Pseudococcus calceolariae, a mealybug commonly found in New Zealand vineyards. To help inform alternative GLRaV-3 control strategies, this study evaluated the three-way interaction between the mealybug, its plant host and the virus. The retention and transmission of GLRaV-3 by P. calceolariae after access to non-Vitis host plants (and a non-GLRaV-3 host) White clover (Trifolium repens L. cv. “Grasslands Huia white clover”), Crimson clover (T. incarnatum), and Nicotiana benthamiana  was investigated. For all experiments, P. calceolariae first instars with a 4 or 6 days acquisition access period on GLRaV-3-positive grapevine leaves were used. GLRaV-3 was detected in mealybugs up to 16 days on non-Vitis plant hosts but not after 20 days. GLRaV-3 was retained by second instars (n = 8/45) and exuviae  following a 4 days acquisition period on infected grapevines leaves and an 11 days feeding on non-Vitis plant hosts. Furthermore, GLRaV-3 was transmitted to grapevine (40−60%) by P. calceolariae second instars after access to white clover for up to 11 days; 90% transmission to grapevine was achieved when no alternative host feeding was provided. The 16 days retention period is the longest observed in mealybug vectoring of GLRaV-3. The results suggest that an alternative strategy of using ground-cover plants as a disrupter of virus transmission may be effective if mealybugs settle and continue to feed on them for 20 or more days.Grapevine leafroll-associated virus 3 (GLRaV-3), an economically significant pathogen of grapevines, is transmitted by  Grapevine leafroll-associated virus 3 (GLRaV-3) is the main and most widespread etiological agent of grapevine leafroll disease (GLD) worldwide . GLD negPseudococcus calceolariae and P. longispinus  are commonly found in New Zealand vineyards. Two cosmopolitan species are especially problematic because they transmit GLRaV-3: gispinus .Pseudococcus calceolariae and P. longispinus are both polyphagous, feeding on numerous plant species throughout New Zealand, including horticultural crops such as apple and pear, and ground-cover species such as white clover, red clover, and doves foot . This response helps facilitate biological control later in the growing season . Thus, Ioncluded . TherefoP. calceolariae after feeding on non-Vitis plant hosts, (ii) whether GLRaV-3 is retained in second instar P. calceolariae and exuviae molted by the first instar P. calceolariae after feeding on non-Vitis plant hosts, and (iii) whether GLRaV-3 is transmissible by viruliferous first and second instar P. calceolariae after feeding on non-Vitis plant hosts. White clover (Trifolium repens L. cv. Grasslands Huia white clover), Crimson clover (Trifolium incarnatum) and Nicotiana benthamiana were used as non-Vitis plant hosts, of which only N. benthamiana has been demonstrated as an alternative host for GLRaV-3.Preliminary retention and transmission experiments were carried out in 2015, with retention experiments repeated in 2019–2020 (2020 Retention experiments). The aims of this study were to determine (i) the GLRaV-3 retention period in viruliferous first instar V. vinifera cv. Pinot noir and V. vinifera cv. Sauvignon blanc grapevines infected with GLRaV-3 group I maintained in controlled growth rooms (23°C with a 16 h light and 8 h dark cycle). For GLRaV-3-negative plant material, Cabernet franc were sourced from a New Zealand grapevine collection  and Merlot from Riversun Nursery . The virus status of all plants was confirmed by enzyme-linked immunosorbent assay (ELISA) or immunocapture RT-qPCR (Trifolium repens L. cv. Grasslands Huia White clover) plants were grown from seed and were used as the non-Vitis host plant.For the retention and transmission experiments in 2015, GLRaV-3-positive leaf material was obtained from  RT-qPCR . White cTrifolium repens L. cv. Grasslands Huia white clover), Crimson clover (Trifolium incarnatum) and Nicotiana benthamiana plants were grown from seed and used as supplied to P. calceolariae. These the non-Vitis host plants were maintained within a glasshouse (at average 24°C with 16 h light and 8 h dark cycle) within separate, species-specific units with adequate space between plants. To ensure each donor leaf was infected with GLRaV-3 and sufficient GLRaV-3 was acquired by mealybugs, the petiole from each grapevine donor leaf was collected and tested by RT-qPCR , Auckland campus, within vented, plastic containers held under controlled laboratory conditions (22°C with 16 h light and 8 h dark cycle). The colonies were sustained on seed potatoes over multiple generations without exposure to grapevines or GLRaV-3.Retention experiments were performed in both 2015 and 2020 as detailed below and summarized in Pseudococcus calceolariae eggs were collected from the colony and placed on a moist, black filter paper for emergence of first instars. Less than 24 h old nymphs were transferred to excised GLRaV-3-positive Cabernet franc grapevine leaves for an acquisition access period (AAP) or to GLRaV-3-free grapevine leaves for a mock AAP. Excised leaves were kept in good condition within vertically-orientated Petri dishes with the petiole extending through a fitted foam plug placed through the base of both lid and dish into a tube of water. After a 4 days AAP, P. calceolariae nymphs were transferred with a paint brush to White clover (Virus/White clover), GLRaV-3-free grapevine leaves (Virus/Grapevine), or maintained on the virus-infected leaves as a positive control (Virus). After a 4 days mock AAP, nymphs were transferred to White clover (No virus/White clover) or maintained on the uninfected grapevine leaves as a negative control (No virus). For all five treatments , first instar P. calceolariae were collected after 1, 2, 3, and 4 days feeding on grapevine or the non-Vitis host. To minimize disruption to mealybug feeding they were collected from separate grapevine or White clover leaves each day. Second instar and exuviae were collected from four treatments . For the Virus treatment, late first instar mealybugs close to molt (AAP 10–12 days) were transferred onto moist filter paper in a Petri dish to ensure that no nascent second instar mealybugs had an opportunity to feed on GLRaV-3-positive leaves. A maximum of 10 first instar mealybugs were transferred into each Petri dish, thereafter the plates were checked daily for exuviae and second instar mealybugs. The second instar mealybugs with recently removed exuviae were identified based on their shiny brown skin and the absence of the wooly appearance characteristic of first instars. For treatments Virus/White clover, No virus/White clover or No virus, second instar and exuviae were sampled directly from White clover or virus-free grapevine leaves, respectively. GLRaV-3 status in mealybugs and exuviae was detected by one-step RT-qPCR as described previously . Each grapevine leaf was maintained in a Petri dish as described above. After 24 h the unhatched eggs were removed, leaving the newly emerged nymphs on the leaf surface . After a 6 days AAP on virus donor grapevine leaves  viruliferous mealybugs were transferred most delicately onto recipient plants by cutting donor grapevine leaf pieces harboring ∼40 mealybugs (per non-Vitis host plant) and placing the mealybug-loaded leaf piece(s) onto each non-Vitis host plant. The grapevine leaf pieces were removed once they were mealybug free. Mealybugs remained on each non-Vitis host for up to 40 days, with harvesting of subsets at 5, 10, 16, 20, and 40 days. Once the mealybug collection was completed, the remaining mealybugs were killed by spraying the plants with insecticide .N. benthamiana, five mealybugs from each non-Vitis host plant per time point were collected, with individual mealybugs placed into Eppendorf tubes. Mealybug males in cocoons were also included in the sample collection. No mealybugs were present on N. benthamiana plants after 16 days. At 35 days post-inoculation, Crimson clover plants were treated with Confidor® for an infestation of aphids, mites and whiteflies that killed 14 plants. After the final mealybug sample collection on day 40, all the plants were treated with a mixture of Movento®, Avid®, and Confidor® with Partner®. At Day 0 (after 6 days AAP), three mealybugs from each donor leaf were collected into individual Eppendorf tubes. The individual donor petioles and the individual mealybug samples were tested for GLRaV-3 as described previously . Following either a 5 or 11 days non-Vitis plant feeding on White clover, P. calceolariae nymphs were inoculated on GLRaV-3-free Merlot grapevine plants, as first (Treatment 1) or second instars (Treatment 2), respectively. As a positive control, P. calceolariae nymphs were maintained on GLRaV-3-positive leaves for a total of 10 or 16 days AAP, and thereafter transferred to GLRaV-3-free Merlot grapevine plants as either first (Treatment 3) or second instars (Treatment 4), respectively. For the inoculation of GLRaV-3-free grapevine plants, large healthy leaves, two or three nodes above the grapevine graft union, were selected. White clover or GLRaV-3 positive leaves with 25–50 mealybugs were secured to the underside of the grapevine leaf with Blu-Tack (Bostik). Mealybugs were maintained for a 7–10 days inoculation access period (IAP) on GLRaV-3-free grapevines, thereafter plants were sprayed with Movento®. Plants were physically separated during the IAP. Grapevines were pruned and maintained in the glasshouse for 5 months and were sprayed with insecticides once a month. Previous research by After a 4 days AAP on excised GLRaV-3-positive grapevine leaves, Vitis), treatment, source plant and alternate host. The false discovery rate correction (fdr) was applied when performing multiple post-hoc pairwise comparisons , the 2015 retention dataset was blocked based on expected positive samples  and expected negative samples (No virus and No virus/White clover). The following variables were compared: GLRaV-3 status, days feeding on alternate host  and between Day 2 (15%) and both Days 3 and 4 (p < 0.001). When feeding on a non-Vitis host plant (Virus/White clover) or on GLRaV-3-free grapevine leaves (Virus/Grapevine), P. calceolariae nymphs retained GLRaV-3 for at least 4 days (p > 0.05). The percentages of viruliferous mealybugs varied for each of the GLRaV-3 retention treatments  on Days 3 and 4 post initial AAP . Similarly, at Day 4, there was evidence of a difference between the Virus (85%) and both Virus/White clover (23%) and Virus/Grapevine (30%) treatments (p < 0.001). All first instar mealybugs sampled from negative control treatments for all blocks (No virus and No virus/White clover treatments) tested negative for GLRaV-3. GLRaV-3 was successfully detected in 17% of second instar P. calceolariae from the Virus treatment. One second instar P. calceolariae, which fed on GLRaV-3-free grapevine leaves, tested positive for GLRaV-3 , suggesting contamination during the virus retention assay or total RNA extraction procedure. GLRaV-3 was not detected in second instar P. calceolariae from the No virus/White clover and Virus/White clover treatments. Composite exuviae samples (10 exuviae per tube) were tested for GLRaV-3. GLRaV-3 was successfully detected in 100% of exuviae samples from the Virus treatment. GLRaV-3 was not detected in exuviae from the No virus, No virus/White clover, and Virus/White clover treatments.Samples positive for GLRaV-3 generated Ct values that ranged between 24 and 38. The percentage of single, viruliferous mealybugs was 7, 15, 81, and 84% after 1, 2, 3, and 4 days post initial AAP, respectively . Based ot 4 days . For thetial AAP . At Day Vitis host plant. Samples positive for GLRaV-3 generated Ct values that ranged between 31.5 and 35. Notably, there was a gradual reduction of mealybugs collected per time point as the experiment progressed. This result was likely because of poor mealybug settlement on the non-Vitis plant host (particularly for N. benthamiana) and the natural attrition of Crimson and White clover plants and the mealybugs they supported. The percentage of mealybugs that tested positive for GLRaV-3 by RT-qPCR reduced gradually, with no GLRaV-3 detected from 20 days on any of the non-Vitis host plants . There was evidence of a statistically significant difference at Day 10 between Crimson clover and N. benthamiana (p < 0.01). At Day 16, there was evidence of a statistically significant difference between both clover species and N. benthamiana (p < 0.001). There was no evidence of any statistically significant difference for the N. benthamiana treatment between the different time points (p > 0.05).A total of 1,288 mealybugs was tested by RT-qPCR as single mealybug samples comprising five time points; 5, 10, 16, 20, and 40 days on a non-t plants . The perP. calceolariae nymphs when they were transferred directly (Treatment 3) or indirectly  to recipient grapevines (p = 0.23).GLRaV-3 was successfully transmitted by apevines . GreaterVitis host White clover, Treatment 2) to recipient grapevines (p = 0.015) there was no evidence of a difference in transmission success between Block 1 and Block 2 for Treatment 4 (p > 0.05). GLRaV-3 was not detected in any of the negative controls grown in parallel to the inoculated grapevines (seven plants).Overall for Block 1 and Block 2, GLRaV-3 was successfully transmitted by second instar mealybugs when transferred directly (Treatment 4) or indirectly .A subset of RNA extracted from mealybugs was tested for P. calceolariae within the mealybug populations in this study. In the 2015 retention experiment, P. calceolariae nymphs were given a 4 days AAP on GLRaV-3-positive grapevine leaves and showed an increased percentage of viruliferous mealybugs over time for the positive control (Virus). In the 2020 retention experiment, a 6 days AAP was used and resulted in 81% acquisition of GLRaV-3. Neither the 2015 nor the 2020 experiments resulted in a drop in GLRaV-3 Ct values over time of mealybug feeding on the non-Vitis host, thereby supporting a non-propagative interaction between vector and virus. Variability in virus acquisition of GLRaV-3 by mealybugs has been reported previously. P. longispinus first instar nymphs transmitted GLRaV-3 after an AAP of 1.5 h. P. ficus fed on GLRaV-3 positive grapevine leaves for a 7 days AAP. P. longispinus adults did not acquire GLRaV-3 in less than 24 h. P. ficus was able to transmit GLRaV-3 after a 15-min AAP and a 15 min IAP, and for P. longispinus, GLRaV-3 was transmitted after an AAP of 10 min and an IAP of 1 h.This study demonstrated that more than 4 days is needed to ensure optimal virus acquisition by individual The 2015 experiment used a single set of excised GLRaV-3-positive Cabernet franc grapevine leaves on which all mealybugs fed prior to distribution to White clover or GLRaV-3-free grapevine leaves for collection at Days 1–4 . The VirP. calceolariae was at least 16 days, i.e., between when the first instar acquired the virus and subsequently fed on either White clover, Crimson clover or N. benthamiana. In the 2015 experiments, GLRaV-3 was detected from 23.7% of the mealybugs collected after 4 days of feeding on White clover. Furthermore, GLRaV-3 was detected from 17% of second instar citrophilus mealybugs left on GLRaV-3 positive grapevine leaves for up to 9 days then removed to Petri dishes before the mealybugs molted to become second instar mealybugs. Moreover, GLRaV-3 was transmitted to grapevines even after the inoculating mealybugs were sustained on White clover plants for 11 days. The 2020 experiments extended past 4 days access to a non-Vitis host and GLRaV-3 was detected in P. calceolariae after 16 days feeding on White clover or N. benthamiana, but at 20 days and beyond, no GLRaV-3 was detected in the test mealybugs.This study demonstrated that the GLRaV-3 retention period in N. benthamiana plants dramatically declined during the 2020 experiment. This result was most likely because of the poorer mealybug settlement on N. benthamiana compared with the clover plants. The poor mealybug settlement and consequent lack of feeding may have led to the slightly higher number of GLRaV-3 positive mealybugs from N. benthamiana plants compared with the clover as the virus would have had less opportunity to be transmitted from the mealybug into the solanaceous plant. GLRaV-3 detection in N. benthamiana plants only occurs months after inoculation  or GLRaV-3-free grapevine, and for 2 days when starving. By contrast, P. longispinus retained GLRaV-3 for at least 3 days when fed on GLRaV-3-free grapevine or starving  and the decrease in percentage viruliferous mealybugs over time on clover or N. benthamiana and the eventual lack of detection of GLRaV-3 in individual mealybugs by 20 days, supports the non-circulative and non-propagative descriptors for the transmission of GLRaV-3.Collectively, based on the acquisition and retention times from previous studies, GLRaV-3 transmission by mealybugs has been described as non-circulative, non-propagative, and semi-persistent, i.e., GLRaV-3 does not breach the gut barrier of the mealybug and is retained in the stylet or foregut prior to transmission . Thus, GPl. citri. When dissected after feeding on GLRaV-3 infected grapevine leaves, in all cases GLRaV-3 was detected in the salivary glands, mid-gut, hindgut, Malpighian tubes, bacteriome, and exuviae, and in some instances, in the reproductive apparatus, suboesophageal ganglion, and mouth apparatus. The virus was not detected in bundles or replication sites in Pl. citri, suggesting GLRaV-3 does not replicate inside the insect  is transmitted by Bemisia tabaci (whitefly) in a non-circulative, semi-persistent transmission and LIYV was shown to localize in the anterior of the foregut or cibarium  and in second instar mealybugs (n = 8/45), even when first instars that had been feeding on GLRaV-3-positive grapevine leaves were transferred to Petri dishes prior to molt so that mealybugs could not feed as second instars prior to virus testing. This raises the question of whether the detected GLRaV-3 is present as a whole virion and is therefore still infectious. Similar feeding/dissection studies of P. calceolariae will validate the GLRaV-3 binding locations for this particular mealybug species and virus, and may verify or refute the previously reported circulative manner of the GLRaV-3-vector interaction. These experiments could also be used to reveal any differences between the transmission of GLRaV-3 genetic variants  plants. Future transmission studies will benefit from transferring mealybugs from plants to Petri dishes prior to molt, similar to the 2015 retention experiment. Furthermore, as noted above, the GLRaV-3-P. calceolariae relationship would benefit from investigation by feeding/dissection studies including second instar and later life stages. In particular, the use of electron microscopy to view immunologically tagged GLRaV-3 virions within mealybugs at different time points in the life-cycle and on a non-Vitis plant, in combination with transmission studies, would help elucidate where the infectious virion is located within the vector. The underlying question still remains, at what point in time does the GLRaV-3 virion lose its infection capacity following mealybug acquisition?The ability of second instar Previous studies have hypothesized that the GLRaV-3 genetic variant has an effect on the efficiency of virus transmission by mealybugs . It is aMealybug instars influence the spread of GLRaV-3 within and between vineyards by crawling between vines, movement via vineyard management activities and aerial dispersal . DispersP. calceolariae after either feeding on a non-Vitis mealybug host or GLRaV-3-free grapevine leaves for up to 16 days. These results demonstrated the insidious nature of GLRaV-3 in vineyards and they go some way to explaining issues around virus persistence, spread, and barriers to effective management. Measures to control GLRaV-3 spread that do not rely on insecticides only could include the use of ground-cover plants that host the mealybug vector but not the virus, thereby interrupting the GLRaV-3 transmission pathway from grapevine to grapevine. However, alternative hosts like clover must be a sink for mealybugs for a period of time sufficient to transition viruliferous individuals to non-viruliferous. In New Zealand, this novel addition to the integrated response to GLRaV-3 management is being evaluated (V. Bell unpublished data), and comes at a time when the wine sector is actively reducing its reliance on insecticides.Here, we have shown that GLRaV-3 can be retained and transmitted by first or second instar The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MS, KC, and RM: conceptualization and supervision. BM, RG, RR, KC, VD, and CM: data generation and curation. BM, KC, RG, and KR: formal analysis. KC, RM, and VB: Funding acquisition. BM, MS, RG, RR, VD, CM, KR, VB, KC, and RM: methodology, writing—review, and editing. BM, MS, KC, RM, and RG: writing—original draft. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Picornaviridae family. The NGS data analysis identified a number of viruses that were missed by the diagnostic panel , and these failures could be attributed to PCR primer/probe binding site mismatches. Unexpected viruses identified included parvovirus B19, enterovirus D68, coxsackievirus A16 and A24 and rubella virus. The regular application of such viral NGS could help evaluate assay performance, identify molecular causes of missed diagnoses and reveal gaps in the respiratory virus set used for local screening assays. The results can provide actionable information to improve the local pneumonia diagnostics and reveal locally important viral pathogens.Pneumonia remains a major cause of mortality and morbidity. Most molecular diagnoses of viruses rely on polymerase chain reaction (PCR) assays that however can fail due to primer mismatch. We investigated the performance of routine virus diagnostics in Kilifi, Kenya, using random-primed viral next generation sequencing  on respiratory samples which tested negative for the common viral respiratory pathogens by a local standard diagnostic panel. Among 95 hospitalised pneumonia patients and 95 household-cohort individuals, analysis of viral NGS identified at least one respiratory-associated virus in 35 (37%) and 23 (24%) samples, respectively. The majority  belonged to the  Polymerase chain reaction (PCR) methods are frequently used for viral diagnosis but costs and logistics limit the number of pathogens included in diagnostic panels. Additionally, mismatches in primers can cause missed diagnoses with evolving RNA pathogens3. Other viruses which are not included in the diagnostic panels due to costs may also contribute to missed diagnoses. Studies in Kilifi  to investigate viral aetiologies of pneumonia in paediatric patients and in the community identified respiratory viruses with these methods9; however, a proportion of samples collected from symptomatic children were negative for any respiratory viruses in the diagnostic screening panel.Pneumonia is a leading cause of illness globally11 without prior knowledge or specific PCR primers. The data generated from agnostic viral metagenomics of clinical samples were used to control the quality of the diagnostics (e.g. determine the presence of unanticipated viruses and variants of unexpected viruses on diagnostic panel), in order to help define the sensitivity of the diagnostic panels, reveal additional common respiratory pathogens, and improve future diagnostic assays for the region. These data will also help infectious disease clinicians to suspect potential causes that are not routinely included in the diagnosis if the clinical presentations warrant such a diagnosis.We evaluated the ability of next-generation sequencing (NGS) to identify viruses missed by the diagnostic panel by evaluating samples from patients with respiratory disease which tested negative for common respiratory viral pathogens. A random priming (agnostic) NGS method was employed directly on clinical samples to detect viruses present in the samples by viral metagenomicsAs shown in Table The number of respiratory viruses (with contigs ≥ 1000 nt) that had been missed by the standard diagnostic panel included 28 human rhinovirus (HRV) in 27 samples , one human parainfluenza virus 1 (HPIV-1) , three RSVB , and six human metapneumovirus (HMPV)  , species B , species C , and species D . The Parvoviridae Human bocavirus (HBoV) and parvovirus B19 (B19) were identified in four and one sample  infection is frequent in young childrenes Table , panel BEnterovirus genus, Picornaviridae family . Apart from HRV (N = 28), viruses from the Enterovirus species were not included in the routine screening.Among 64 viruses detected, the majority were from the Enteroviruses identified were Rhinovirus species A, B and C, with 28 complete or partial genomes identified. All three sets of diagnostic primers used at the time showed multiple mismatches with the genome target sites that could account for the 28 missed HRV cases 14. The 22-month old patient infected with CV-A16 was hospitalized at KCH with pneumonia, but presented no clinical HFMD symptoms, and was discharged home after 3 days. Phylogenetically, the patient’s CV-A16 virus genome was closely related to a CV-A16 strain identified from an Ethiopian child in April 2016 , enterovirus 71 (EV-71) and several additional 16  was identified in six samples, all from a 2-month period (8 April thorugh 3 June 2010) in the household study Fig. . The sixThe detection of human poliovirus type 2 was likely due to viral shedding after oral poliovirus vaccination. The child in whom this isolate was detected was 6 weeks old at the time of sampling, and had received a dose of oral polio vaccine (OPV) 4 days prior. The child was hospitalised with cough and difficulty breathing, and was discharged home after 3 days. The human poliovirus 2 genome obtained was identical to the vaccine strain Sabin 2 (GenBank AY184220). Health authorities were informed about this finding.Four EV-D68 were identified in the hospitalised cohort over a 5-month period (8 April through 318 July 2010). Two of these patients were co-infected with additional Enterovirus strains, CV-A16 or HRV-A. The four children with EV-D68 were hospitalised with severe pneumonia for 3–6 days, and were discharged with no report of neurological symptom.Phylogenetic analysis Fig. , panel CMatonaviridae family, Rubivirus genus, infecting people of any age. However, primary RVi infection during the first trimester of pregnancy may result in congenital rubella syndrome (CRS) or miscarriage. Common sequelae of CRS include deafness, glaucoma and retinopathy and heart defects. Rubella infections can be prevented by highly effective rubella vaccine. In Kenya, national rubella vaccination was not implemented until October 2016 and there was no surveillance of RVi prevalence or CRS incidence17.Rubella is a contagious typically mild disease caused by rubella virus (RVi), a single-stranded RNA virus in the RVi was detected in two KCH patients through this NGS study Table . They weWe identified parvovirus B19 (B19) in a 5-month old hospitalised patient with very severe pneumonia and anaemia. Tests for malaria were negative, and the patient was discharged after 3 days. The identified B19 virus genome belonged to genotype 1A, similar to other global B19 sequences as shown in the phylogenetic tree comparing the local B19 genome to global sequences  type 1 was identified in a hospitalised child with malnutrition, severe pneumonia and diarrhoea, and in three children with upper respiratory infections from different households. These 4 HBoV1 genomes clustered in 2 sub-lineages within genotype 1 when compared with all global sequences as shown in Supplementary Fig. Viruses detected at low frequency included HPIV-1 (one case), human parechovirus (one case), human herpesvirus 5 , human herpes simplex virus , Dengue Virus type 2  and echovirus E1 (one case).The date of collection of specimens that were test negative using the routine viral panel assay, and their NGS viral detection results, are plotted by time Fig. . The varCategory 1 The virus was in the diagnostic panel but locally circulating strains differed at diagnostic primer/probe sites. This is remedied by updating the diagnostic panel with locally appropriate primers, as being done recently2. Category 2 The virus was detected but below the cut-off for a positive diagnosis. The current cut-off for positive diagnosis is PCR Ct < 35.0; yet the assays did not show Ct-values of negative diagnoses, hence limiting the interpretation of findings here. Further studies are needed to determine assay sensitivities to update positive/negative cutoffs. Category 3 The virus was not in the diagnostic test panel. Due to practical constraints, it is not feasible to include all potential pathogens in a diagnostic panel, however findings from NGS analyses may support the decision to modify diagnostic panels accordingly. A limitation of this NGS approach is the threshold below which a virus does not yield identifiable sequences. Although 30.5% of the samples returned a viral diagnosis, 69.5% failed to yield a classifiable viral sequences. We expect that future improvements in NGS methods will increase the fraction of new diagnoses allowed by these methods.Respiratory infections are a leading cause of morbidity and mortality worldwide, sensitive and accurate viral diagnostics are crucial for guiding clinical care. In this study, we investigated clinical respiratory samples from a single location in Kenya that had failed to return a diagnosis with the local PCR diagnostic panel. We used randomly primed (unbiased) metagenomic sequencing to increase the viral detection potential. The objectives of this study were twofold. Firstly, we were interested in the number of missed virus diagnoses in respiratory cases. Secondly, we sought respiratory infections in Kilifi whose agents were not included in the routine screening panel. Here we have demonstrated the utility of direct deep sequencing of clinical respiratory samples to identify virus genomes circulating in a resource-limited country. When applying this simple strategy of random-primed viral NGS in respiratory samples testing negative in local diagnostic panels, viral sequences were identified and the approach revealed several categories of missed diagnostics. 18. Co-infections with multiple viruses were also detected which may also account for disease severity. Finally, the methods yielded complete or nearly complete genome sequences for respiratory viruses circulating in Kilifi providing a valuable sequence resource for improving local PCR diagnostic assays.The unexpected/not-tested viruses , providing additional possible causative agents explaining the symptoms and could help in the local guidelines and policy for disease management and practice. Common viruses were found that had been missed by diagnostics . In many cases these detected virus sequences showed nucleotide changes in the primer and/or probe target sites that may account for the missed detection. Although rhinoviruses are the most common virus diagnosis in the Kilifi setting, they were frequently missed by the diagnosticsAlthough viral NGS would be expensive to apply for all diagnostics, the data from this study can inform an optimum pace of applying agnostic viral NGS to improve local diagnostics. The concept of the idea is illustrated in Supplementary Fig. The data and analyses presented here provide a description of circulating respiratory viruses from two cohorts  from one region in coastal Kenya. Albeit small sample size and from a single location, the study included patients with a wide range of symptoms ranging from runny nose, sneezing to severe and very severe pneumonia, with all case types failing to yield a diagnosis for an aetiological pathogen. The study setting and methodologies, i.e. the combination of agnostic viral NGS on samples that had also been subjected to the local viral diagnostic panel, are the strength of the study. This combination allowed us to make important conclusions about the number and type of viruses missed by the local viral diagnostic panel and therefore provides useful information for improving local viral diagnostics.5 and a household cohort investigating Who-Acquires-Infection-From-Whom (WAIFW)9 from Kilifi, coastal Kenya. The hospital surveillance enrolled paediatric patients presenting with severe or very severe pneumonia based on clinical symptoms as previously defined5, and included children aged 1 day to 59 months and excluded babies with neonatal tetanus. For the KCH surveillance, naso- and oropharyngeal swab (NP/OP) samples were collected as soon as possible after hospital admission as previously described19. Ninety-five samples which had tested negative for the 15 respiratory viruses ses see “” section9. Ninety-five samples from household members with symptoms of upper respiratory tract infection, which had tested negative for viral pathogens diagnosis, were chosen and represent the mild spectrum of respiratory infections in this study.For the household cohort, members of households in a rural coastal in Kilifi were enrolled and an NP swab was collected from all members irrespective of respiratory symptoms at regular twice-weekly visits from December 2009 to June 201020: respiratory syncytial virus (RSVA and B), influenza virus A, B, and C, human rhinovirus (HRV), human coronavirus , adenovirus (AdV), human parainfluenza virus (HPIV1–4), and human metapneumovirus (HMPV). Primers, probes and target genes used for the PCR assays are summarised in Supplementary Table 5. The multiplex real-time PCR with or without the IFAT assay is referred to as “the standard diagnostic panel” throughout the manuscript.All samples were screened by multiplex real-time PCR for 15 respiratory virus targets21. Briefly, the method includes centrifugation and DNase treatment to remove free non-encapsidated DNA, reverse transcription with non-ribosomal random hexamers avoiding rRNA targets followed by sequencing on Illumina HiSeq 2500, generating 2–3 million 250 nt paired-end reads/sample.Total nucleic acid extraction and dsDNA conversion were performed as previously described22) were de novo assembled (SPAdes v.3.1023). Virus contigs were identified with UBLAST24 using virus family protein databases. Final quality control of genomes included checking open reading frames (ORFs), and comparison with reference sequences retrieved from GenBank. Stringent criteria for calling a sample positive for required greater than or equal to 1000 nt .Quality controlled reads  with 100 pseudoreplicates. Bootstrap values of ≥ 70% were considered statistically significant and were shown for major nodes. Tree was visualised in FigTree v.1.4.3 (http://tree.bio.ed.ac.uk/software/figtree). The tree was mid-point rooted for clarity and horizontal branch lengths were drawn to the scale of nucleotide substitutions per site.Global reference sequences were retrieved from GenBank, coding regions from reference and assembled genomes were extracted, and aligned using MAFFTThe study was approved by the Kenyan Medical Research Institute Scientific and Ethics Review Unit (KEMRI-SERU) and the Coventry Research Ethics Committee (United Kingdom), and all methods were performed in accordance with the relevant guidelines and regulations by KEMRI-SERU and the Coventry Research Ethics Committee. Written informed consent was obtained for all eligible participants before sample collection. For children (< 18 years), informed consent was given by parents or guardians.Supplementary Legends.Supplementary Information 1.


Approximately 8.9 million children in Sub-Saharan Africa have disabling hearing loss, accounting for 11% of the global child healthcare hearing costs. For children living in Low- and Middle-Income Countries (LMICs), 75% of hearing loss is preventable.We evaluate the overall intervention and expansion costs of a humanitarian, pediatric hearing health and screening program in Malindi, Kilifi County, Kenya. A cost analysis is conducted from the provider perspective, identifying the mean cost incurred for each case of newly identified hearing loss. Estimates were made for 3 different cost scenarios. A one-way sensitivity analysis and probabilistic sensitivity analysis using Monte Carlo simulation determined the impact of variations in individual cost parameters. These results were used to project scale-up costs to achieve sub-county expansion of the program.155 children ages 5 to 16 years old were screened, of which 5.8% were diagnosed with hearing impairment. The total cost for implementation in four schools was $6,783 USD, thus a mean cost of $212 per diagnosis of hearing loss. The highest proportion of costs were recurrent costs of resident travel (27.9%), capital costs for providing audiometric testing (25.3%), and equipment maintenance (18.7%). Expansion of an exclusively CHW-run program across all 77 primary public schools in Malindi is projected to be $130,573 .We provide relevant cost-estimation for an expansion of an intervention which identified higher than average rates of hearing loss. Humanitarian aid plays a key role in the sustainability and feasibility of expanding this program. An estimated 466 million people worldwide have disabling hearing loss, including 34 million children . In chilThe prevalence of disabling hearing loss in children 0 to 14 years old in Sub-Saharan Africa is 8.9 million and accounts for 11% of global child healthcare hearing costs . ModeratHalf of all cases of childhood hearing loss can be prevented through public health measures and implementation of screening and intervention programs play an important role . DespiteTo help address this need, we developed a school-based pediatric hearing screening program to improve access to hearing and health services in low resource settings ,11. The Research procedures were reviewed by the Vanderbilt University Medical Center’s Institutional Review Board. Procedures for the school-based intervention took place in October 2018 and are described briefly below. Additional details of the intervention’s recruitment, screening protocols, and clinical effectiveness are available in a previously public .The hearing health initiative was conducted as part of an annual, two-week surgical training camp organized by the Department of Otolaryngology at Vanderbilt University Medical Center (VUMC), local Non-Governmental Organizations (NGOs) and private and district government hospitals in Malindi, Kenya. Team members were trained to conduct hearing screenings and video-otoscopy via a smart phone-based platform during a two-hour training session by a U.S surgical resident. They then traveled to four semi-rural schools within the Malindi sub-county, which were contacted beforehand to introduce the screening process, request permission and arrange for a suitable site . ChildreThis study was conducted from a provider perspective , with taWe evaluated intervention costs under three different scenarios: 1) Program costs including involvement of one VUMC resident but excluding humanitarian grants and discounts, 2) program costs including both resident involvement with humanitarian grants and discounts, and 3) program costs excluding resident involvement and excluding humanitarian grants and discounts. The first indicates gross cost of the program with resident involvement. The second is the baseline cost scenario describing the program as implemented in 2018, which included humanitarian grants and discounts. The final scenario is used for estimating future expansion and maintenance costs and represents our long-term sustainability goal for this project. As part of this final model, humanitarian aid that is currently provided was removed for a more conservative estimate. A process map providing a program overview and sources of where a cost was accrued is described in Capital cost estimates included all medical costs and associated hearing screening equipment . Actual costs were calculated utilizing receipts of purchase for the five sets of audiometry software and two video otoscopes.Recurrent costs were divided into medical and non-medical costs. All costs can be found in We conducted one-way sensitivity analysis to determine the robustness of our results to variations in individual model parameters. Our base case comprised a program that was solely CHW-driven and required no U.S. surgical resident involvement throughout the sub-county of Malindi. Anticipated sources of variation include ‘economic variation’ to account for currency fluctuations that would affect all elements of program costs (± 5%), capital costs (± 20%), equipment maintenance costs (± 20%), medical supplies (± 20%), and staff salaries (± 20%). Lastly, we conducted a probabilistic sensitivity analysis using Monte Carlo simulation to estimate overall model uncertainty. We simultaneously conducted 10,000 random draws from triangle probability distributions for each variable with the ranges listed above and recalculated both intervention costs and cost-effectiveness for each iteration within the model.All scale-up estimates for a self-sustained program model are based on the actualized program in 2018, with the removal of resident participation costs and without incorporation of humanitarian discounts Model 3, . This coThe hearing health initiative was implemented at four primary schools in the Malindi sub-county participated in the program . A totalThe program’s cost estimates under various funding scenarios are provided in Results of one-way sensitivity analyses for self-sustained program , Model 3Using the per-school costs calculated in this study for the self-sustained model , Model 3Early identification and intervention in pediatric hearing loss is vital to prevent additional morbidity and mitigate costs of hearing disability at both the individual and the community levels. The prevalence of hearing loss in Sub-Saharan Africa accounts for an estimated 20.7 billion dollars annually and highlights the need for early hearing loss identification and aural rehabilitation . ThroughClinically, 5.8% of study participants had hearing impairment. The prevalence of hearing loss in our study population is much higher than a comparable prevalence rate in Sub-Saharan Africa of 1.9% . This inLooking toward expanding the program, this report provides stakeholders with key information on the costs associated with resources needed to identify hearing loss for this population in this region. The calculations presented here estimate the cost of continuing the current protocol without resident involvement to be $2880, serving four schools at approximately $720 per school. The estimate of $130,515 for a sub-county scale-up is further examined in sensitivity analyses to consider the effects of variations in cost-influential variables, providing a range of $119,352 to $142,240. Similar calculations done which account for humanitarian aid that is currently well established and would likely continue with expansion of this program cut the scale up cost in more than half to $55,440. Though the model utilized provides a more conservative estimate for an idealized program, these findings highlight the importance of sustaining ongoing local community partnerships to reduce costs and keep a program such as this sustainable.This finding is further reinforced when looking at the estimated costs for the different models. Program costs for Model 2, show lower total costs compared to Model 3 despite having to account for resident involvement. This is likely because changes in capital costs were shown to have the greatest potential impact on overall costs and many of the humanitarian aid currently in places is targeted at mitigating these costs. Changes to program structure in terms of recurrent costs, such as purchase of ground transportation rather than rental may also help to further decrease costs in an expanded version of the program. Additionally, headphone calibration facilities that are now available in the United States may present cost savings, as hearing screening tools had to be sent for calibration in South Africa during the intervention.We estimated average costs in lieu of actual costs for some non-recurrent medical expenses due to a lack of data availability. Costs within this study were estimated under the assumptions of a model largely supported by an NGO and humanitarian grants. In LMICs, NGOs are crucial to providing healthcare for vulnerable populations and to supplement gaps in services left by the public health sector . It is pCurrent guidelines on costing analysis promote the adoption of a societal perspective which includes healthcare and household costs. In practice, estimating healthcare costs in LMICs can be challenging to due issues of data availability and the lack of both standardized electronic health records and staff support for data extraction. The analysis completed here is based on screening and intervention conducted solely in school-aged children during school hours, lasting about 30 min, thereby limiting the amount of household costs. There were no wages lost or travel expenses incurred by caregivers to bring the child for diagnostic testing and patient-related costs were minimal thus excluded from our analysis. However, as future iterations of the program ensure continued follow-up for these patients, there may be additional household costs incurred.More broadly, our study was conducted at four primary schools within Malindi it is important to consider the generalizability of our results to the rest of Kenya and beyond. We conducted sensitivity analyses to account for major sources of potential variation in program costs such as economic uncertainty, equipment costs, and equipment maintenance. However, given the diversity of local economic conditions within LMICs, our simulations may not represent the full range of potential costs. Estimates as their purchase and maintenance contributed to the highest proportion of costs.The utilization of a formal cost-effective analysis would be helpful in comparing the ongoing practices for providing hearing health in this region to the costs of expansion seen in this program. Cost-effectiveness could potentially be determined in further analysis by calculating cost of treatment for hearing-related Quality Adjusted Life Years (QALYs) gained. This would also help to generalize the findings and create a standard that allows comparison between similar interventions. Scale-up costs that expand to county and national estimates could also further serve to describe the broad scope a program such as this one could have.In anticipation of future expansion of a previously developed hearing health initiative for LMICs, we evaluated the costs associated with our current hearing screening program, providing stakeholders and potential investors with estimates relevant to scaling-up. Humanitarian aid plays a key role in the sustainability and feasibility of expanding this program. Other LMICs may benefit from a similar program designed to reduce barriers to hearing health access. A formal cost-effectiveness analysis is needed to determine cost-saving strategies and compare the outcomes of this program to the long term costs of ongoing and preventable pediatric hearing loss in this region. Given the substantial costs attributable to hearing loss, we believe initiatives such as these will ultimately prove to be financially feasible for these communities in addition to the myriad benefits associated with preventing and rehabilitating childhood hearing loss.


A eusocial colony typically consists of two main castes: queens that reproduce and sterile workers that help them. This division of labor, however, is vulnerable to genetic elements that favor the development of their carriers into queens. Several factors, such as intracolonial relatedness, can modulate the spread of such caste‐biasing genotypes. Here we investigate the effects of a notable yet understudied ecological setting: where larvae produced by hybridization develop into sterile workers. Using mathematical modeling, we show that the coevolution of hybridization with caste determination readily triggers an evolutionary arms race between nonhybrid larvae that increasingly develop into queens, and queens that increasingly hybridize to produce workers. Even where hybridization reduces worker function and colony fitness, this race can lead to the loss of developmental plasticity and to genetically hard‐wired caste determination. Overall, our results may help understand the repeated evolution toward remarkable reproductive systems  observed in several ant species. Eusociality is characterized by a striking division of reproductive labor between two castes: queens and workers , as well as their interactions with potential costs and benefits of hybridization, for instance, owing to hybrid incompatibilities or hybrid vigor.m∈{1,2,…} of males. Each of these mates can either be an allo‐ (with probability η) or a con‐specific male (with complementary probability 1−η). Once mated, queens found monogynous colonies  and lay a large number of eggs. A proportion f of these eggs are diploid  and (1−f) are haploid . Assuming random egg fertilization, a queen therefore produces on average fη hybrid and f(1−η) nonhybrid females. We assume that a hybrid female can only develop as a worker, while a nonhybrid female can either develop as a worker (with probability ω) or as a queen (with complementary probability 1−ω). Overall, a colony thus consists of fη hybrid and f(1−η)ω nonhybrid sterile workers, as well as f(1−η)(1−ω) virgin queens and (1−f) males that are available for reproduction at the next generation.We consider a large population of annual eusocial haplodiploids with the following life‐cycle Fig. . First, e≥0 (so that the effective workforce of a colony is efη+f(1−η)ω). When e=1, hybrid workers have the same working efficiency as nonhybrid workers. By contrast, when e<1, hybrid workers are less efficient, for instance, due to outbreeding depression. This can also reflect other potential costs associated with hybridization, such as the production of sterile or nonviable hybrid queens . We nonetheless allow for differential contribution to the workload between hybrid and nonhybrid workers, with the contribution of hybrid workers weighted by a parameter  Umphrey .ω that a larva develops as a worker to vary. We assume that this probability is under individual genetic control  and that it evolves via random mutations with weak additive phenotypic effects  and where hybridization is fixed at a given level .We first investigate the evolution of caste determination by allowing the probability  Boomsma , we thuse=1). Equation (η=0), a larva will develop into a worker with a probability of 1/3 at equilibrium , royal cheating is increasingly favored and larvae become increasingly likely to develop as queens rather than workers , the population evolves toward a complete loss of nonhybrid workers via the fixation of increasingly caste‐biasing royal cheats alleles (ω→0). In this case, nonhybrid females eventually all develop into queens that rely on sperm parasitism to produce workers.Our analyses Appendix  reveal tω*=13−e2e, modulates the effect of hybridization on the evolution of caste determination  but hindered otherwise (e<1). Nevertheless, even when hybridization is extremely costly (0<e≪1), complete worker‐loss can evolve . We assume that this probability η is under individual queen control  and like caste determination, evolves via rare mutations with weak additive phenotypic effects  while the probability ω to develop as a worker stabilizes for its baseline equilibrium  and hybridization stabilizes at an intermediate equilibrium , the coevolution of genetic caste determination and hybridization can lead to worker‐loss in our model.We find that depending on the efficiency ω=1/3, η=0), we tracked these different fitness effects along a typical evolutionary trajectory that leads to worker‐loss  their direct fitness effects on the reproductive success of the individuals that express them; and (2) of their indirect fitness effects on other related individuals that can also transmit them , and monandry (m=1) mediate within‐colony conflicts and therefore should be relevant to the evolutionary arms race leading to worker‐loss , evolutionary dynamics do not necessarily lead to either complete worker‐loss or hybridization avoidance. For intermediate values of e  and larvae retain developmental plasticity  the evolution toward such intermediate state always happens when hybrid workers outperform regular workers , our mathematical analysis further shows that partial hybridization and larval plasticity is not evolutionary stable . Beyond this special case, the evolution of worker‐loss is impeded by low polyandry and impossible under monandry in our model. This is because with a low number of mates, a queen runs the risk of being fertilized by only one type of male. Under complete worker‐loss (when the population is fixed for ω=0), a queen mated to only conspecific males produces only larvae destined to be queens but no workers to ensure their survival and thus has zero fitness.In the special case of monandry and overperforming hybrid workers  and if they develop into workers, they have the same working efficiency as nonhybrid workers .Our finding that monandry inhibits the emergence of worker‐loss contrasts with the observation that several ant species, notably of the genus ω=0) evolutionary stable ? And (2) when can hybridization evolve when absent in the population ? When these two conditions are met, evolution will tend to favor the emergence and maintenance of worker‐loss  when is worker‐loss .We additionally computed the level of hybridization favored by selection when the population has evolved worker‐loss (and this is an evolutionarily stable state). We find that hybridization increases as queens mate with fewer males and as parthenogenesis increases Fig. , so muchIn sum, our analyses indicate that worker‐loss readily evolves when queens can hybridize with a lineage of males by whom fertilization leads to the production of workers. This evolution in our model occurs through a sequence of substitutions of alleles that increasingly bias the development of their carrier toward the queen caste, that is, “royal cheats”. Hybridization, or sperm parasitism, allows royal cheats to fix in the population by providing a way for colonies to compensate for the reduced workforce. In fact, when queens are capable of recognizing genetic differences among males and when royal cheats are present in the population, selection favors hybridization by queens to regain control over caste allocation in their colony. This in turn promotes greater cheating by larvae, which favors greater hybridization by queens and so on. This evolutionary arms race, fueled by intracolonial conflicts, eventually leads to complete intraspecific worker‐loss: a state where larvae have lost their developmental plasticity and develop as workers or queens depending only on whether they are the product of hybridization or not, respectively.Temnothorax curvispinosus supports that caste determination is influenced by additive effects in this species (Linksvayer Pogonomyrmex rugosus (Schwander and Keller Of course, our analyses are based on several idealized assumptions. In particular, we assumed that the probability for larvae to develop as workers is under complete larval genetic control. Typically the developmental fate of female larvae also depends on various environmental factors created by adult colony members, such as food quality and quantity Brian , or mechPogonomyrmex harvester ants (Helms Cahan et al. Our result that sperm parasitism favors the emergence of worker‐loss via the fixation of royal cheats may be relevant to unorthodox reproductive systems found in ants. Of particular interest is social hybridogenesis, whereby females produced through regular intralineage mating or thelytokous parthenogenesis develop into queens, while workers emerge from eggs fertilized by allospecific males (Helms Cahan et al. Solenopsis xyloni; Helms Cahan and Vinson Pogonomyrmex harvester ants; Anderson et al. Here, we have shown mathematically that social hybridogenesis could also result from additive genetic effects on caste development and queen‐larvae conflicts within colonies. This theory, previously described verbally in Anderson et al. , 2008, mWasmannia auropunctata (Fournier et al. Vollenhovia emeyri (Ohkawara et al. Paratrechina longicornis (Pearcy et al. W. auropunctata; Rey et al. η=1, Fig. Our model may also be relevant to other unorthodox systems of reproduction such as those found in populations of e<1, e.g., because hybridization leads to an inefficient workforce due to hybrid incompatibilities in workers; or increased efforts in mate‐finding and mating, Maroja et al. Our formal approach is especially useful in a context where hybrid vigor in workers has been raised to explain the evolutionary origin of social hybridogenesis and other hybridization‐dependent systems (Julian and Cahan Cataglyphis (out of 11 species tested, Kuhn et al. Messor (out of 9, Romiguier et al. More generally, our results suggest that natural selection can lead to an association between hybridization and caste determination. To date, such associations have been reported in only 18 distinct ant species or populations (Helms Cahan et al. Pogonomyrmex (Rheindt et al. Messor (Norman et al. Cataglyphis ants (Kuhn et al. In addition to showing that hybrid vigor is not necessary for the emergence of intraspecific worker‐loss, our model highlights several factors that can facilitate such evolution. The first of these is polyandry, which favors sperm parasitism and worker‐loss by minimizing the risks associated with hybridization. Interestingly, even though polyandry is generally rare in social insects (Strassmann Solenopsis species with this reproductive system (Helms Cahan and Vinson Messor barbarus and cf. structor; Romiguier et al. Although not considered in our study for simplicity, another factor that can minimize the risks associated with hybridization in monandrous species is polygyny, whereby related queens form multi‐queen nests. Such social organization allows both intra‐ and interspecifically mated queens to be part of the same colony, which can then produce both queens and workers. Polygyny should therefore further facilitate hybridization. Although this may have played a role in the evolution of social hybridogenesis in the polygynous versus con‐specific males evolves (Fig. Solenopsis or European Temnothorax; Feldhaar et al. More important for the evolution of worker‐loss in our model is that queens hybridize often enough. This readily happens when the propensity of queens to mate with allo‐ ves Fig. . In thisves Fig. . Alternaves Fig. . Under cves Fig. , for insves Fig. . In inteves Fig. . Such a Whether it occurs randomly or not, hybridization requires pre‐zygotic barriers to be sufficiently low. Various mechanisms, such as secondary contacts or high dispersal ability, are known to lower these barriers (de Aguiar et al. Teleutomyrmex schneideri, for instance, queens do not themselves produce workers but rather infiltrate the colony of a host and trick host workers into caring for their progeny (Hölldobler and Wilson At a broader level, our results suggest that worker‐loss can readily evolve when a source of workers that is impervious to royal cheats can be exploited by queens. Besides sperm parasitism, other forms of parasitism can provide such a source of workers and have been associated with worker‐loss (Nonacs and Tobin Intracolonial conflicts are inevitably part of the social lives of nonclonal organisms. Here we have shown that such conflicts readily lead to an association between interspecific sperm parasitism and intraspecific worker‐loss via the fixation of royal cheats. This association is especially relevant to the evolution of reproductive systems that like social hybridogenesis rely on hybridization. Beyond these unorthodox systems and sperm parasitism, the fixation of royal cheats and loss of intraspecific workers may be connected to other forms of antagonistic interspecific relationships such as social parasitism. More broadly, our model illustrates how the unique conflicts that are inherent to eusocial life can lead to evolutionary arms races, with implications for elaborate reproductive systems and novel ecological interactions between species.AW, JR, and CM conceived the study. AW performed the analysis and wrote the first draft of the manuscript under the guidance of JR and CM. All authors contributed to the final version.https://zenodo.org/record/5167179.A Mathematica notebook that reproduces our results and a R file implementing our simulations are available here: Associate Editor: C. MoreauTable S1: Colonial investment in males, queens and workers.Figure S1: Properties of the internal singular strategy under monoandry and low polyandry.Figure S2: Polymorphism under monandry is due to positive correlational selection. A.Figure S3: Non‐linear effects of investment in workers.Click here for additional data file.


Ambient temperature and humidity strongly affect inactivation rates of enveloped viruses, but a mechanistic, quantitative theory of these effects has been elusive. We measure the stability of SARS-CoV-2 on an inert surface at nine temperature and humidity conditions and develop a mechanistic model to explain and predict how temperature and humidity alter virus inactivation. We find SARS-CoV-2 survives longest at low temperatures and extreme relative humidities (RH); median estimated virus half-life is >24 hr at 10°C and 40% RH, but ∼1.5 hr at 27°C and 65% RH. Our mechanistic model uses fundamental chemistry to explain why inactivation rate increases with increased temperature and shows a U-shaped dependence on RH. The model accurately predicts existing measurements of five different human coronaviruses, suggesting that shared mechanisms may affect stability for many viruses. The results indicate scenarios of high transmission risk, point to mitigation strategies, and advance the mechanistic study of virus transmission. For viruses to transmit from one host to the next, virus particles must remain infectious in the period between release from the transmitting host and uptake by the recipient host. Virus environmental stability thus determines the potential for surface (fomite) transmission and for mid-to-long range transmission through the air. Empirical evidence suggests that virus environmental stability depends strongly on ambient temperature and humidity, particularly for enveloped viruses; examples among enveloped viruses that infect humans include influenza viruses , endemicIn late 2019, a new zoonotic coronavirus now called SARS-CoV-2 emerged; it has since caused a global pandemic (COVID-19) and is poised to become an endemic human pathogen. Many countries in the Northern Hemisphere experienced a substantial uptick in transmission with the arrival of their late autumn and winter. Epidemiologists had anticipated such a seasonal increase  based onVirus transmission is governed by many factors, among them properties of the virus and properties of the host population. But anticipating seasonal changes in transmission and preventing superspreading events both require an understanding of virus persistence in the environment, since ambient conditions can facilitate or impede virus spread. Empirical evidence suggests that SARS-CoV-2, like other enveloped viruses, varies in its environmental stability as a function of temperature and humidity , but theMoreover, despite years of research on virus environmental stability, there do not exist mechanistically motivated quantitative models for virus inactivation as a function of both temperature and humidity. Existing predictive models for the environmental stability of SARS-CoV-2  and otheWe measured the environmental stability of SARS-CoV-2 virus particles (virions) suspended in cell culture medium and deposited onto a polypropylene plastic surface at nine environmental conditions: three relative humidities  at each of three temperatures . We first quantified viable (infectious) virus titer over time and estimated virus decay rates and corresponding half-lives in each condition using a simple Bayesian regression model . We quantified the evaporation of the suspension medium and compared virus stability during the sample evaporation phase—while substantial water loss was ongoing—to virus stability after a quasi-equilibrium phase was reached—when further evaporation was not evident over the timescale of the experiment.We then created a mechanistic biochemical model of virus inactivation kinetics, drawing upon existing hypotheses for how temperature and humidity affect the inactivation chemistry of virus particles in microdroplets . We fit Our mechanistic model is based on a simple premise: virus inactivation in the environment is a chemical reaction and so obeys the laws of chemical kinetics. Reactions proceed faster at higher temperatures and higher solute concentrations. Solutes will be more concentrated when there is more evaporation; this occurs when the ambient relative humidity is lower. But below a threshold relative humidity, the efflorescence relative humidity (ERH), droplets may crystallize; this is also expected to change reaction kinetics. These principles apply across reactions. We do not need to know the exact identities and concentrations of non-virus reactants  involved to make mechanistic predictions about how the inactivation reaction rate will vary with temperature and humidity.Our model encodes these principles. We estimated its three central parameters from our data.Our data suggest that SARS-CoV-2 environmental persistence could vary meaningfully across the range of temperatures and humidities encountered in daily life, with posterior median [95% credible interval] half-lives as long as 27 hr   and as short as 1.5 hr  , once droplets reach quasi-equilibrium with the ambient air conditions .Minimal virus decay occurred during the evaporation phase , when exOverall, virus decay became markedly faster as temperature increased for all humidities, with decay at 27°C roughly five to ten times faster than decay at 10°C. Across temperatures, virus decay was relatively rapid at 65% RH and tended to be slower either at lower (40%) or higher (85%) humidities, or when excess water was present during the evaporation phase , Table 1Many viruses, including SARS-CoV-2, exhibit exponential decay on surfaces and in aerosols . We drewWe model virus inactivation at quasi-equilibrium on inert surfaces as a chemical reaction with first-order reaction kinetics; that is, the quantity of virus is the limiting reactant of the rate-determining step. This reflects the empirical pattern of exponential decay and is consistent with the fact that virions will be numerically rare in microdroplets compared to other reactants.We characterize the temperature dependence of this reaction with the Arrhenius equation, which describes a reaction rate  . Prior wThe exact quasi-equilibrium state reached will depend on the solutes present, since different solutes depress vapor pressure to different degrees. In electrolyte solutions like bodily fluids or cell culture media, efflorescence is also important. Below a threshold ambient humidity—the efflorescence relative humidity (ERH)—electrolytes effloresce out of solution, forming a crystal . Below tDuring the evaporation phase prior to quasi-equilibrium, reactants are less concentrated and decay is expected to be slower, as observed from our data . If smalWe partition inactivation at quasi-equilibrium into two humidity regimes, effloresced and solution, according to whether the ambient RH is below the ERH (effloresced) or above (solution). In either case, we approximate virus inactivation as a first-order reaction with inactivation rate pressure , we use We model the effloresced and solution inactivation rates Given our assumption of first-order kinetics, an n-fold increase in the non-virion reactant concentrations should translate directly into an n-fold increase in the inactivation rate. Lower relative humidity leads to higher quasi-equilibrium concentration and thus increases virus inactivation rate, until the ERH is reached. Below the ERH, inactivation rates may again be low due to crystallization, depending on We estimated ion data .The relationship between RH and quasi-equilibrium concentration factor depends on complex evaporative kinetics that will vary among media. For this reason, we do not attempt to predict it from first principles, but instead measure it directly and use the fitted curve to extrapolate to unmeasured RH conditions. We use this approach for the results presented in the main text; we refer to it as the ‘main model’.To check robustness of the main model results, we also estimated a version of the model without this theoretical curve–using only directly-measured equilibrium concentration factors. This model (referred to as the ‘directly-measured concentration model’) yielded similar results to the main model; see Appendix, Mechanistic model versions for details.We also considered a four-parameter variant of the model with distinct activation energies below the ERH .We discuss additional practical implications for the empirical study of virus environmental stability in the Appendix .Despite years of research on virus stability as a function of temperature and humidity and plausible hypotheses about the underlying chemistry, proposed mechanisms have lacked explicit quantitative support. By encoding the underlying chemistry into a mathematical model and estimating parameters using modern computational techniques, we provide such support, with critical insights for the control of an ongoing pandemic. Our empirical results provide mechanistic insight into transmission risks associated with cold and climate-controlled indoor settings, while our modeling work allows for explicit quantitative comparison of the aerosol and fomite risks in different environments, and suggests that simple, general mechanisms govern the viability of enveloped viruses: hotter, more concentrated solutions are favorable to chemical reactions—and therefore unfavorable to viruses.We used SARS-CoV-2 strain HCoV-19 nCoV-WA1-2020  at each of three temperatures . These controlled environmental conditions were produced in incubators (MMM Group CLIMACELL and Caron model 6040) with protection from UV-B or UV-C exposure. We prepared 216 disks corresponding to three replicates per eight post-deposition time-points  for the nine conditions. At each time-point, samples were collected by rinsing the disks with 1 mL of DMEM and stored at −80°C until titration.We measured virus stability on polypropylene  as previously described . We prep2O respectively before use. We measured medium mass We measured the evaporation kinetics of suspension medium under the same temperature and humidity conditions as the virus stability experiments. We placed 50 μL aliquots of supplemented DMEM onto polypropylene disks in a Electro-Tech Systems 5518 environmental chamber. The polypropylene disks were rinsed three times 1M sulfuric acid, ethanol and DI HWe quantified the stability of SARS-CoV-2 under different conditions by estimating the decay rates of viable virus titers. We inferred individual titers using a Bayesian model we have previously described . BrieflyWe estimated parameters of our mechanistic models by predicting titers based on those models and then applying the same Poisson single-hit observation process to estimate parameters from the data. See Appendix (Mechanistic model estimation) for a complete description, including model priors.We estimated evaporation rates and corresponding drying times by modeling mass loss for each environmental condition We drew posterior samples using Stan , which iTo test the validity of our model beyond the measured environmental conditions (i.e. beyond 10–27°C and 40–85% RH), we compiled data from 11 published studies on human coronaviruses, including SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-OC43, and HCoV-299E, under 17 temperature-RH conditions. We generated estimates of half-lives and uncertainties  and compWe also included data from SARS-CoV-1 and MERS-CoV collected by our group during previous studies . Those dBioRender.com.We created plots in R using ggplot2 , ggdist  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:eLife. There is little doubt that this will be a very worthy and meaningful contribution to the literature. Even further, this work may transcend the COVID-19 pandemic, and serve as grand example of how one can rigorously integrate fields like biophysics with epidemiology.The study offered a provocative, highly technical, and courageously cross-disciplinary approach to understanding environmental survival in viruses, including SARS-CoV-2. We applaud the rigor and care in the work, and thank you for submitting it to Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor.Thank you for submitting your article "Mechanistic theory predicts the effects of temperature and humidity on inactivation of SARS-CoV-2 and other enveloped viruses" for consideration by Please address the changes as outlined by the reviewers below, as they will make for an improved manuscript. We encourage the authors to address them, and more broadly, make this article as clear as possible. Both reviewers provided some insight into this, and strongly encourage the authors to pay close attention to this.Reviewer #1:In this manuscript, Morris and colleagues offer a mechanistic model, featuring chemistry and physics details, that explain the effects of temperature and humidity.I found this manuscript to be highly relevant for any virus, but given the context-the COVID-19 pandemic-I find the results to be timely, relevant, and fantastically well conducted. The interaction between the epidemiological knowledge and biophysical precision is clear, and the implications are also very clear. The manuscript is well-written and organized.I believe that this manuscript should be published, would make a worthy contribution to great body of SARS-CoV-2 and extended COVID-19 literature.The recommended changes have to do with subtle editorial points, and efforts to make this work as clear as possible.1. Figure 1: I believe that the authors should check the labels. I could be wrong, but I believe the labels in the captions are wrong.2. Equations:The interaction between the text and the appendix could be improved, and I challenge the authors to better integrate this information. The authors should read the equations from the perspective of the reader: can the reader fully grasp the flow of information from the equations as provided. I am of a mixed opinion on this, but given the overall excellence of the manuscript, I encourage the authors to make this work as clear and digestible as possible, as it has the potential to be a defining piece of literature.Reviewer #2:This is a very well-done and thorough analysis of SARS-CoV-2 decay rates, with new theory and models being developed that also apply to other viruses. The methodology is solid, the analyses extensive, and the authors fully achieve their goal of further understanding the decay characteristics of SARS-CoV-2 and other viruses in the environment. The main novel contribution is the detailed mechanistic model relating decay rates to temperature and relative humidity. I believe this is a major contribution toward better understanding and predicting environmental virus decay dynamics.eLife. Overall, I think another pass-through the main manuscript with an eye to making it as easy and stand-alone to follow as possible for the 'casual reader/browser' would be useful.I consider this paper perfectly suited for  Reviewer #1:In this manuscript, Morris and colleagues offer a mechanistic model, featuring chemistry and physics details, that explain the effects of temperature and humidity.I found this manuscript to be highly relevant for any virus, but given the context-the COVID-19 pandemic-I find the results to be timely, relevant, and fantastically well conducted. The interaction between the epidemiological knowledge and biophysical precision is clear, and the implications are also very clear. The manuscript is well-written and organized.I believe that this manuscript should be published, would make a worthy contribution to great body of SARS-CoV-2 and extended COVID-19 literature.The recommended changes have to do with subtle editorial points, and efforts to make this work as clear as possible.1. Figure 1: I believe that the authors should check the labels. I could be wrong, but I believe the labels in the captions are wrong.2. Equations:The interaction between the text and the appendix could be improved, and I challenge the authors to better integrate this information. The authors should read the equations from the perspective of the reader: can the reader fully grasp the flow of information from the equations as provided. I am of a mixed opinion on this, but given the overall excellence of the manuscript, I encourage the authors to make this work as clear and digestible as possible, as it has the potential to be a defining piece of literature.We have revised the manuscript in light of the decision comments, with the aim of improving readability and clarity. Edits include collating supplementary figures as figure supplements to the main figures, where appropriate, nudging points in figures to avoid overplotting and improve readability, and clarifying distinctions between the different measurements and model fits used.


Metarhizium brunneum and the CO2 producing yeast Saccharomyces cerevisiae. Adding caseins altered the porous structure of beads. FC decreased the bead density from (1.0197 ± 0.0008) g/mL to (1.0144 ± 0.0008) g/mL and the pore diameter by 31%. In contrast to casein, FC enhanced the water absorbency of alginate/starch beads by 40%. Furthermore, incorporating FC quadrupled the spore density on beads containing M. brunneum and S. cerevisiae, and simultaneous venting increased the spore density even by a factor of 18. Moreover, FC increased the total CO2 produced by M. brunneum and S. cerevisiae by 29%. Thus, our findings suggest that rehydration is enhanced by larger capillaries, resulting in an increased water absorption capacity. Our data further suggest that gas exchange is improved by FC. Therefore, our results indicate that FC enhances the fungal activity of both fungi M. brunneum and S. cerevisiae, presumably leading to an enhanced attract-and-kill efficacy for pest control.The control of root-feeding wireworms has become more challenging as synthetic soil insecticides have been progressively phased out due to environmental risk concerns. Innovative microbial control alternatives such as the so-called attract-and-kill strategy depend on the rapid and successful development of dried encapsulated microorganisms, which is initiated by rehydration. Casein is a functional additive that is already used in food or pharmaceutical industry due to its water binding capacity. Cross-linked forms such as formalin-casein (FC), exhibit altered network structures. To determine whether FC influences the rehydration of alginate beads in order to increase the efficacy of an attract-and-kill formulation for wireworm pest control, we incorporated either casein or FC in different alginate/starch formulations. We investigated the porous properties of alginate/starch beads and subsequently evaluated the activities of the encapsulated entomopathogenic fungus The online version contains supplementary material available at 10.1007/s11274-021-03121-3. Coleoptera: Elateridae) are widespread pests that damage a variety of crops, including grain i.e. wheat, barley, maize and most vegetables such as carrot and potato. Since the mid-twentieth century wireworm damage is becoming an increasing concern in both conventional and organic potato cultivation , aiming to quickly provide a sufficient amount of attractive CO2 and entomopathogenic conidia.The first part of this work elucidates the influence of casein and FC on the porous properties and the rehydration of alginate/starch beads. The second part clarifies how casein and FC, and consequently the bead properties, influence the fungal activity of All chemical compounds used in this study were acquired from Carl Roth GmbH  and concentrations are given as (w/w), unless stated otherwise.Casein from bovine milk  was grinded in a ball mill and sieved subsequently to reach a particle size ≤ 150 µm. Formalin-casein  was supplied in the appropriate particle size and was therefore only sieved to select particles ≤ 150 µm.S. cerevisiae H205 was provided ready for use by Deutsche Hefewerke GmbH .M. brunneum strain CB15 was obtained from Prof. Dr. Stefan Vidal . To produce aerial conidia (AC) used for inoculation of submerged cultures, freeze-dried biomass was thawed in Potato Dextrose Broth (PDB) and cultivated on Potato Dextrose Agar (PDA) in the dark at 25 °C for 7 days. Plates were sealed with Parafilm M  to avoid moisture loss. Then, the pre-culture was inoculated with AC resulting in a final concentration of 1 × 106 AC/mL and cultivated in liquid medium modified according to Krell et al. , 7.0% polyethylene glycol 200) at 25 °C and 150 rpm in 250 mL baffled shake flasks for 48 h. Cultures were inoculated with 5.0% (v/v) vegetative pre-culture and cultivated for 72 h. The mycelial biomass was harvested by vacuum filtration through filter paper  and rinsed with 0.9% NaCl solution to remove blastospores and residual cultivation medium.l et al.   was dissolved in ultrapure water to a final concentration of 4.0% and autoclaved for 6 min at 121 °C. The encapsulation suspensions were prepared as follows.The encapsulation suspension for the control beads was produced by thoroughly mixing 2.0% calcium alginate with 20.0% native corn starch  for 10 min until suspended. Finally, water was added to reach final concentrations.Encapsulation suspension with casein additives was produced by mixing 2.0% sodium alginate with 13.3% native corn starch for 5 min and then with 6.7% either casein or formalin-casein for 5 min until suspended. Because the suspension became viscous, the water required to reach final concentrations was added shortly before dripping.M. brunneum mycelial biomass was added and gently stirred for 2 min. Water was added to reach the final concentrations.The encapsulation suspension for the control Kill beads was produced by mixing 2.0% sodium alginate with 20.0% native corn starch for 10 min until suspended. Then, 1.0% M. brunneum mycelial biomass was added and gently stirred for 2 min. The water required to reach final concentrations was added shortly before dripping.The encapsulation suspension with either casein or FC was produced by mixing 2% sodium alginate with 13.3% native corn starch for 5 min, and subsequently with 6.7% either casein or formalin-casein for 5 min until suspended. Then, 1.0% S. cerevisiae for 5 min until blended and then with 20.0% native corn starch for 10 min. Then, 1.0% M. brunneum mycelial biomass was added and gently stirred for 2 min. Finally, water was added to reach final concentrations.The encapsulation suspension for the control A&K beads was produced by mixing 2.0% sodium alginate with 16.7% The encapsulation suspensions with casein or FC were prepared the same way, but only 13.3% native corn starch was mixed for 5 min before 6.7% either casein or FC was added and mixed for another 5 min.2 solution by means of a single syringe pump , with a 0.9 mm cannula . The pumping speed was set to 4 mL/min. After 20 min, beads were separated and washed with ultrapure water. Moist beads were dried under a laminar flow at room temperature  for 24 h. Alginate/starch beads were subsequently dried over silica to reach a final water activity of 0.2, unless specified otherwise. Water activities were controlled with a water activity meter  at 25 °C.Beads were prepared by dripping the suspension into a sterile 180 mM CaClThe C/N ratio of alginate beads was analyzed with a C/N elemental analyzer  to assess the nutritive influence of casein and FC. Dried calcium alginate beads with starch or starch and either casein or FC were grinded with a ball mill to obtain a homogenous composition of the sample matrix. The amount of 5 g of each matrix was weighed in tin containers and loaded into an automatic sampler. Each sample matrix was measured twice.The particle projected area diameters derived from digital images of the beads were determined using the open source image processing program Fiji   in small, closed Petri dishes. Rehydration Afterwards the samples were transferred into special airtight containers for water activity measurements and stored until measured. Water activities were determined with a water activity meter  at 25 °C. Water activity and temperature stable time were set to 2 min and 1 min, respectively.The bead density ads see ‘’, \documWe used differential scanning calorimetry (DSC) to assess the pore volume distributions and therefore how casein or FC influence the pore sizes in calcium alginate/starch beads. DSC measurements were modified according to the measurements of pore sizes in silica gels by Kazuhiko Ishikiriyama et al. , b and MTm is described by the Gibbs -Thomson equation:–6 m3/mol, Hm is the heat of fusion of water which is 6.02 kJ/mol.Dried beads were rehydrated on water agar in Petri dishes and then immersed in excessive water overnight to remove residual ions and to fill the pores with water. Accurately weighed samples (3–6 mg) were sealed in 40 µL aluminum pans with pins. Because of their weight and size, rehydrated beads needed to be cut to fit into the aluminum pans. The samples were measured against an empty reference pan. Nitrogen served as purge gas at 50 ml/min. The measurements were performed over a temperature range from 233.15 to 283.15 K and melting curves were measured to determine the pore volume distributions. Samples were rapidly cooled to 233.15 K, held isothermally for 30 min, and subsequently heated to 183.15 K at a low scanning rate of 0.5 K/min to avoid thermal and time delays. After DSC measurements, samples were dried at 60 °C for 3 days and the dry weight or rather the mass of the water was determined. Measurements were conducted three times with three different beads for each kind of formulation. Resulting peaks were analyzed according to Maloney  with theHs is the temperature dependent specific heat of fusion of the ice-water transition concerning freezable pore water which was determined according to Ishikiriyama et al. .The spore forming activity of Beads were produced as described previously see ‘’ with bi2 release measurements from beads’). It should be noted that over time mycelial growth was favored and spore densities may be underestimated, since spores adhered in the dense mycelium.The spore density was evaluated again after 21 days, in order to compare the sporulation with that in a vented system .To assess how casein and FC influenced the metabolic activity of 2 accumulation and inhibition during cultivation. Additionally, the pressured air was humified using a gas washing bottle to prevent the beads and the water agar from drying. The relative humidity reached approximately 80%. By means of a compressed air distributor, 18 vessels were vented and cultured at once  simultaneously. The biomass from the same culture or batch was used for each kind of formulation. The spore density on beads was determined after the experiment was terminated, i.e. after 21 days.In order to determine the CO2 productivity of control alginate/starch beads without M. brunneum or S. cerevisiae biomass, was previously evaluated and was negligible low  and therefore, in subsequent experiments neglected. Due to surprising results, the experiment was repeated for Kill beads and A&K beads with and without FC.The CO. For non-normally distributed data sets, Kruskal–Wallis tests with multiple comparisons between groups were calculated.Statistical analyses were conducted with SPSS Statistics v. 27  with a significance level of p < 0.05, except for spore density analysis of sealed and vented cultures, which were conducted with a significance level of p < 0.01. All data are presented as mean values ± standard deviations (SD). Percentage data on the water activity and the rehydration factor were arcsine transformed prior to analysis of normality and homogeneity of variance with Shapiro–Wilk's and Levene's test, respectively. To analyze the influence of the treatments or the influence of treatment and time, one-way or repeated measures (RM) analysis of variance (ANOVA) with Bonferroni post hoc tests were conducted for normally distributed and homogeneous data. Welch's correction was applied for nonhomogeneous variances. The sphericity of the matrix assumption was assessed with the Mauchley sphericity test and F values were corrected accordingly using the Greenhouse–Geisser approachFirst, we investigated plain calcium alginate/starch beads without any fungal biomass in terms of their nutrient content, water absorbency and pore structure.Beads of different compositions appeared relatively homogeneous, rigid and did not disintegrate. The sphericity factor of dried and rehydrated beads were as follows, 0.076 ± 0.010 and 0.082 ± 0.003 for alginate/starch beads, respectively, 0.066 ± 0.003 and 0.071 ± 0.008 with FC, respectively, and 0.072 ± 0.010 and 0.065 ± 0.001 with casein, respectively (three replicates with 89–112 beads per replicate). The circularity of beads of different composition, either dried or rehydrated, was consistently between 0.88–0.89.We analyzed the formulation matrices with a C/N elemental analyzer to assess the nutritional influence of casein and FC. As expected, alginate/starch beads contained almost no nitrogen. Despite equal proportions of casein and FC in the mixture, incorporating FC, however, doubled the nitrogen content compared to casein, resulting in higher C/N ratios of 17.33 compared to 9.72, respectively .In order to study whether casein or FC influence the water absorption and water activity of alginate/starch beads, dried beads were rehydrated on water agar over a period of 48 h.w 0.174–0.175, 0.239–0.256 and 0.165–0.178, respectively. The water absorption of beads with either casein or FC differed significantly, also across time . Interestingly, casein initially decelerated the water absorption compared to alginate/starch beads with or without FC, finally reaching a rehydration factor of alginate/starch beads only . Casein initially decreased water activity of beads, reaching only 0.848 ± 0.019 after 15 min, compared to 0.953 ± 0.007 for FC and 0.954 ± 0.003 for alginate/starch. Regarding rehydration factor or water activity, all formulations reached equilibrium after 2 h.The bead composition and the interaction of bead composition and time also affected the water activity significantly  and the median pore size , but not the total pore volume .The formulation composition significantly affected the bead density . Additional data are given in Online Resource 1 Table A2.The pore volume distributions revealed a broader spectrum of pores for FC with more pronounced shoulders and peaks were visibly shifted to larger pore sizes for both casein and FC Fig. . ConsequIn sum, FC had a greater impact on porosity and density than casein, resulting in less dense beads with larger pores.M. brunneum, or both S. cerevisiae and M. brunneum, in Kill beads or A&K beads, respectively, by determining the spore formation and CO2 release.Next, we assessed the activity of either M. brunneum on beads. Moreover, it should be investigated to what extent a possible effect can be observed on beads containing additionally encapsulated S. cerevisiae. Therefore M. brunneum was individually encapsulated (Kill beads) or co-encapsulated with S. cerevisiae (A&K beads) in alginate/starch beads with either casein or FC. Beads were dried to aw 0.29–0.46. Both casein and FC increased spore density on Kill and A&K beads, but only FC led to a significant difference on both  and also across time  .2 release (see ‘CO2 release from beads’) the different Kill and A&K formulations from the same batch previously described, were aerated during cultivation. The sporulation differed quite obviously in the course of the experiment and it became apparent that to some extent spores were formed earlier ·106 spores/mm2 .By aeration, the spore density was increased on both Kill beads and A&K beads but only when FC was incorporated Fig. . As a re5 to 0.5 ± 0.5 105 spores/mm2). However, this was not seen for A&K beads, confirming the inconsistency of results mentioned previously. Overall, enhanced mycelial growth could be observed to some extent, but a positive and significant change in sporulation due to aeration was observed exclusively for FC. These results were in line with the drying kinetics of alginate/starch beads, which showed faster drying when FC was incorporated .Taken together, incorporating FC and subsequently venting the cultures increased the spore density even by the factor 18. Surprisingly, aeration had a negative impact on spore formation on Kill beads when casein was added , whereas casein even decreased it by 19% (from 142 g/L to 115 g/L). Thus, A&K beads containing casein produced only 63% of the CO2 that A&K beads containing FC released.Plain A&K beads reached their maximum productivity fastest, after 3 days, but collapsed also, recovering after 4 days. The CO2 yield even by 34% from 92 mL/g to 123 g/L . FC increased the CO2 productivity of M. brunneum and probably S. cerevisiae over a period of 20 days.The replica confirmed these surprising results and FC increased the total CO2 productivity, it led to a more continuous production and hence to an increased total CO2 release.Even though incorporating FC did not reach the maximum CO2 productivity of plain A&K beads were somehow caused by fluctuating water content or water activity. Beads were placed on semi selective water agar in Petri dishes and measured daily for 8 days. However, water absorption remained unchanged after 24 h for all formulations as they reached maximal water activity possible, which equaled that of moist beads .We investigated whether the fluctuations in COM. brunneum and S. cerevisiae, leading to enhanced spore formation and CO2 productivity.In this study, we found that FC influences the porous properties of alginate/starch beads, thus enhancing their water absorbency. Moreover, FC increases the fungal activity of encapsulated Metarhizium anisopliae (M. anisopliae), and according to Braga et al.  due to logistic reasons, and M. brunneum growth is known to be temperature dependent. This indicates that gas exchange like oxygen supply plays an important role which might be favored in the more porous structure conveyed by FC.FC enhanced the activity of S. cerevisiae was co-encapsulated, whereas casein’s impact was slightly improved on average, though spore yields were inconsistent. It is conceivable that S. cerevisiae compensated for the influence of FC on the porous structure due to its relatively high proportion in the matrix. We observed, that S. cerevisiae proliferation was favored on/in beads containing casein. However, stronger proliferation of S. cerevisiae coincided with poor sporulation of M. brunneum. There might have been an interaction between casein and S. cerevisiae, which led to inhibition of spore formation. It has already been reported that yeast, among them S. cerevisiae, are capable of decreasing the growth of filamentous spoilage fungi  n Walker . It is wOverall, we have not yet resolved, how the fungus develops inside the beads and how growth is influenced, since we investigated sporulation only.S. cerevisiae and the entomopathogenic fungus Beauveria bassiana enhances the CO2 release of moist alginate/starch beads compared to the respective single encapsulation  who also studied, the CO2 release of dried alginate/starch beads, but depending on amyloglucosidase content. There, maximum CO2 concentration was reached after 7 days. This is in line with our results obtained from A&K beads containing casein or FC. However, fluctuations in CO2 concentrations were not observed, even in the absence of amyloglucosidase Below is the link to the electronic supplementary material.