Arun Rajamohan
@ars.usda.gov
Research Entomologist, Insect Genetics and Biochemistry, USDA-ARS-Edward T Schafer Agricultural Research Center
USDA-ARS
RESEARCH, TEACHING, or OTHER INTERESTS
Agricultural and Biological Sciences, Animal Science and Zoology
Scopus Publications
Scopus Publications
Shawna Pantzke, Beth Ferguson, Arun Rajamohan, Joseph P Rinehart, Deirdre Prischmann-Voldseth, and Jarrad R Prasifka
Oxford University Press (OUP)
Abstract Most natural mortality of the red sunflower seed weevil, Smicronyx fulvus LeConte (Coleoptera: Curculionidae), occurs while larvae overwinter in the soil. To test the hypothesis that S. fulvus mortality is related to low temperatures, experiments were used to (i) evaluate the temperature at which larvae freeze (= supercooling point [SCP]), (ii) assess possible vertical movement between entry into the soil in fall and adult emergence in summer, and (iii) determine if realistic soil temperatures could explain patterns of overwintering mortality. Mean SCP for groups of S. fulvus larvae differed between years and months, but only ranged from −20.93 to −22.68 °C. Most overwintering larvae were found within 6 cm of the soil surface, but larvae appeared to move 1–2 cm deeper between pairs of successive sample dates (September to January, January to April). Significant larval mortality that occurred between January and April 2021 was tentatively attributed to a period in February where daily minimum soil temperatures ranged from −8 to −12 °C. When overwintering under control conditions (constant 4 °C) was interrupted with week-long exposure to −4, −8, or −12 °C in a cold bath, significant S. fulvus mortality was seen for temperatures at or below −8 °C. Combined results suggest that mortality of overwintering S. fulvus is likely caused by continuous exposure to low temperatures that may not be cold enough to freeze larvae. Additionally, the shallow overwintering by S. fulvus supports the idea that routine farm management, including tillage and herbicide incorporation, may help limit populations of this sunflower pest.
Claire Campion, Arun Rajamohan, and Michael E. Dillon
Elsevier BV
Arun Rajamohan, Jarrad R. Prasifka, and Joseph P. Rinehart
MDPI AG
Embryos of the sunflower moth, Homoeosoma electellum (Hulst), were cryopreserved after modification to the method that was previously described for Pectinophora gossipiella. The workflow to develop the protocol consisted of methods to weaken the embryonic chorion followed by the application of various methods to disrupt the sub-chorionic wax layer. These steps were necessary to render the embryos permeable to water and cryoprotectants. Initially, the embryos were incubated at 21° and 24 °C, and the development of the double pigment spots/eyespot and eclosion were tracked every two hours. The embryos at 24 °C showed eyespots as early as 30 h, while in the case of the embryos that were incubated at 21 °C, there was a developmental delay of approximately 20 h. The embryos at 24 °C showed peak eclosion between 55 and 70 h, and the embryos at 21 °C eclosed between 80 and 100 h of development. Estimating this range is crucial for the purposes of stage selection and treatment initiation for cryopreservation protocol development for the embryos. The control hatch percentage at either developmental temperature was >90%, and the sodium hypochloride, 2-propanol and alkane-based treatments reduced the embryo hatchability to <10%. Hence, a modified surfactant—hypochlorite mixture—was used to destabilize the chorion and solubilize the hydrophobic lipid layers. Water permeability assessments using the dye-uptake method show that polysorbate 80 in combination with sodium hypochlorite alone is capable of permeabilizing the embryo as efficiently as sequential hypochlorite—alkane treatments, but with significantly higher hatch rates. A vitrification medium consisting of ethane diol and trehalose was used to dehydrate and load the embryos with the cryoprotective agent. The median hatch rates after vitrification were 10%, and maximum was 23%.
Korie M. DeBardlabon, Arun Rajamohan, and Joseph P. Rinehart
Elsevier BV
Claire Campion, Arun Rajamohan, and Joseph P. Rinehart
Elsevier BV
George D Yocum, Arun Rajamohan, and Joseph P Rinehart
Oxford University Press (OUP)
Abstract Interrupting the spring incubation of Megachile rotundata (F.) with a period of low-temperature storage for synchronizing the bees’ emergence with crop bloom is an essential part of M. rotundata management. Previously, we demonstrated that bees exposed to thermoperiods (TPs) during low-temperature storage have higher survival rates than bees exposed to constant temperatures. But changing the temperature in the large mass of bees commonly found in most commercial settings would place considerable stress on the chambers’ refrigeration system. Reducing the difference between a TP’s cryophase and thermophase would decrease the stress on the refrigeration system. Therefore, we investigated a range of TPs with cryophases (12 h) of 6, 12, or 15°C and thermophases (12 h) of 15 or 18°C and compared the survival rates of these bees against bees exposed to constant temperatures of 12, 15, or 18°C. For eye-pigmented pupae, the TP 6–18°C and the control fluctuating thermal regime (FTR; 6°C with a daily 1-h pulse at 20°C) had the highest survival rates for the 2 yr tested. For the constant-temperature storage protocols, constant 15 and 18°C were either equivalent or lower survival than the control FTR. For emergence-ready adults, the 6–18°C TP had the highest survival rates. The constant 15°C and the control FTR had equivalent survival rates. Under the current constraints imposed by a commercial chamber’s refrigeration system, interrupting M. rotundata spring incubation by exposing the developing bees to constant temperatures of 15–18°C is currently the best option for commercial operations.
Jacob B. Campbell, Andrew Dosch, Catherine M. Hunt, Ellen M. Dotson, Mark Q. Benedict, Arun Rajamohan, and Joseph P. Rinehart
Elsevier BV
Arun Rajamohan, Robert G. Danka, Brandon K. Hopkins, and Joseph P. Rinehart
Elsevier BV
George D Yocum, Joseph P Rinehart, Arun Rajamohan, Julia H Bowsher, Kathleen M Yeater, and Kendra J Greenlee
Oxford University Press (OUP)
AbstractInsects exposed to low temperature stress can experience chill injury, but incorporating fluctuating thermoprofiles increases survival and blocks the development of sub-lethal effects. The specific parameters required for a protective thermoprofile are poorly understood, because most studies test a limited range of thermoprofiles. For example, thermoprofiles with a wave profile may perform better than a square profile, but these two profiles are rarely compared. In this study, two developmental stages of the alfalfa leafcutting bee, Megachile rotundata, eye-pigmented pupae, and emergence-ready adults, were exposed to one of eight thermoprofiles for up to 8 weeks. All the thermoprofiles had a base of 6°C and a peak temperature of either 12°C or 18°C. The duration at peak temperature varied depending on the shape of the thermoprofile, either square or wave form. Two other treatments acted as controls, a constant 6°C and a fluctuating thermal regime (FTR) with a base temperature of 6°C that was interrupted daily by a single, 1-h pulse at 20°C. Compared with constant 6°C, all the test thermoprofiles significantly improved survival. Compared with the FTR control, the thermoprofiles with a peak temperature of 18°C outperformed the 12°C profiles. Bees in the eye-pigmented stage exposed to the 18°C profiles separated into two groups based on the shape of the profile, with higher survival in the square profiles compared with the wave profiles. Bees in the emergence-ready stage exposed to 18°C profiles all had significantly higher survival than bees in the FTR controls. Counter to expectations, the least ecologically relevant thermoprofiles (square) had the highest survival rates and blocked the development of sub-lethal effects (delayed emergence).
George D. Yocum, Anna K. Childers, Joseph P. Rinehart, Arun Rajamohan, Theresa L. Pitts-Singer, Kendra J. Greenlee, and Julia H. Bowsher
The Company of Biologists
Our understanding of the mechanisms controlling insect diapause has increased dramatically with the introduction of global gene expression techniques, such as RNA-seq. However, little attention has been given to how ecologically relevant field conditions may affect gene expression during diapause development because previous studies have focused on laboratory reared and maintained insects. To determine whether gene expression differs between laboratory and field conditions, prepupae of the alfalfa leafcutting bee, Megachile rotundata, entering diapause early or late in the growing season were collected. These two groups were further subdivided in early autumn into laboratory and field maintained groups, resulting in four experimental treatments of diapausing prepupae: early and late field, and early and late laboratory. RNA-seq and differential expression analyses were performed on bees from the four treatment groups in November, January, March and May. The number of treatment-specific differentially expressed genes (97 to 1249) outnumbered the number of differentially regulated genes common to all four treatments (14 to 229), indicating that exposure to laboratory or field conditions had a major impact on gene expression during diapause development. Principle component analysis and hierarchical cluster analysis yielded similar grouping of treatments, confirming that the treatments form distinct clusters. Our results support the conclusion that gene expression during the course of diapause development is not a simple ordered sequence, but rather a highly plastic response determined primarily by the environmental history of the individual insect.
Arun Rajamohan, Joseph P. Rinehart, and Roger A. Leopold
Elsevier BV
Bryan R. Helm, Garett Slater, Arun Rajamohan, George D. Yocum, Kendra J. Greenlee, and Julia H. Bowsher
The Company of Biologists
In holometabolous insects, larval nutrition affects adult body size, a life history trait with a profound influence on performance and fitness. Individual nutritional components of larval diets are often complex and may interact with one another, necessitating the use of a geometric framework for elucidating nutritional effects. In the honey bee, Apis mellifera, nurse bees provision food to developing larvae, directly moderating growth rates and caste development. However, the eusocial nature of honey bees makes nutritional studies challenging, because diet components cannot be systematically manipulated in the hive. Using in vitro rearing, we investigated the roles and interactions between carbohydrate and protein content on larval survival, growth, and development in A. mellifera. We applied a geometric framework to determine how these two nutritional components interact across nine artificial diets. Honey bees successfully completed larval development under a wide range of protein and carbohydrate contents, with the medium protein (∼5%) diet having the highest survival. Protein and carbohydrate both had significant and non-linear effects on growth rate, with the highest growth rates observed on a medium-protein, low-carbohydrate diet. Diet composition did not have a statistically significant effect on development time. These results confirm previous findings that protein and carbohydrate content affect the growth of A. mellifera larvae. However, this study identified an interaction between carbohydrate and protein content that indicates a low-protein, high-carb diet has a negative effect on larval growth and survival. These results imply that worker recruitment in the hive would decline under low protein conditions, even when nectar abundance or honey stores are sufficient.
Antonios A. Augustinos, Arun Rajamohan, Georgios A. Kyritsis, Antigone Zacharopoulou, Ihsan ul Haq, Asya Targovska, Carlos Caceres, Kostas Bourtzis, and Adly M. M. Abd-Alla
Public Library of Science (PLoS)
The Mediterranean fruit fly, Ceratitis capitata, is one of the most serious pests of fruit crops world-wide. During the last decades, area-wide pest management (AW-IPM) approaches with a sterile insect technique (SIT) component have been used to control populations of this pest in an effective and environment-friendly manner. The development of genetic sexing strains (GSS), such as the Vienna 8 strain, has been played a major role in increasing the efficacy and reducing the cost of SIT programs. However, mass rearing, extensive inbreeding, possible bottleneck phenomena and hitch-hiking effects might pose major risks for deterioration and loss of important genetic characteristics of domesticated insect. In the present study, we present a modified procedure to cryopreserve the embryos of the medfly Vienna 8 GSS based on vitrification and used this strain as insect model to assess the impact of the cryopreservation process on the genetic structure of the cryopreserved insects. Forty-eight hours old embryos, incubated at 24°C, were found to be the most suitable developmental stage for cryopreservation treatment for high production of acceptable hatch rate (38%). Our data suggest the absence of any negative impact of the cryopreservation process on egg hatch rate, pupation rates, adult emergence rates and stability of the temperature sensitive lethal (tsl) character on two established cryopreserved lines (flies emerged from cryopreserved embryos), named V8-118 and V8-228. Taken together, our study provides an optimized procedure to cryopreserve the medfly Vienna 8 GSS and documents the absence of any negative impact on the genetic structure and quality of the strain. Benefits and sceneries for utilization of this technology to support operational SIT projects are discussed in this paper.
Arun Rajamohan, Joseph P. Rinehart, and Roger A. Leopold
Elsevier BV
Arun Rajamohan, Joseph P. Rinehart, and Roger A. Leopold
Oxford University Press (OUP)
ABSTRACT
Embryos of Lucilia (Phaenicia) sericata (Meigen) (Diptera: Calliphoridae), the green blowfly, were successfully cryopreserved by vitrification in liquid nitrogen and stored for 8 yr. Embryos incubated at 19°C for 17 h after oviposition were found to be the most appropriate stage to cryopreserve. Removal of the embryonic surface water was done using 2-propanol before the alkane treatment to permeabilize the embryo. Exposure to 2-propanol for >10 s caused necrotic tissue damage in the embryos. Among the alkanes used, hexane was found to be a superior permeabilizing solvent compared with heptane or octane, with embryo hatching rates on par with the controls. Treatment with the vitrification solution for <12 min was insufficient to vitrify the embryos. Treatment time in the solution beyond 15 min reduced embryo viability. However, the percentage of embryos vitrifying upon exposure to liquid nitrogen vapor remained constant after 12 min of treatment. Long-term storage was initiated in 2004, and the mean hatch percentage recorded then for the short-term cryopreserved embryos was 9.51%. When the long-term stored samples were retrieved in 2012, 8.47% of the embryos hatched, 66.36% larvae pupariated, and 36.96% of the pupae eclosed. Recent optimization of the technique has resulted in a hatch rate of 34.08 ± 15.5%, of which 67.5% of the larvae pupariated and 72% of the pupae eclosed to normal flies.
Arun Rajamohan, Joseph P. Rinehart, Stephen P. Foster, and Roger A. Leopold
Oxford University Press (OUP)
ABSTRACT
The aim of this study was to develop a method to cryopreserve the embryos of the pink bollworm moth, Pectinophora gossypiella (Saunders). Previously developed dipteran cryopreservation protocols were not directly adaptable to use with the embryos of this lepidopteran species. Physiochemical and electron microscope observations revealed substantial differences in the structure of the chorion, wax layer, and vitelline membrane complex when comparing the cryopreservable embryonic stages of P. gossypiella and dipteran embryos. Thus, the initial steps dealing with dechorionation and permeabilization were ineffective and had to be altered. Exposure to the sodium hypochlorite-based chorion removal step decreased P. gossypiella embryo viability to a very low level. Survival increased and permeability was evident when an alkane wash was used as the first step in the procedure. After the alkane treatment with a surfactant yielded the maximum exchange of cryoprotectant with water as evidenced by a significant lowering of the supercooling point of the cryoprotectant-loaded embryos. The remainder of the cryopreservation and storage recovery protocol for P. gossypiella was similar to those developed for dipteran embryos. Survival of recovered, hatched embryos to adulthood was ≈7%.
Stephanie Sobek, Arun Rajamohan, Daniel Dillon, Robert C. Cumming, and Brent J. Sinclair
Wiley
1 The emerald ash borer Agrilus planipennis (Coleoptera: Buprestidae) (EAB), an invasive wood‐boring beetle, has recently caused significant losses of native ash (Fraxinus spp.) trees in North America. Movement of wood products has facilitated EAB spread, and heat sanitation of wooden materials according to International Standards for Phytosanitary Measures No. 15 (ISPM 15) is used to prevent this. 2 In the present study, we assessed the thermal conditions experienced during a typical heat‐treatment at a facility using protocols for pallet wood treatment under policy PI‐07, as implemented in Canada. The basal high temperature tolerance of EAB larvae and pupae was determined, and the observed heating rates were used to investigate whether the heat shock response and expression of heat shock proteins occurred in fourth‐instar larvae. 3 The temperature regime during heat treatment greatly exceeded the ISPM 15 requirements of 56 °C for 30 min. Emerald ash borer larvae were highly tolerant of elevated temperatures, with some instars surviving exposure to 53 °C without any heat pre‐treatments. High temperature survival was increased by either slow warming or pre‐exposure to elevated temperatures and a recovery regime that was accompanied by up‐regulated hsp70 expression under some of these conditions. 4 Because EAB is highly heat tolerant and exhibits a fully functional heat shock response, we conclude that greater survival than measured in vitro is possible under industry treatment conditions (with the larvae still embedded in the wood). We propose that the phenotypic plasticity of EAB may lead to high temperature tolerance very close to conditions experienced in an ISPM 15 standard treatment.
Roger A Leopold, Arun Rajamohan, Todd E Shelly, and Alfred M Handler
Oxford University Press (OUP)
Abstract This study evaluates characteristics commonly used to define insect quality or fitness by using a complement of three species of tephritid fruit flies (Diptera: Tephritidae) obtained from cryopreserved embryos. Anastrepha ludens (Loew), Anastrepha suspensa (Loew), and Ceratitis capitata (Wiedemann) were used to assess embryo to adult emergence and adult longevity, flight ability, mating ability, fecundity, and genetic variability after cryopreservation. With the three species tested, embryo survival was reduced by 50–70% whereas adult eclosion seemed unaffected by cryogenic treatment. Laboratory cage survival of cryopreserved A. ludens paralleled that of controls when tested with or without food and water posttreatment. With C. capitata, field cage survival was also similar when the adult progeny of cryopreserved parents was compared with that of untreated flies of the same age. Assessment of flight ability of cryopreserved A. ludens over a 19-d period by using a flight mill showed no statistical difference when compared with the untreated groups over the same time period. Flight ability within field cages for newly emerged progeny of cryopreserved C. capitata also mirrored that of the controls. Observed matings occurring within laboratory cages containing equal numbers of A. ludens males and females did not differ from comparable control groups. Furthermore, male progeny obtained from cryopreserved C. capitata parents competed equally with untreated males for mates while housed in field cages. A laboratory analysis of fertility and fecundity of A. suspensa revealed that males mated with control females were unaffected by cryopreservation as embryos, whereas cryopreserved females exhibited a significantly reduced fecundity when mated with control males. The fecundity of C. capitata progeny of cryopreserved parents also did not differ from control levels while caged under laboratory conditions. A random amplified polymorphic DNA assay of the genetic diversity of A. ludens comparing cryopreserved males with control males showed that the coefficient of similarity was ≥85%. This study indicates that embryo cryopreservation had little or no effect on the reproduction, longevity and flight of the species tested and can be used to support maintenance of insect stocks and control programs supported by the mass-rearing process.
Brent J. Sinclair, Allen G. Gibbs, Wah-Keat Lee, Arun Rajamohan, Stephen P. Roberts, and John J. Socha
Public Library of Science (PLoS)
Although the biochemical correlates of freeze tolerance in insects are becoming well-known, the process of ice formation in vivo is subject to speculation. We used synchrotron x-rays to directly visualise real-time ice formation at 3.3 Hz in intact insects. We observed freezing in diapausing 3rd instar larvae of Chymomyza amoena (Diptera: Drosophilidae), which survive freezing if it occurs above −14°C, and non-diapausing 3rd instar larvae of C. amoena and Drosophila melanogaster (Diptera: Drosophilidae), neither of which survive freezing. Freezing was readily observed in all larvae, and on one occasion the gut was seen to freeze separately from the haemocoel. There were no apparent qualitative differences in ice formation between freeze tolerant and non-freeze tolerant larvae. The time to complete freezing was positively related to temperature of nucleation (supercooling point, SCP), and SCP declined with decreasing body size, although this relationship was less strong in diapausing C. amoena. Nucleation generally occurred at a contact point with the thermocouple or chamber wall in non-diapausing larvae, but at random in diapausing larvae, suggesting that the latter have some control over ice nucleation. There were no apparent differences between freeze tolerant and non-freeze tolerant larvae in tracheal displacement or distension of the body during freezing, although there was markedly more distension in D. melanogaster than in C. amoena regardless of diapause state. We conclude that although control of ice nucleation appears to be important in freeze tolerant individuals, the physical ice formation process itself does not differ among larvae that can and cannot survive freezing. This suggests that a focus on cellular and biochemical mechanisms is appropriate and may reveal the primary adaptations allowing freeze tolerance in insects.
ARUN RAJAMOHAN and BRENT J. SINCLAIR
Wiley
Abstract Chill‐susceptible insects are able to improve their survival of acute cold exposure over both the short term (i.e. hardening at a relatively severe temperature) and longer term (i.e. acclimation responses at milder temperatures over a longer time frame). However, the mechanistic overlap of these responses is not clear. Four larval stages of four different strains of Drosophila melanogaster are used to test whether low temperature acclimation (10 °C for 48 h) improves the acute cold tolerance (LT90, ∼2 h) of larvae, and whether acclimated larvae still show hardening responses after brief exposures to nonlethal cold or heat, or a combination of the two. Acclimation results in increased cold tolerance in three of four strains, with variation among instars. However, if acclimation is followed by hardening pre‐treatments, there is no improvement in acute cold survival. It is concluded that short‐term thermal responses (e.g. hardening) may be of more ecological relevance to short‐lived life stages such as larvae, and that the mechanisms of low temperature hardening and acclimation in D. melanogaster may be antagonistic, rather than complementary.
Arun Rajamohan and Brent J. Sinclair
Elsevier BV
Brent J. Sinclair and Arun Rajamohan
Cambridge University Press (CUP)
AbstractWe tested the hypothesis that slow rewarming would improve the ability ofDrosophila melanogasterMeigen (Diptera: Drosophilidae) larvae to survive acute low-temperature exposure. Four larval stages (1st, 2nd, and 3rd instars, including wandering-stage 3rd instars) of four wild-type strains were exposed to –7 °C for periods of time expected to result in 90% mortality. Larvae were then directly transferred to their rearing temperature (21 °C) or returned to this temperature either in a stepwise fashion (pausing at 0 and 15 °C) or by slow warming at 1 or 0.1 °C/min. We observed a reduced rapid cold-hardening effect and no general increase in survival of acute chilling in larvae rewarmed in a stepwise or slow fashion, and we hypothesize that slow rewarming may result in accumulation of chill injuries.
A. Rajamohan and R.A. Leopold
Elsevier BV