Stock Colony Research Articles

Fat Accretion and Growth in the Rat

The normal course of fat accretion in male rats from birth to 3 years is described. The relative amount of fat and relative size of fat pads increased steadily throughout life (except for a brief cessation in fat accretion at weaning), in a manner closely related to body weight and independent of age. Several new rat strains with extremes of growth or fat accretion, fed the same diet, were used to define and illustrate courses of fat accretion: very large and very small non-obese strains and a hereditarily obese rat. Rats of different known strains fed a variety of more refined diets show a course of fat growth different from that obtained with stock diets. Several types of obesity are discussed: insulin obesity, nutritional obesity, and hereditary obesity. Hereditary size and growth of the rat were readily (in a few generations) altered up or down, by consistently using the largest or the smallest 25% of the population for breeders. It is probable that the large increase in size of some laboratory rats over the past 40 years has been accomplished not only by improvement in stock diets and colony conditions, but also by selection for increased hereditary size.

Inheritance of Two Traits With High Plasticity, Developmental Speed, and Body Size, in Tenebrio molitor (Coleoptera: Tenebrionidae).

The study of inheritance of quantitative traits of high plasticity in insects has been limited. The heritability of larval development time and body weight in Tenebrio molitor L. was determined using the method of parent-offspring regression. The parental group of adults obtained from a cohort from one day of oviposition from a stock colony was divided into 28 class groups according to their larval development time and pupal weight. The progeny resulting from these parental classes was grouped in experimental units and allowed to develop to the pupal stage. Means of larval development time and pupal weight of the progeny were compared with their parental class levels using linear regression. The selection of larval development time and pupal weight in the parental classes had a significant impact on the means of larval development time and pupal weight of the progeny. The regression coefficients for larval development time and pupal weight were 0.626 ± 0.02 and 0.408 ± 0.02, respectively. These values represent the proportion of genetic determination of these two traits based on the principles of the method of parent-offspring regression. The apparent independence of larval development time and pupal weight based on their poor linear correlation is discussed.

Establishment and Maintenance of Gnotobiotic American Cockroaches (<em>Periplaneta americana</em>)

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches (Periplaneta americana). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 °C for approximately 4−6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana).

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches (Periplaneta americana). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 °C for approximately 4-6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.

RNA viruses in the house dust mite Dermatophagoides pteronyssinus, detection in environmental samples and in commercial allergen extracts used for in vivo diagnosis.

Allergy to house dust mites (HDM), the most important source of indoor allergens worldwide, is diagnosed and treated using natural extracts from cultures that can contain immunoactive components from the HDM microbiome, including mite-infecting viruses. This study aimed to contribute to the discovery and characterization of RNA viruses from Dermatophagoides pteronyssinus, followed by their detection in different mite-derived sources. Viruses were assembled after in silico metatranscriptomic analysis of D.pteronyssinus RNA samples, visualized by electron microscopy, and RNA detected by direct RT-PCR or data mining. Mite culture performance was evaluated in vivo. Seven RNA viruses were identified in our laboratory stock colony. Picornavirus-like viral particles were detected in epithelial cells of the digestive system and in fecal pellets. Most of these viruses could be persistently transmitted to an inbred virus-free colony by inoculating fecal material from the stock colony. Upon viral infection, no significant effect could be seen on mite population growth. Transcriptomic screening confirmed the presence of homolog sequences to these viruses in independent laboratory stocks of D.pteronyssinus and in other Astigmata mites. Noteworthy, RNA from most of the viruses could be detected by RT-PCR on house dust samples, reference standards, and/or commercial diagnostic D.pteronyssinus extracts. Our results show that viral infections are common and widespread in D.pteronyssinus, both in natural and culture-based growth conditions. Potential effects on the mites themselves and consequences toward allergenicity in humans whether exposed naturally or after immunotherapy are discussed.

Description of a new species of Acotyledon Oudemans (Acari: Acaridae) feeding on eggs of the Sitotroga cerealella (Olivier) (Lepidoptera: Gelechiidae), with a method for determining sex in deutonymphal stages

A new species, Acotyledon hookisetoses sp. nov. (Acari: Acaridae), collected from the stock colony of Sitotroga cerealella reared on wheat grains, is described, and illustrated from adults and deutonymphs. Sex-specific morphological differences at the deutonymphal stage are reported and discussed.

Colony aging affects the reproductive performance of Swiss Webster females used as recipients for embryo transfer.

The objective was to evaluate the influence of colony aging in a Swiss Webster (SW) outbred stock used as recipients for embryo transfer. In the first study, a retrospective analysis was performed throughout several generations during a 38-month period in 2,398 embryos transferred to 108 SW recipients. A decrease in the percentage of live pups from transferred embryos was found at the end of the period. Impairment occurred due to the incidence of maternal cannibalism that increased from 0% to 67-100% (P<0.05), while pregnancy rate (pregnant/transferred recipients) and number of pups per delivered female were not affected throughout the period (P=NS). A following study was carried out to compare the reproductive performance of SW stock vs. B6D2F1 hybrid females in a 5-year interval. The study was conducted on a total of 893 embryos transferred to 40 females (20 SW and 20 B6D2F1) in Year #1, and 514 embryos transferred to 30 females (15 SW and 15 B6D2F1) in Year #5. No cases of maternal cannibalism were found on Year #1 in any of the strains (0/10 and 0/10). However, an incidence of 44,4% (4/9) was seen on Year #5 for SW, while for B6D2F1 the incidence was 0% (0/12) (P<0.05). Further examination of the uterus showed endometrial cysts and abnormal implantation sites in SW on Year #5 but not in B6D2F1 females. In conclusion, this study reports an impairment of the reproductive performance of an early aged SW outbred stock colony mainly due to the occurrence of maternal cannibalism. This finding has important implications for embryo transfer programs conducted in mouse facilities.

Genomes, expression profiles, and diversity of mitochondria of the White-footed Deermouse Peromyscus leucopus, reservoir of Lyme disease and other zoonoses

The cricetine rodents Peromyscus leucopus and P. maniculatus are key reservoirs for several zoonotic diseases in North America. We determined the complete circular mitochondrial genome sequences of representatives of 3 different stock colonies of P. leucopus, one stock colony of P. maniculatus and two wild populations of P. leucopus. The genomes were syntenic with that of the murids Mus musculus and Rattus norvegicus. Phylogenetic analysis confirmed that these two Peromyscus species are sister taxa in a clade with P. polionotus and also uncovered a distinction between P. leucopus populations in the eastern and the central United States. In one P. leucopus lineage four extended regions of mitochondrial pseudogenes were identified in the nuclear genome. RNA-seq analysis revealed transcription of the entire genome and differences from controls in the expression profiles of mitochondrial genes in the blood, but not in liver or brain, of animals infected with the zoonotic pathogen Borrelia hermsii. PCR and sequencing of the D-loop of the mitochondrion identified 32 different haplotypes among 118 wild P. leucopus at a Connecticut field site. These findings help to further establish P. leucopus as a model organism for studies of emerging infectious diseases, ecology, and in other disciplines.

Colonialism ties and stock markets: Evidence from Sub-Saharan Africa

This paper investigates the relationship among stock markets from the perspective of colonialism in sub-Saharan Africa. I posit that colonialism ties may induce former colonizers to invest more in their former colonies’ stock markets, as they have similar legal systems, accounting standards, and economic systems, among other factors. Using monthly data from 1999 to 2016, I use the cointegration and error-correction model techniques and show that there is a stable, long-run relationship between colonizer and colony stock markets. I also find a long-run relationship between colonizer and non-colony stock markets. This mixed evidence suggests that there may be other motives for investment in Africa.

Histopathological Changes in the Muscle of the Desert Locust, Schistocerca gregaria (orthoptera: acrididae)Treated with Insect Growth Regulator (IGR), Lufenuron

The present study aimed to minimize the pollution effects of traditional insecticides on the environment by development and synthesis of bio-insecticide and to evaluate insect growth regulators , Lufenuron as insecticidal agents. Stock colony of Schistocerca gregaria was used in this study which were kept in cages and fed on leaves of Castor plant. Treatment of the fifth nymphal instar of locusts by using insect growth regulator, Lufenuron were carried out. Results showed that, the treatment with different concentrations of Lufenuron (50, 75 and 100 ppm) resulted in nymphal mortality of 0.0, 12.33 and 21.01% respectively and percentages of adult malformed with 89.99, 73.34 and 72.33 respectively comparing with control. The current study showed that Lufenuron caused malfunction and decomposition of the components in the muscles of the desert locust.

Experimental and field investigation of non-biting flies as potential mechanical vectors of Echinococcus granulosus eggs

Synanthropic fly species can be potential mechanical vectors of many infectious agents. The potential of the flies to carry Echinococcus granulosus eggs is not fully documented. The purpose of the present study was to determine the possible role of non-biting flies to carry taeniid eggs. A total of 210 flies were collected from seven selected sites in areas of Kerman city, southeastern Iran from November 2016 to May 2017. Adult flies were live-caught using sweeping nets. Flies were placed individually in small glass bottles and transported to the laboratory. All the flies were killed by deep freezing and then identified to the species level using both morphological and molecular methods. The flies were homogenized in test tubes and genomic DNA was extracted and amplified by PCR. PCR protocols were used both to identify the live-caught flies to the species level, and for the detection of E. granulosus. The laboratory reared second generation flies were experimentally exposed to dog feces manually spiked by Echinococcus eggs. Two runs of experiments with 1–3 h of exposure were designed. For each experiment 20 flies were selected from the stock colony and were starved for three days. After each experiment, the flies were frozen for further molecular studies. The dominant fly species were Musca domestica and Lucilia sericata. No eggs were found on the body surface and/or guts of live-caught flies. After the first hour of exposure, 60%, of the flies of both species were found to harbor Echinococcus eggs. However, in the case of L. sericata 50% of the flies harbored Echinococcus eggs after 3 h of exposure. Results of the present study indicate the probable role of synanthropic flies in harboring Echinococcus eggs and mechanical transmission of cystic echinococcosis. When the helminth eggs are susceptible to desiccation grooming flies can remove many of eggs from exterior surfaces of them. Despite this result the role of synanthropic flies in the transmission of certain helminthiases should not be discounted because of their vagility and feeding mechanisms.

Laboratory colonization and mass rearing of phlebotomine sand flies (Diptera, Psychodidae).

Laboratory colonies of phlebotomine sand flies are necessary for experimental study of their biology, behaviour and mutual relations with disease agents and for testing new methods of vector control. They are indispensable in genetic studies and controlled observations on the physiology and behaviour of sand flies, neglected subjects of high priority. Colonies are of particular value for screening insecticides. Colonized sand flies are used as live vector models in a diverse array of research projects, including xenodiagnosis, that are directed toward control of leishmaniasis and other sand fly-associated diseases. Historically, labour-intensive maintenance and low productivity have limited their usefulness for research, especially for species that do not adapt well to laboratory conditions. However, with growing interest in leishmaniasis research, rearing techniques have been developed and refined, and sand fly colonies have become more common, enabling many significant breakthroughs. Today, there are at least 90 colonies representing 21 distinct phlebotomine sand fly species in 35 laboratories in 18 countries worldwide. The materials and methods used by various sand fly workers differ, dictated by the availability of resources, cost or manpower constraints rather than choice. This paper is not intended as a comprehensive review but rather a discussion of methods and techniques most commonly used by researchers to initiate, establish and maintain sand fly colonies, with emphasis on the methods proven to be most effective for the species the authors have colonized. Topics discussed include collecting sand flies for colony stock, colony initiation, maintenance and mass-rearing procedures, and control of sand fly pathogens in colonies.

Land-use change reduces habitat suitability for supporting managed honey bee colonies in the Northern Great Plains

Human reliance on insect pollination services continues to increase even as pollinator populations exhibit global declines. Increased commodity crop prices and federal subsidies for biofuel crops, such as corn and soybeans, have contributed to rapid land-use change in the US Northern Great Plains (NGP), changes that may jeopardize habitat for honey bees in a part of the country that supports >40% of the US colony stock. We investigated changes in biofuel crop production and grassland land covers surrounding ∼18,000 registered commercial apiaries in North and South Dakota from 2006 to 2014. We then developed habitat selection models to identify remotely sensed land-cover and land-use features that influence apiary site selection by Dakota beekeepers. Our study demonstrates a continual increase in biofuel crops, totaling 1.2 Mha, around registered apiary locations in North and South Dakota. Such crops were avoided by commercial beekeepers when selecting apiary sites in this region. Furthermore, our analysis reveals how grasslands that beekeepers target when selecting commercial apiary locations are becoming less common in eastern North and South Dakota, changes that may have lasting impact on pollinator conservation efforts. Our study highlights how land-use change in the NGP is altering the landscape in ways that are seemingly less conducive to beekeeping. Our models can be used to guide future conservation efforts highlighted in the US national pollinator health strategy by identifying areas that support high densities of commercial apiaries and that have exhibited significant land-use changes.

Discriminatory abilities of facultative slave-making ants and their slaves.

Intra-colony odor variability can disturb ants’ ability to discriminate against intruders. The evolutionary relevance of this phenomenon can be revealed by studies on colonies of slave-making ants in which the parasite, and not the host, is subject to selection pressures associated with living in a mixed colony. We examined how the European facultative slave-making species Formica sanguinea and its F. fusca slaves perform in discriminating ants from alien colonies. Results of behavioral assays showed that slave-maker ants respond with hostility to conspecific individuals from alien colonies but are relatively tolerant to alien slaves. Furthermore, the behavior of slaves indicated a limited ability to discriminate ants from alien parasitic colonies. The subdivision of colony fragments into mixed and species-separated groups demonstrated that contact with the parasite is necessary for F. fusca slaves to be re-accepted by former nestmates after a period of separation from the stock colony. The results presented in this paper are consistent with the following hypotheses: (1) F. sanguinea ants, as opposed to their slaves, are adapted to discriminate alien individuals in the conditions of odor variability found in a mixed-species colony, (2) the recognition of slaves by F. sanguinea ants involves a dedicated adaptive mechanism that prevents aggression toward them, (3) the odor of slaves is strongly influenced by the parasite with beneficial effect on the colony integrity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00040-016-0493-z) contains supplementary material, which is available to authorized users.

Laboratory and field tests of Carbaryl 5% against fleas in Lushoto district, Tanzania.

Background and Objective: Lushoto district in north-eastern Tanzania has been an active focus of plague disease since 1980 and many pesticides were used to control rodents and fleas from 1980 to 2003 when outbreaks occurred yearly. For over seven years ago commercial Carbaryl 5% powder has been used for controlling fleas in the area. However, there is no current research to substantiate its effectiveness either in the laboratory or in the field. Methods: Immature stages of Xenopsylla brasiliensis were collected from two villages in Lushoto and transported to Sokoine University of Agriculture, Morogoro and reared in an insectary to stock colony. Known weights of commercial Carbaryl 5% powder were thoroughly mixed with known weights of clean fine sand as to obtain a final concentration of 0.05% of Carbaryl+sand mixture. The same concentration was suspended in 50cc distilled water and pieces of filter paper were soaked in the solution, left at room temperature until the suspension was fully adsorbed and the papers were left to dry. Adult fleas of mixed ages and of both sexes were obtained from the stock colony and exposed to both Carbaryl+sand mixture and Carbaryl-adsorbed filter papers for various periods of time. Field trials were carried out at Manolo and Viti villages where house and rodent flea indices were determined before and after dusting with commercial Carbaryl 5% powder. Results: In the laboratory tests, 100% mortality occurred at 90 minutes exposure in Carbaryl+sand mixture experiments and at 35 minutes exposure to Carbaryl-adsorbed filter papers. LT 50 in both sets of exposure was 48.2 min and 23.1 min in Carbaryl+sand mixture and Carbaryl-coated filter papers tests respectively. In field trials, X. brasiliensis and Pulex irritans were the most abundant flea species. Post-dusting flea populations were significantly lower in treated than in control houses (p=0.028). House flea indices dropped from 7.7 to 0.33 and 37.8 to 0 in Viti and Manolo villages respectively at 3 months post-dusting. Conclusion: Commercial Carbaryl 5% powder in current use was still effective against potential flea vectors in Lushoto.

Contamination Delays the Release of Laricobius osakensis for Biological Control of Hemlock Woolly Adelgid: Cryptic Diversity in Japanese Laricobius spp. and Colony-Purification Techniques

Laricobius osakensis (Coleoptera: Derodontidae) was imported from Japan to the United States in 2006 for study in quarantine facilities as a potential biological control of Hemlock Woolly Adelgid. Laricobius osakensis was released from quarantine in 2010, but it was soon discovered that the colony also contained a cryptic species, Laricobius naganoensis. This led to the placement of L. osakensis back into quarantine and development of a method to purify the colony. To distinguish the two species, we designed a restriction fragment length polymorphism (RFLP) assay using mitochondrial DNA sequences and developed a non-lethal testing method. Twenty-one diagnostic nucleotide sites separated the two species, and they both exhibited extraordinary intra-specific haplotype diversity. Sequencing the ITS2 nuclear region did not produce evidence of hybridization between the species in the field or in the lab colony. Splitting the colony into small groups and testing their species composition was successful in isolating L. osakensis. Efforts should be made to maintain high genetic diversity in L. osakensis colonies. Continued genotyping of new colony stock will be necessary to fully characterize the diversity within both species.

Resistance of citrus genotypes to Phyllocnitis citrella Stainton (Lepidoptera: Gracillariidae)

The development and reproduction of the citrus leafminer (CLM), Phyllocnistis citrella Stainton, were evaluated in six citrus genotypes in order to identify genotypes with resistance traits that could be applied in a program for the development of citrus varieties resistant to the citrus leafminer. Tests were conducted under controlled laboratory conditions (25 ± 1ºC, 70 ± 10% RH, and 14h photophase). Seedlings of each genotype tested were infested with eggs obtained from a stock colony of CLM maintained on 'Cravo' lemon (Citrus limonia L. Osbeck), and the duration and survival of the eggs, larval and pupal stages, pupal size and weight, fecundity and longevity of adults, and sex ratio were evaluated. No influence was observed on the duration and survival of eggs, larvae and pupae of P. citrella. However, pupae obtained in the hybrid C x R(4) were significantly smaller and lighter than pupae from the remaining treatments. Adult females from the hybrids C x R(4) and C x R(315) were the least fecund. However, the lowest value for the corrected reproductive potential (CRP) was recorded in the hybrid C x R(315), suggesting that this genotype is the least favorable for the development and reproduction of CLM. On the other hand, the highest CRP value obtained in the 'Rugoso' lemon confirms the susceptibility of this genotype, indicating it as the most suitable for CLM.

Biology of Leipothrix dipsacivagus (Acari: Eriophyidae), a candidate for biological control of invasive teasels (Dipsacus spp.)

The present study describes key aspects of the biology of Leipothrix dipsacivagus, an eriophyid mite that is under study as a biological control candidate of Dipsacus fullonum and D. laciniatus (Dipsacaceae). Preliminary host-specificity tests have shown that it can develop and reproduce only on Dipsacus spp. (teasels). Studies were conducted in a laboratory at 26 ± 2(o)C with 16 h of light per day. Mites for the stock colony were collected from D. laciniatus in Klokotnitsa, Bulgaria and reared on rosettes of D. laciniatus in the laboratory. Unfertilized L. dipsacivagus females reared in isolation from the juvenile stage produced male offspring only, while progeny of fertilized females were of both sexes, suggesting arrhenotokous parthenogenesis with haplodiploid sex determination. Experiments were designed to compare male progeny from fertilized females to males from unfertilized females and to compare males and females from fertilized females. Male progeny of virgin mothers had significantly longer durations of active immature stages and total egg-to-adult period than male progeny of fertilized females. Female progeny had significantly longer durations of egg incubation, active immature stages and egg-to-adult period than male progeny from fertilized mothers. Adult longevity was significantly greater in females than in males. Fertilized females produced significantly more eggs per day and overall than virgin females. The results of this study suggest that fertilization status of L. dipsacivagus females can affect both their own fecundity and the development of their male progeny.

Growth of Chrysomya megacephala (Fabricius) Maggots in a Morgue Cooler*

In estimating the postmortem interval (PMI) using maggots obtained during autopsy, the forensic entomologist makes decisions regarding the effects of low-temperature storage of the body on the insects. In this case report, a corpse was found in an abandoned house in the residential area of Bukit Mertajam, Penang, Malaysia. The maggots were found to be alive inside the mouth of the deceased although the corpse had been in the morgue cooler for 12 days. The maggots were reared and identified as Chrysomya megacephala (Fabricius). The emerged adult flies were kept as a stock colony, and the duration of development under the indoor fluctuating temperature regime was studied. The total duration of developmental process of this species was 9.5 ± 0.5 days, and the PMI estimated was 3.2 ± 0.6 days. This case report demonstrates the survival of Ch. megacephala maggots for 12 days and their growth inside the morgue cooler.

Desempenho do parasitóide Nasonia vitripennis (Walker)(Hymenoptera, Pteromalidae) utilizando como hospedeiro Cochliomyia macellaria (Fabricius) (Diptera, Calliphoridae), sob diferentes tempos de exposição

The post-embryonic development, the productivity of the host pupae, the rate of parasitism and the sex ratio of Nasonia vitripennis reared in pupae of Cochliomyia macellaria were studied. Different exposition times were used: 24, 48, 72, 96 hours. Nulliparous females originating from the stock colony were individualized in test tubes covered with hydrophobic cotton and containing the host pupae. Each treatment was constituted by 10 replicates. After exposition, the hymenopterans were discarded and the host pupae were individualized in test tubes until the emergency of the adults of C. macellaria or N. vitripennis. Samples of host pupae not exposed to parasitism and receiving the same experimental treatment, were used as control. The parasitoids showed a slower development in relation to the increase exposition time. The 72 hours exposition was the one which enabled higher productivity. The sex ratio tended towards a deviation for the birth of females after 48 hour. The rate of parasitism increased up to 72 hours. The rate of dipteran's emergence showed a decrease until 72 hours.