatch, 24 neonates from each female line were placed on non-Bt diet, and 24 neonates were placed on Cry1Ac-containing diet (1 μg of Cry1Ac/g diet). The concentration of Bt Cry1Ac protoxin was 1.0 μg/g diet. The composition of the diet was previously described . RearingH. armigera at a transcriptomic level, we collected samples of the HaDV2-negative and HaDV2-positive individuals from single pairs of H. armigera and performed RNA-seq, using larvae at 24, 48, and 72 hr after hatching. Briefly, newly hatching larvae were fed on filtered liquid with/without HaDV2 as described previously (To determine the effect of HaDV2 on eviously . Sampleseviously . The Trieviously  was usedeviously  with no eviously . Quantiteviously  with defeviously  was usedeviously .8 copies/μL) or filtered-liquid from non-infected individuals (control). One hundred N-strain neonates were placed in each treatment Petri-dish for 2 days to ensure that larvae ingested the treated diet. They were then transferred to a 24-well plate  containing the artificial diet containing 1 µg/mL Cry1Ac toxins. After 48 hr, the larvae were collected and stored at −80°C for transcriptome sequencing. Twenty larvae were pooled together as a sample. A total of 12 samples were sequenced including three replications of four treatments .Offspring from a single uninfected breeding pair were reared to produce the N-strain (uninfected) laboratory culture. Neonate N-strain larvae were first orally inoculated with either filtered-liquid containing HaDV2   , was use In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Your work, which will be of interest to a broad audience interested in microbe-insect interactions and how they may affect adaptation to pesticides, nicely indicates that infection with a mutualistic virus can enhances fitness of a moth, and that selection pressure represented by transgenic crops may be driving the spread of mutualistic infections in moth populations.Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Detlef Weigel as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Rapid spread of a symbiotic virus in a major crop pest following wide-scale adoption of Bt-cotton in China" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential Revisions:1) Change the title to more precisely reflect the most certain conclusions of the study.2) Clearly distinguish between resistance (the evolution of increased tolerance due to genetic changes in the pest population) and other mechanisms of increased tolerance (e.g. virus infection).3) Provide the minimum detection limit for the densovirus, to ensure that latent infection is not missed.4) In the Discussion, consider and evaluate an alternative hypothesis; that increased virus infection and increased tolerance of Bt cotton are parallel but independent responses to the stress of feeding on Bt cotton.5) Redraw Figure 1 with the more appropriate units of LC50 rather than resistance ratio.6) Clarify questions about the identity and uniqueness of the virus.7) In the Discussion, use existing data to estimate the potential impact on reduction of pest control efficacy by Bt cotton due to densovirus infection in relevant cotton-growing areas of China; and compare this impact to estimated reduction in efficacy due to cadherin mutations and due to mutations in the ABC transporter.8) In the Discussion, describe the ideal approach/dataset that would conclusively show that infection by the densovirus causes (not merely is associated with) the increased tolerance of Bt cotton, and evaluate this approach with respect to feasibility. Likewise evaluate the approaches presented in the manuscript relative to this ideal approach.9) In the Discussion, discuss the feasibility or infeasibility of manipulating the virus infection status to increase the pest control efficacy by Bt cotton.Reviewer #1:Previous reports have provided evidence identifying infection of cotton bollworm with a densovirus as resulting in increased fitness. In the current manuscript, the relevance of this infection towards field resistance to transgenic Bt corn is evaluated by comparing its incidence between regions in China growing non-Bt versus Bt cotton. A clear correlation emerges with infection rates being higher in Bt versus non-Bt cotton growing areas, although its effect on resistance to Cry1Ac and Bt cotton is not as clear.Strengths:The manuscript presents evidence for the spread of densovirus infection in field bollworm populations, and that this spread seems to occur at a faster rate in areas of China where Bt cotton is grown versus non-Bt cotton areas.Life table comparisons clearly show increased fitness in bollworms infected with the virus.The study capitalizes on availability of an impressive collection of samples with distinct geographic and historic origin to address relevant evolutionary questions.Weaknesses:The suggested role for densovirus infection in resistance to Cry1Ac and Bt cotton is supported by association and the data presented does not necessarily support causation. In fact, the confidence intervals in all the comparisons from bioassays overlap substantially and the resulting resistance ratio is not a good estimate of any significant differences the infection may have on ability to survive Cry1Ac. Infection by a virus is expected to activate the immune system, so the larvae used in bioassays should be considered as "primed" and the slight reduction in susceptibility should not be considered as an effect of the virus itself.The life table data clearly shows that fertility and fecundity are probably the most relevant aspects affecting fitness of infected insects. These differences in reproduction  could explain why infection is rapidly spreading in the wild. However, most of the research and analyses are focused on the possibility that the viral infection may make the insects more able to survive Cry1Ac or Bt cotton. There are no conclusive data supporting this hypothesis in the current version, other than increased infection rates in Bt-cotton growing areas. This could be explained by effects on reproduction rather than enhanced survival.Related to this aspect, there should be a more clear distinction between the densovirus increasing fitness versus increasing resistance, the data supports the former but is not so clear in the later.It would be useful to provide a map detailing regions were moths were collected.The putative effect of infection on susceptibility could be strengthened by comparing effects of treating the larvae with a non-pathogenic microbe other than the densovirus. One would expect a similar priming of the immune response, making the larva less susceptible to subsequent exposure to Cry1Ac.The text may be misleading as written as in several statements suggests densovirus infection is related to resistance to Cry1Ac.Resistance is not due to the densovirus infection, but to other mechanisms.More focus needs to be put on the effect of viral infection increasing reproductive output rather than larval growth. It is plausible that the former will have a more relevant role on the spread of infection. The observed higher frequency of infection in Bt cotton areas could be due to participation of infection in survival (as hypothesized) or may be a result of initial selection of individuals that were infected and subsequent spread due to higher reproductive output.Reviewer #2:H. armigera and HaDV2 is more a mutualistic relationship, thus, the authors should consider changing the titles of the manuscript and the supplementary data. This is an exciting study and is well written and will be of general interest to the field.The various pathogenic, parasitic, symbiotic, and mutualistic interactions between insects and the microbes they interact with represents a rich area of research. This study by Xiao et al. represents a very interesting example of such a relationship. Overall the study is well designed and executed. The approach they utilize to test their hypothesis is valid and they combined both laboratory and field collected insects to address the question. The RNAseq analysis also provides potential insights into possible mechanisms by which the virus HaDV2 enables enhanced resistance to Bt Cry1Ac. The RNAseq data also represent one of the minor issues. The authors focused on analyzing only development and immune systems, however, they do not report on any other significantly different changes in gene expression other than reporting that there were 1573 significant differences. The authors should at least provide some holistic analysis and report the data in the supplemental results. Focusing on development and immune systems is valid and rationally supported but a complete analysis should be presented. The relationship between Heliothis armigera and the development of increased resistance to the Bt toxin Cry1Ac in transgenic cotton. Previous studies from this group had shown that infection of H. armigera with the densovirus H. armigera densovirus-1 (HaDV2) enhanced resistance to baculovirus infections and potentially increased resistance to Cry1Ac with no detrimental impacts to fitness. In this study they test the hypothesis that the large adoption of transgenic Bt-cotton has selected for H. armigera infected with HaDV2. To test this they examined different H. armigera strains with and without HaDV2 and observed a significant increase in resistance in all strains. Previous studies has shown Bt resistance has fitness costs, they showed that resistant strains had improved reproductive rate if the strain was infected with HaDV2 apparently compensating for the fitness costs of resistance. Analysis of field collected larvae from Bt-cotton and non-Bt-cotton regions over the last 10 years showed a direct correlation with HaDV2 infection. To address the question of molecular basis for the enhanced resistance to Bt cotton when infected with HaDV2, resistant and non-resistant strains with or without HaDV2 infection were analysed by RNAseq. Differences in gene expression was bioinformatically analyzed. Focusing on biochemical pathways related to development and immune systems a number of pathways were identified which could account for the observed enhanced fitness of Bt resistant larvae. They conclude the selection of the this mutualistic virus has been selected for by the widespread use of Bt-cotton.In this study Xiao et al., report on the association of an insect virus with the lepidopteran host Additional guidance for Essential Revisions:1) Title: The word "symbiotic" should be removed from the title. Since viruses are not free-living, they are symbiotic by definition . Whether the word "mutualistic" can be used in its place will depend on whether the authors can demonstrate a causal link between presence of the virus and greater fitness (not sufficiently shown in the current version). "Densovirus" should replace "virus" in the title.H. armigera resistance to the granulovirus. When discussing the response of the insect to the virus, words like "tolerance", "virulence", or "susceptibility" must be used. If an individual insect's tolerance to virus infection increases as a result of feeding on Bt cotton, this is not resistance. Previous publications from this group also misused the word "resistance", but that is not a sufficient reason for its continued misuse here.2) Use of the word "resistance" must be confined to genetic changes in a population which increase its tolerance to an external stress as a result of previous exposure to that stress. "Resistance" cannot be used as a synonym for "tolerance". There is no evidence presented in the manuscript for By the same token, any increase in an individual insect's tolerance to Bt cotton caused by exposure to the virus, or genes upregulated in response to the virus, is not resistance, and must not be referred to as such. "Enhanced resistance" or "increase in resistance level" may not be used. Instead, two components of tolerance should be discussed, one due to genetic resistance to Cry toxin caused by a specific mechanism (e.g. mutations in the cadherin or ABC transporter), and the other due to the difference between presence or absence of the virus.not to harbor the virus must be explicitly stated here.3) Detection limit for latent virus: Has the virus actually spread, or has the stress of Bt cotton converted it from a universally-present latent form to an active form in some stressed individuals? A latent virus might be present in all individuals--can the authors rule out this possibility? The authors need to give the detection limit of their PCR technique, in term of virus genomes per single-copy gene per cell of the insect. The reference cited  describes the use of a standard curve for copy number estimation but does not describe the detection limit. The criterion for when a field-collected insect is declared 4) Parallel response to stress: Apart from the issue of latent viruses; the higher incidence of virus in areas of Bt cotton than non Bt-cotton, and the increase in virus incidence with the amount and proportion of Bt-cotton over time, could be partly or completely explained by increased stress on the insects feeding on Bt cotton, that weakens their defenses against the virus and allows the virus to increase within the insect. How can the authors rule this out? Even if this is only a partial explanation, how much of the higher incidence of virus could be explained by this factor, rather than a protective role of the virus against Bt toxin?5) In Figure 1, the slope is dependent on which strain is chosen for the susceptible reference, and the labelling of the axes confounds the two components of tolerance. The figure should be redrawn with LC50 as the units on both axes. The scale may be either linear or logarithmic.Helicoverpa armigera densovirus strain SDBH2010-1". Please provide enough information in the Supplement to remove any doubt about which virus is being discussed. Since the GenBank record names a specific strain, can we assume that there are many different strains infecting H. armigera in nature? This is also implied by the switch from -1 to -2 in the abbreviation. If there is an HaDV2, does that mean there is also a HaDV1? Which of them can be detected by the PCR method? Which of them are being counted in the increase?6) Uniqueness of the virus: Two abbreviations for the virus are used: HaDNV-1 and HaDV2; mostly HaDV2. The study by Xu et al. uses HaDNV-1. The GenBank record  calls it "7) Estimation of potential impact: This will require assumptions about the relationship between the reproductive rate and crop damage caused by the pest. Clearly state these assumptions, possibly using two or three different relationships, so that their robustness can be evaluated.8) Ideal demonstration of causality: In the laboratory, this would involve infecting a virus-free strain of bollworm, and then curing the strain of the virus so that pre-infection, infection, and post-infection performance could be compared. Is this even possible in the laboratory? In the field?9) Evidence was presented in earlier work that densovirus infection can reduce baculovirus infection. Does a latent or active baculovirus infection have any prospects to reverse the effect of densovirus on tolerance of Bt-cotton? Essential Revisions:1) Change the title to more precisely reflect the most certain conclusions of the study.We changed the title to “Rapid spread of a densovirus in a major crop pest following wide-scale adoption of Bt-cotton in China”.2) Clearly distinguish between resistance (the evolution of increased tolerance due to genetic changes in the pest population) and other mechanisms of increased tolerance (e.g. virus infection).We have adjusted several expressions in the article in order to better distinguish resistance and tolerance. .3) Provide the minimum detection limit for the densovirus, to ensure that latent infection is not missed.Accepted, we added the detection limit of PCR for HaDV2.“the virus detection limits were tested with DNA extracted from HaDV2-positive individuals and the plasmid generated with the fragments amplified with the primers for detection of HaDV2 and pEASY-T1 cloning vector . This showed that the detection limit for HaDV2 with PCR method was > 61.85 copies/µL or 0.13 ng /µL DNA .” .4) In the Discussion, consider and evaluate an alternative hypothesis; that increased virus infection and increased tolerance of Bt cotton are parallel but independent responses to the stress of feeding on Bt cotton.Accepted, we provided additional experiment and added some discussion about the relationship between HaDV2 infection and the tolerance of Bt cotton as follows:Methods: To test whether the Bt could change the replication rate of HaDV2 in their hosts, newly-hatched larvae from single pair matings, which both the parents and larvae were with low dose of HaDV2, fed on artificial diets with either Bt (0.5 µg/g) or without Bt and quantified larval growth and virus titres 8 days later using the qPCR method . H. armigera larvae were significantly lighter  and had lower HaDV2 viral loads in individuals feeding on diet containing Bt than the ones without Bt , suggesting that Bt decreased the replication rate of HaDV2 by suppressing the growth of H. armigera larvae . .Results: At 8 days post-hatching, H. armigera as very low-titre covert infections, and that the higher incidence of virus in areas of Bt-cotton was due to the stress associated with larvae feeding on plants expressing Bt toxin. To test the hypothesis, using larvae with low dose of HaDV2 from single pair, we quantified the copy numbers of HaDV2 within the cotton bollworms feeding on artificial diets with or without Bt toxin. Interestingly, the results showed that feeding on the Bt toxin resulted in lower titres of HaDV2, which might be due to the Bt suppressing the growth of H. armigera larvae. These results suggest that the higher infection rate of HaDV2 might be due to lower mortality by higher Bt tolerance and the increased reproduction of the cotton bollworms with HaDV2 than those without. .Discussion: Using the PCR method, it is not possible to discount the possibility that HaDV2 was widespread in field populations of 5) Redraw Figure 1 with the more appropriate units of LC50 rather than resistance ratio.H. armigera strains with or without HaDV2 infection. Accepted. The figure 1 were redrew by LC50 of 6) Clarify questions about the identity and uniqueness of the virus.Helicoverpa armigera. At the beginning, densovirus was abbreviated as “DNV” . For distinguishing the densovirus with ambisense genome in H. armigera which was named as HaDNV , we named the densovirus we find as HaDNV-1 . However, the ICTV changed the abbreviation of densovirus as “DV” and named the HaDNV as HaDV1 in 2014 . Accordingly, we changed HaDNV-1 to HaDV2 and using HaDV2 stand for the densovirus we found in newly published paper . We added the illustration .Accepted . HaDNV-1 and HaDV2 are pseudonyms for the same virus we found in Cotmore SF, Agbandje-McKenna M, Chiorini JA, Mukha DV, Pintel DJ, Qiu J, Soderlund-Venermo M, Tattersall P, Tijssen P, Gatherer D, Davison AJ 2014. The family Parvoviridae. Arch Virol 159: 1239-1247. doi: 10.1007/s00705-013-1914-1El-Far M, Szelei J, Yu Q, Fediere G, Bergoin M, Tijssen P 2012. Organization of the Ambisense Genome of the Helicoverpa armigera Densovirus. J Virol 86: 7024.Tijssen P, van den Hurk J, Kurstak E 1976. Biochemical, biophysical, and biological properties of densonucleosis virus. I. Structural proteins. J Virol 17: 686-691.Xu P, Cheng P, Liu Z, Li Y, Murphy RW, Wu K 2012. Complete genome sequence of a monosense densovirus infecting the cotton bollworm, Helicoverpa armigera. J Virol 86: 10909.Xu P, Graham RI, Wilson K, Wu K 2017a. Structure and transcription of the Helicoverpa armigera densovirus (HaDV2) genome and its expression strategy in LD652 cells. Virol J 14: 23.Xu P, Liu Y, Graham RI, Wilson K, Wu K 2014. Densovirus Is a Mutualistic Symbiont of a Global Crop Pest (Helicoverpa armigera) and Protects against a Baculovirus and Bt Biopesticide. PLoS Pathog 10: e1004490.Xu P, Yuan H, Yang X, Graham RI, Liu K, Wu K 2017b. Structural proteins of Helicoverpa armigera densovirus 2 enhance transcription of viral genes through transactivation. Arch Virol 162: 1745-1750.7) In the Discussion, use existing data to estimate the potential impact on reduction of pest control efficacy by Bt cotton due to densovirus infection in relevant cotton-growing areas of China; and compare this impact to estimated reduction in efficacy due to cadherin mutations and due to mutations in the ABC transporter.H. armigera but with significant fitness costs (e.g. lower reproduction) , however, HaDV2 infection could enhance host reproduction and tolerance to Cry1Ac in both susceptible and resistant strains of H. armigera, increasing larval survival rate. .Accepted. We added to the discussion. The Bt resistance induced by ABCC2 and cadherin is due to genomic heritable variation , while the Bt tolerance induced by HaDV2 appears to be due to the regulation of immune and other pathways. ABCC2 and cadherin can directly mediate high levels of resistance to Bt toxin in 8) In the Discussion, describe the ideal approach/dataset that would conclusively show that infection by the densovirus causes (not merely is associated with) the increased tolerance of Bt cotton, and evaluate this approach with respect to feasibility. Likewise evaluate the approaches presented in the manuscript relative to this ideal approach.We have added a new section to the Discussion:H. armigera to Bt in the field, probably via increasing larval growth rates and via priming of immune defences. Demonstrating causation conclusively in the field will prove difficult however. One possibility would be to plant some Bt cotton in areas that are currently Bt-free and to compare larval growth patterns and HaDV2 infection levels in neighbouring Bt and non-Bt areas, mirroring the ‘natural’ field experiments we report here. Even this approach, though, would not demonstrate causality conclusively. We envisage that HaDV2 infection levels will eventually go to fixation (or close to it) in both Bt- and non-Bt areas, due to the growth benefits associated harbouring this mutualist but that the time to get to this point will take longer in non-Bt areas, due to the lower fitness benefits. Likewise, we envisage that the spread of HaDV2 infection would slow down in Bt-areas if Bt-cotton were to be replaced by conventional cotton or a GM variety that did not rely on Bt toxins.” .“Whilst data from the field are inevitably correlational, when combined with the laboratory experiments, we feel that the most parsimonious explanation for the observations reported here is that HaDV2 directly enhances tolerance of 9) In the Discussion, discuss the feasibility or infeasibility of manipulating the virus infection status to increase the pest control efficacy by Bt cotton.H. armigera because of the lack of suitable anti-virals (against HaDV2) and because of the efficient vertical and horizontal transmission of this virius in the field.” .Accepted, we added the discussion as follows: “Although we show that HaDV2 increases the tolerance of its host to Bt, it is not feasible to manipulate HaDV2 infection status in the field in order to enhance the capacity of Bt to manage Reviewer #1:Previous reports have provided evidence identifying infection of cotton bollworm with a densovirus as resulting in increased fitness. In the current manuscript, the relevance of this infection towards field resistance to transgenic Bt corn is evaluated by comparing its incidence between regions in China growing non-Bt versus Bt cotton. A clear correlation emerges with infection rates being higher in Bt versus non-Bt cotton growing areas, although its effect on resistance to Cry1Ac and Bt cotton is not as clear.Strengths:The manuscript presents evidence for the spread of densovirus infection in field bollworm populations, and that this spread seems to occur at a faster rate in areas of China where Bt cotton is grown versus non-Bt cotton areas.Life table comparisons clearly show increased fitness in bollworms infected with the virus.The study capitalizes on availability of an impressive collection of samples with distinct geographic and historic origin to address relevant evolutionary questions.Weaknesses:The suggested role for densovirus infection in resistance to Cry1Ac and Bt cotton is supported by association and the data presented does not necessarily support causation. In fact, the confidence intervals in all the comparisons from bioassays overlap substantially and the resulting resistance ratio is not a good estimate of any significant differences the infection may have on ability to survive Cry1Ac. Infection by a virus is expected to activate the immune system, so the larvae used in bioassays should be considered as "primed" and the slight reduction in susceptibility should not be considered as an effect of the virus itself.The life table data clearly shows that fertility and fecundity are probably the most relevant aspects affecting fitness of infected insects. These differences in reproduction  could explain why infection is rapidly spreading in the wild. However, most of the research and analyses are focused on the possibility that the viral infection may make the insects more able to survive Cry1Ac or Bt cotton. There are no conclusive data supporting this hypothesis in the current version, other than increased infection rates in Bt-cotton growing areas. This could be explained by effects on reproduction rather than enhanced survival.Related to this aspect, there should be a more clear distinction between the densovirus increasing fitness versus increasing resistance, the data supports the former but is not so clear in the later.It would be useful to provide a map detailing regions were moths were collected.The putative effect of infection on susceptibility could be strengthened by comparing effects of treating the larvae with a non-pathogenic microbe other than the densovirus. One would expect a similar priming of the immune response, making the larva less suceptible to subsequent exposure to Cry1Ac.H. armigera with Wolbachia but we failed due to very low infection rate of Wolbachia in field populations of H. armigera. We didn’t find any other non-pathogenic microbes in field populations of H. armigera but if we do, the proposed experiment would be feasible.Many thanks for the suggestions. That might be interesting and we also tried to construct strains of The text may be misleading as written as in several statements suggests densovirus infection is related to resistance to Cry1Ac.Resistance is not due to the densovirus infection, but to other mechanisms.H. armigera to Cry1Ac toxin. But Resistance is not due to the HaDV2 infection. In order to avoid misleading readers, we have made necessary revisions to the relevant descriptions in the results and Discussion section of manuscript . Please see details in the revised manuscript with tracking changes.Accepted. HaDV2 infection could increases the tolerance level of More focus needs to be put on the effect of viral infection increasing reproductive output rather than larval growth. It is plausible that the former will have a more relevant role on the spread of infection. The observed higher frequency of infection in Bt cotton areas could be due to participation of infection in survival (as hypothesized) or may be a result of initial selection of individuals that were infected and subsequent spread due to higher reproductive output.H. armigera, but it is worth noting that the reproductive rate of HaDV2-positive individuals is ~20% greater than that of HaDV2-negative individuals and that survival from egg to adult is also ~20% higher for individuals carrying HaDV2, suggesting that both life-history traits contribute to the enhanced 0R of HaDV2-infected individuals.We cannot be sure about the relative importance of increased growth, survival or reproduction in determining the spread of HaDV2 in Additional guidance for Essential Revisions:1) Title: The word "symbiotic" should be removed from the title. Since viruses are not free-living, they are symbiotic by definition . Whether the word "mutualistic" can be used in its place will depend on whether the authors can demonstrate a causal link between presence of the virus and greater fitness (not sufficiently shown in the current version). "Densovirus" should replace "virus" in the title.Accepted. We changed the title to “Rapid spread of a densovirus in a major crop pest following wide-scale adoption of Bt-cotton in China”.2) Use of the word "resistance" must be confined to genetic changes in a population which increase its tolerance to an external stress as a result of previous exposure to that stress. "Resistance" cannot be used as a synonym for "tolerance". There is no evidence presented in the manuscript for H. armigera resistance to the granulovirus. When discussing the response of the insect to the virus, words like "tolerance", "virulence", or "susceptibility" must be used. If an individual insect's tolerance to virus infection increases as a result of feeding on Bt cotton, this is not resistance. Previous publications from this group also misused the word "resistance", but that is not a sufficient reason for its continued misuse here.By the same token, any increase in an individual insect's tolerance to Bt cotton caused by exposure to the virus, or genes upregulated in response to the virus, is not resistance, and must not be referred to as such. "Enhanced resistance" or "increase in resistance level" may not be used. Instead, two components of tolerance should be discussed, one due to genetic resistance to Cry toxin caused by a specific mechanism (e.g. mutations in the cadherin or ABC transporter), and the other due to the difference between presence or absence of the virus.Accepted. We changed “resistance” to “tolerance” and changed several descriptions in this paper in order to better distinguish resistance and tolerance .3) Detection limit for latent virus: Has the virus actually spread, or has the stress of Bt cotton converted it from a universally-present latent form to an active form in some stressed individuals? A latent virus might be present in all individuals--can the authors rule out this possibility? The authors need to give the detection limit of their PCR technique, in term of virus genomes per single-copy gene per cell of the insect. The reference cited  describes the use of a standard curve for copy number estimation but does not describe the detection limit. The criterion for when a field-collected insect is declared not to harbor the virus must be explicitly stated here.Accepted, please see the answer for Essential Revisions Question 3.4) Parallel response to stress: Apart from the issue of latent viruses; the higher incidence of virus in areas of Bt cotton than non Bt-cotton, and the increase in virus incidence with the amount and proportion of Bt-cotton over time, could be partly or completely explained by increased stress on the insects feeding on Bt cotton, that weakens their defenses against the virus and allows the virus to increase within the insect. How can the authors rule this out? Even if this is only a partial explanation, how much of the higher incidence of virus could be explained by this factor, rather than a protective role of the virus against Bt toxin?Accepted, Accepted, please see the answer for Essential Revisions Question 4.5) In Figure 1, the slope is dependent on which strain is chosen for the susceptible reference, and the labelling of the axes confounds the two components of tolerance. The figure should be redrawn with LC50 as the units on both axes. The scale may be either linear or logarithmic.Please see the answer for Essential Revisions Question 5.6) Uniqueness of the virus: Two abbreviations for the virus are used: HaDNV-1 and HaDV2; mostly HaDV2. The study by Xu et al., uses HaDNV-1. The GenBank record  calls it "Helicoverpa armigera densovirus strain SDBH2010-1". Please provide enough information in the Supplement to remove any doubt about which virus is being discussed. Since the GenBank record names a specific strain, can we assume that there are many different strains infecting H. armigera in nature? This is also implied by the switch from -1 to -2 in the abbreviation. If there is an HaDV2, does that mean there is also a HaDV1? Which of them can be detected by the PCR method? Which of them are being counted in the increase?Accepted, please see the answer for Essential Revisions Question 6.7) Estimation of potential impact: This will require assumptions about the relationship between the reproductive rate and crop damage caused by the pest. Clearly state these assumptions, possibly using two or three different relationships, so that their robustness can be evaluated.0, for HaDV2-infected insects was around 40% higher than that of their non-infected counterparts, which probably sets an upper limit to the potential impact of HaDV2 infection. Following Himler (2011), a simple population growth model based on these R0 estimates suggests that HaDV2 would spread to close to fixation in Bt-cotton growing areas in 20-30 generations  .Estimating the potential impact of HaDV2 and its interaction with Bt-cotton would probably require an extensive agronomic modelling exercise, which is beyond the scope of the present study, but it is worth noting that the estimated fundamental net reproductive rates, R8) Ideal demonstration of causality: In the laboratory, this would involve infecting a virus-free strain of bollworm, and then curing the strain of the virus so that pre-infection, infection, and post-infection performance could be compared. Is this even possible in the laboratory? In the field?Right, we also tried to compare pre-infection (HaDV-), infection (HaDV2 infecting) and post-infection (HaDV2+) with samples collected at 24h, 48h and 72h after HaDV2 exposure. We conducted an RNA sequencing experiment may provide some answers to this question. The principal component analysis of the transcriptome with differentially expressed gene data clearly distinguished HaDV2-positive from -negative individuals at three different time points: 24 h, 48 h and less so at 72 h after HaDV2 inoculation, suggesting that the HaDV2 has a major effect on the gene expression profiles of their hosts. However, we thought our results only stand for pre-infection and post-infection. It’s difficult to determine the samples which were being infected by HaDV2. See pages 9-10, lines 212-234 for details.9) Evidence was presented in earlier work that densovirus infection can reduce baculovirus infection. Does a latent or active baculovirus infection have any prospects to reverse the effect of densovirus on tolerance of Bt-cotton?Many thanks for the suggestion. The covert infection of NPV was naturally occurred . It’s interested to run a novel investigation to determine whether latent or active baculovirus infection has any prospects to reverse the effect of HaDV2 on tolerance of Bt by series bioassays.Kemp EM, Woodward DT, Cory JS 2011. Detection of single and mixed covert baculovirus infections in eastern spruce budworm, Choristoneura fumiferana populations. J Invertebr Pathol 107: 202-205.Murillo R, Hussey MS, Possee RD 2011. Evidence for covert baculovirus infections in a Spodoptera exigua laboratory culture. J Gen Virol 92: 1061-1070.Williams T, Virto C, Murillo R, Caballero P 2017. Covert Infection of Insects by Baculoviruses. Front Microbiol 8: 1337.