TY - BOOK

T1 - Viral and fungal pathogens in house cricket rearing

T2 - Effects of temperature, density and co-infection

AU - Takacs, Jozsef

PY - 2024

Y1 - 2024

N2 - The house cricket, Acheta domesticus, is a commonly reared insect for food and feed purposes. In 1977, a report described a colony collapse, which was caused by the single-stranded DNA virus, the Acheta domesticus densovirus (AdDV) and ever since, disease outbreaks are a continuous threat to A. domesticus mass-rearing. Currently the reasons leading to viral outbreaks are unknown, and in most cases house crickets show no symptoms and only little mortality occurs in the populations. AdDV infection and disease outbreaks in house cricket mass-rearing could be prevented by identifying triggers leading to viral outbreaks and removing or preventing such triggers, by reducing virus spread or by obtaining virus-free crickets. To limit virus spread, a better understanding of how viral levels vary and how the virus is transmitted during the house cricket life cycle is required. The central objective of this thesis was to study the effects of key stressors associated with house cricket rearing that could affect house cricket survival and biomass and/or that could be triggers of AdDV outbreaks. These stressors include cricket density, temperature and exposure to two fungal pathogens. House crickets are usually reared in high densities to maximize cost effectiveness, but this may imply stressful conditions affecting house cricket fitness and creating a highly favourable environment for disease outbreaks. A better understanding of the relation between AdDV levels and these stressors can be used to implement changes in the rearing setups with the aim to reduce the probability of AdDV outbreaks and losses related to this pathogen or other suboptimal conditions. Combined stressors affecting house crickets simultaneously under rearing conditions received little attention so far. In this thesis, several combined stressor experiments were conducted to assess the effects on house cricket survival, biomass and AdDV levels in different tissues.Correlations between house cricket rearing density or temperature and AdDV abundance have been hypothesized, but experimental evidence is lacking. Optimised rearing conditions, including optimal temperature and house cricket density, are key to cost-effective house cricket production, thus these two key factors were selected (Chapter 2). House crickets were subjected to three different temperatures (25, 30, 35 °C) in combination with three different rearing densities (10, 20, 40 crickets per experimental unit). The effects on cricket survival, on male and female individual biomass, on total biomass obtained and on AdDV levels (relative to the starting levels) were recorded and statistically compared. Rearing temperature had a minor effect on cricket survival, which ranged between 80 and 83%. Total cricket biomass increased with higher temperatures and higher densities. Viral abundance in crickets at the end of the rearing period was variable; however, a high rearing density tended to result in a higher AdDV abundance. At 35 °C, a temperature considered suboptimal for house cricket production, viral abundance tended to be lower than at 25 °C or 30 °C. This was the first study to examine the combined effect of these two stressors.In a next study two other stressors were combined, a microbial pathogen and rearing density (Chapter 3). We first compared the virulence of six isolates of Beauveria bassiana and six isolates of Metarhizium brunneum to house crickets. Subsequently, the most virulent isolate from each genus (B. bassiana isolate KVL 97-44 and M. brunneum isolate KVL 20-01) were tested in combination with three different cricket densities (10, 20, 40 crickets per experimental unit). The combined effect of these stressors on cricket survival, individual female and male biomass and the total biomass obtained was evaluated. Both the exposure to fungal pathogens and the high rearing density reduced cricket survival and had a negative impact on the total biomass production. The average individual biomass of females and males in case of B. bassiana exposure was negatively affected, but no effect of M. brunneum exposure was observed. The increased cricket densities had a minor negative impact on individual biomass. The combined effect of the two stressors resulted in a trend towards a lower individual biomass, but with only a minor effect in the case of B. bassiana exposure. Producers need to consider the risk of a higher mortality with higher cricket densities, but the increased losses might be outweighed by the higher total biomass output from the same surface area. Fungal pathogens are a major threat if they appear in the rearing facility, but only if relatively high spore numbers are present, which are less likely to occur when preventive measures and proper sanitation protocols are established. House cricket producers with good hygiene and rearing setups can overcome the threats, but constant monitoring and, if necessary, intervention might be required.Literature suggest that mortality due to AdDV peaks in the late instars of house crickets, but this has not yet been experimentally examined. In Chapter 4, therefore, the changes in viral levels were measured throughout the rearing cycle, using quantitative PCR. Samples were collected 1) simultaneously from different life stages present in the rearing room at a given time and 2) weekly from a single rearing container that was monitored during the successive stages of development. In addition, the tissue tropism of AdDV was examined by determining its presence and levels in different tissues. Viral levels were measured in various tissues from mated and non-mated adult crickets to infer the route of virus transmission. Ovaries of both unmated and mated females and the spermatheca of mated females were collected, while testes and accessory glands were collected from unmated and mated males. Guts were collected from both genders. We found that viral levels are increasing during the life cycle of the house crickets, with older crickets having higher viral levels. Interestingly, for both genders, unmated individuals had significantly lower viral levels than mated individuals. Furthermore, AdDV was present in every tested tissue, but at different levels. In both mated and unmated females, the gut and ovaries showed higher viral levels than the spermatheca of mated females. The dissected male tissues all had similar viral levels. The results presented suggest that AdDV is both horizontally and vertically transmitted among house crickets and provide relevant information for future work on designing optimized preventive measures and on establishing virus-free cricket lines.Although AdDV is considered the most prevalent virus in house cricket rearing, we cannot ignore other viruses to be potentially present in crickets, and the possible effects that those viruses could have on cricket fitness and on disease outbreaks. Various studies identified viruses present in house cricket colonies, but the majority used a primer-based approach for detecting viruses and were limited to viruses already known to be pathogens of house crickets. With the advancement of next generation sequencing (NGS) technologies, virus detection and discovery can be carried out using an unbiased approach. In Chapter 5, DNA and RNA viruses present in house crickets were identified, by analyzing sequencing data from house crickets originating from three different populations. This is the first study to use NGS on pooled samples obtained from various cricket stocks to report both DNA and RNA viruses present. Three putative new RNA viruses are described, which were not yet associated with house crickets before. Two of the new viruses were positive single stranded RNA viruses (+ssRNA) and were identified as the Acheta domesticus virus 2 (AdV2) from the Iflaviridae family and the Acheta domesticus nodavirus 1 (AdNdV1) from the Nodaviridae family. These viruses were so far not associated with house crickets. The third new virus was a double stranded RNA virus (dsRNA) from the Reoviridae family and was identified as Acheta domesticus cypovirus 1 (AdCyp1). As for other DNA viruses, only AdDV was detected and none of the earlier described cricket viruses (Cricket paralysis virus (CrPV) cricket iridovirus (CrIV), the A. domesticus mini ambidensovirus (AdMADV), A. domesticus volvovirus (AdVVV) , A. domesticus iflavirus (AdIV) and the A. domesticus virus (AdV)) were present in either of the tested stocks. Unexpectedly, from the three tested colonies, only one was infected by AdDV, the other two proved to be free of AdDV. In Chapter 6, the findings of the research chapters are integrated. I discuss how cricket rearing is affected by multiple stressors that need to be examined in combination to design optimal cricket rearing strategies. Furthermore, I elaborate on the need for complex diagnostic methods integrating modern technologies and classical bioassays. At the end of the chapter, suggestions for future research are given.

AB - The house cricket, Acheta domesticus, is a commonly reared insect for food and feed purposes. In 1977, a report described a colony collapse, which was caused by the single-stranded DNA virus, the Acheta domesticus densovirus (AdDV) and ever since, disease outbreaks are a continuous threat to A. domesticus mass-rearing. Currently the reasons leading to viral outbreaks are unknown, and in most cases house crickets show no symptoms and only little mortality occurs in the populations. AdDV infection and disease outbreaks in house cricket mass-rearing could be prevented by identifying triggers leading to viral outbreaks and removing or preventing such triggers, by reducing virus spread or by obtaining virus-free crickets. To limit virus spread, a better understanding of how viral levels vary and how the virus is transmitted during the house cricket life cycle is required. The central objective of this thesis was to study the effects of key stressors associated with house cricket rearing that could affect house cricket survival and biomass and/or that could be triggers of AdDV outbreaks. These stressors include cricket density, temperature and exposure to two fungal pathogens. House crickets are usually reared in high densities to maximize cost effectiveness, but this may imply stressful conditions affecting house cricket fitness and creating a highly favourable environment for disease outbreaks. A better understanding of the relation between AdDV levels and these stressors can be used to implement changes in the rearing setups with the aim to reduce the probability of AdDV outbreaks and losses related to this pathogen or other suboptimal conditions. Combined stressors affecting house crickets simultaneously under rearing conditions received little attention so far. In this thesis, several combined stressor experiments were conducted to assess the effects on house cricket survival, biomass and AdDV levels in different tissues.Correlations between house cricket rearing density or temperature and AdDV abundance have been hypothesized, but experimental evidence is lacking. Optimised rearing conditions, including optimal temperature and house cricket density, are key to cost-effective house cricket production, thus these two key factors were selected (Chapter 2). House crickets were subjected to three different temperatures (25, 30, 35 °C) in combination with three different rearing densities (10, 20, 40 crickets per experimental unit). The effects on cricket survival, on male and female individual biomass, on total biomass obtained and on AdDV levels (relative to the starting levels) were recorded and statistically compared. Rearing temperature had a minor effect on cricket survival, which ranged between 80 and 83%. Total cricket biomass increased with higher temperatures and higher densities. Viral abundance in crickets at the end of the rearing period was variable; however, a high rearing density tended to result in a higher AdDV abundance. At 35 °C, a temperature considered suboptimal for house cricket production, viral abundance tended to be lower than at 25 °C or 30 °C. This was the first study to examine the combined effect of these two stressors.In a next study two other stressors were combined, a microbial pathogen and rearing density (Chapter 3). We first compared the virulence of six isolates of Beauveria bassiana and six isolates of Metarhizium brunneum to house crickets. Subsequently, the most virulent isolate from each genus (B. bassiana isolate KVL 97-44 and M. brunneum isolate KVL 20-01) were tested in combination with three different cricket densities (10, 20, 40 crickets per experimental unit). The combined effect of these stressors on cricket survival, individual female and male biomass and the total biomass obtained was evaluated. Both the exposure to fungal pathogens and the high rearing density reduced cricket survival and had a negative impact on the total biomass production. The average individual biomass of females and males in case of B. bassiana exposure was negatively affected, but no effect of M. brunneum exposure was observed. The increased cricket densities had a minor negative impact on individual biomass. The combined effect of the two stressors resulted in a trend towards a lower individual biomass, but with only a minor effect in the case of B. bassiana exposure. Producers need to consider the risk of a higher mortality with higher cricket densities, but the increased losses might be outweighed by the higher total biomass output from the same surface area. Fungal pathogens are a major threat if they appear in the rearing facility, but only if relatively high spore numbers are present, which are less likely to occur when preventive measures and proper sanitation protocols are established. House cricket producers with good hygiene and rearing setups can overcome the threats, but constant monitoring and, if necessary, intervention might be required.Literature suggest that mortality due to AdDV peaks in the late instars of house crickets, but this has not yet been experimentally examined. In Chapter 4, therefore, the changes in viral levels were measured throughout the rearing cycle, using quantitative PCR. Samples were collected 1) simultaneously from different life stages present in the rearing room at a given time and 2) weekly from a single rearing container that was monitored during the successive stages of development. In addition, the tissue tropism of AdDV was examined by determining its presence and levels in different tissues. Viral levels were measured in various tissues from mated and non-mated adult crickets to infer the route of virus transmission. Ovaries of both unmated and mated females and the spermatheca of mated females were collected, while testes and accessory glands were collected from unmated and mated males. Guts were collected from both genders. We found that viral levels are increasing during the life cycle of the house crickets, with older crickets having higher viral levels. Interestingly, for both genders, unmated individuals had significantly lower viral levels than mated individuals. Furthermore, AdDV was present in every tested tissue, but at different levels. In both mated and unmated females, the gut and ovaries showed higher viral levels than the spermatheca of mated females. The dissected male tissues all had similar viral levels. The results presented suggest that AdDV is both horizontally and vertically transmitted among house crickets and provide relevant information for future work on designing optimized preventive measures and on establishing virus-free cricket lines.Although AdDV is considered the most prevalent virus in house cricket rearing, we cannot ignore other viruses to be potentially present in crickets, and the possible effects that those viruses could have on cricket fitness and on disease outbreaks. Various studies identified viruses present in house cricket colonies, but the majority used a primer-based approach for detecting viruses and were limited to viruses already known to be pathogens of house crickets. With the advancement of next generation sequencing (NGS) technologies, virus detection and discovery can be carried out using an unbiased approach. In Chapter 5, DNA and RNA viruses present in house crickets were identified, by analyzing sequencing data from house crickets originating from three different populations. This is the first study to use NGS on pooled samples obtained from various cricket stocks to report both DNA and RNA viruses present. Three putative new RNA viruses are described, which were not yet associated with house crickets before. Two of the new viruses were positive single stranded RNA viruses (+ssRNA) and were identified as the Acheta domesticus virus 2 (AdV2) from the Iflaviridae family and the Acheta domesticus nodavirus 1 (AdNdV1) from the Nodaviridae family. These viruses were so far not associated with house crickets. The third new virus was a double stranded RNA virus (dsRNA) from the Reoviridae family and was identified as Acheta domesticus cypovirus 1 (AdCyp1). As for other DNA viruses, only AdDV was detected and none of the earlier described cricket viruses (Cricket paralysis virus (CrPV) cricket iridovirus (CrIV), the A. domesticus mini ambidensovirus (AdMADV), A. domesticus volvovirus (AdVVV) , A. domesticus iflavirus (AdIV) and the A. domesticus virus (AdV)) were present in either of the tested stocks. Unexpectedly, from the three tested colonies, only one was infected by AdDV, the other two proved to be free of AdDV. In Chapter 6, the findings of the research chapters are integrated. I discuss how cricket rearing is affected by multiple stressors that need to be examined in combination to design optimal cricket rearing strategies. Furthermore, I elaborate on the need for complex diagnostic methods integrating modern technologies and classical bioassays. At the end of the chapter, suggestions for future research are given.

M3 - Ph.D. thesis

BT - Viral and fungal pathogens in house cricket rearing

PB - Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen

ER -