Infrared Absorption Characteristics of Culicoides sonorensis in Relation to Insect Age

Columbia International Publishing American Journal of Agricultural Science and Technology (2014) Vol. 2 No. 2 pp. 49-61 doi:10.7726/ajast.2014.1006

Research Article

______________________________________________________________________________________________________________________________ *Corresponding e-mail: [email protected] 1 Biological and Agricultural Engineering, Kansas State University, Manhattan, Kansas 2 USDA ARS, Arthropod-Borne Animal Diseases Research Unit, Center for grain and Animal Health

Research, Manhattan, Kansas 3 USDA ARS, Engineering and Wind Erosion Research Unit, Center for grain and Animal Health Research

Manhattan, Kansas 49

Infrared Absorption Characteristics of Culicoides sonorensis in Relation to Insect Age

Kamaranga H. S. Peiris 1, Barbara S. Drolet 2, Lee W. Cohnstaedt2, and Floyd E. Dowell3*

Received 27 November 2013; published online 14 June 2014 © The author(s) 2014. Published with open access at www.uscip.us

Abstract Biting midges can transmit diseases that significantly impact livestock in many parts of the world. The age structure of an insect vector population determines its likelihood of transmitting pathogens because the older insects are more likely to be infected than younger ones. Understanding the insect age distribution allows for predictions of their behavior, habitat, vector competence and the vector-borne disease epidemiology. Most insect age grading techniques are laborious and slow, thus we investigated the novel application of mid-infrared (MIR) spectroscopy to determine insect age. Female biting midges (Culicoides sonorensis) were anesthetized with chloroform at 1, 4, 7, 10, 13, and 16 days after eclosion. MIR Attenuated Total Reflectance (ATR) spectra of desiccated insects were collected using a Fourier Transform Infrared spectrometer. Transmission spectra of 1, 7, and 13 day old midges were also taken via potassium bromide (KBr) disks prepared with homogenized desiccated insects in each age group. ATR and transmission spectra had identical bands and provide chemical information about the whole insect. The ratio of absorbance of ATR spectra at 1634/1540 cm-1 showed a systematic change with increasing insect age. A similar trend was also observed in the transmission spectra. These absorption bands may be due to the absorbance of chitins and proteins. Therefore, the observed changes in absorption ratios may reflect qualitative or quantitative changes in insect cuticle and/or body proteins in relation to chronological age. When using absorbance data, insects were classified as young (<= 7 days) with 89.2% accuracy, and old with 63.5% accuracy. These results suggest that infrared spectroscopy may be used to develop a rapid method for age grading of midges. Keywords: Biting midge; Age grading; Infrared spectroscopy

1. Introduction Bluetongue virus (BTV) of domestic and wild ruminants is an arthropod-borne virus belonging to the family Reoviridae and genus Orbivirus. It is an economically important arthropod-borne animal

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disease prevalent in many parts of the world including certain geographical regions of the USA (Tabachnick, 2004; Schmidtmann et al., 2011). Livestock industries suffer significant economic losses due to treatment costs, decreased production, and non-tariff trade restrictions on BTV positive animals and animal germplasm following BTV infection (Tabachnick 1996; Hoar et al., 2003). Several species of biting midges of the genus Culicoides transmit bluetongue in different parts of the world (Tabachnick, 2004) and C. sonorensis Wirth & Jones (formerly C. variipennis) (Diptera: Ceratopogonidae) is the primary vector in North America (Tabachnick 1996; Holbrook et al., 2000). The age structure of an insect vector population determines its capacity to transmit pathogens and as a consequence is an important parameter that influences the epidemiology of vector-borne diseases. Therefore, the age composition of insect populations is monitored to determine their current or potential impact on outbreak epidemiology, as well as to appraise the effectiveness of age-specific insect pest management strategies. A range of techniques based on predictable changes in reproductive systems (Wall et al., 1991; Moon and Krafsur, 1995), somatic changes (Dyce 1969; Tyndale-Biscoe and Kitching, 1974) or cuticular degradation (Snow and Tarimo, 1985) are available for chronological and physiological age-grading of insects. However, each method has its own associated drawbacks. Examination of the stage of ovarian development is a commonly used age-grading technique, however, its success may be affected by factors other than physiological age such as protein intake and delayed oviposition due to site deprivation or unmated status. Measurement of changes in pteridine levels are used for age-grading certain male and female insects but may not be applicable for individual insects. Likewise, age-grading by wing fray may be a convenient technique for age-grading flies but wing damage may also be affected by other factors such as predator attacks and abrasions from habitats. Therefore, it can be beneficial to use two or more complementary age-grading techniques, as inherent problems and inaccuracies of one may be minimized by the strengths of the other (Hayes and Wall, 1999). Because most of these techniques are laborious, spectroscopic methods have also been developed as rapid methods for age determination of numerous insects. Near-infrared spectroscopy has been used to age-grade stored-product beetles (Perez-Mendoza et al., 2004), houseflies (Perez-Mendoza et al., 2002), mosquitoes (Mayagaya et al., 2009), and biting midges (Reeves et al., 2010). Near-infrared spectroscopy uses the overtone and combination bands of fundamental mid-infrared absorption bands of insects. The fundamental infrared absorption bands are found in the mid-infrared region (~ 4000-400 cm-1) therefore it is worth studying the mid-infrared absorption patterns of insects as it may help develop more direct and straightforward method for insect age classification. Study of mid-infrared absorption patterns of different age classes of an insect species may help identify the differences in fundamental infrared absorptions due to subtle changes in chemical composition related to the age of insects. If there are age related differences in mid-infrared absorption patterns, it may be possible to use mid-infrared spectroscopy to develop a rapid, complementary method for age grading of insects. Therefore, this study was conducted to explore the mid-infrared absorption patterns of female C. sonorensis midges in relation to their chronological age using a Fourier Transform Infrared/ Fourier Transform Near-infrared (FTIR/FTNIR) spectrometer. Female insects were used for age determination because only the female biting midges act as potential vectors of the BTV disease in ruminants. If systematic changes in the infrared absorption patterns occur due to age of insects, infrared spectroscopy may be used to develop rapid methods for age grading of C. sonorensis midges.

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2. Materials and Methods 2.1 Insects C. sonorensis adults, from an established colony of midges from Idaho (AK) (Jones and Foster 1974, 1978), were maintained in nylon mesh covered cardboard paper containers at 27°C and provided 10% sucrose ad libitum. Containers were sampled at 1, 4, 7, 10, 13, and 16 days post-eclosion. Midges were anesthetized in the container by placing a cotton plug with chloroform on the net and tightly covering the container for about 30 minutes. Females were separated from males using the morphological characteristics of genitalia and antennae. Female midges were then transferred to a plastic vial with silica gel and paper plug for desiccation. Vials were closed and stored in a dehumidifier chamber (~10 % RH) until used. Infrared spectra of midges were collected using Attenuated Total Reflectance (ATR) and transmission sampling methods. 2.2 ATR spectra of midges Using fresh and desiccated midges, preliminary tests were done to develop a scanning protocol to obtain ATR spectra of midges. Examination of spectra showed that desiccated insects produce better spectra. Therefore in this experiment only desiccated insects were used for collecting ATR spectra. Desiccated insects were scanned to acquire the mid-infrared (MIR) spectra from 4000- 380 cm-1 using the Universal Attenuated Total Reflection (UATR) accessory of the Perkin Elmer Spectrum 400 FTIR/FTNIR spectrometer (Perkin Elmer, Waltham, MA). A single midge was placed at the center of the UATR diamond crystal using a pair of forceps and the whole insect was gradually pressed to a force gauge reading of 145-150 N using the large tip of the pressure arm. The spectrometer conditions used were resolution = 2 cm-1; data interval = 0.5 cm-1; scan mirror speed = 0.1 cm/s; number of scans = 8. Spectra from 32-36 insects were recorded for each age group. A background spectrum was collected with a clean crystal. 2.3 Transmission spectra of midges Transmission spectra of 1, 7, and 13d old female insects were collected using KBr pellets. A sample of 12 desiccated female midges (approximately 1.0 mg) was quickly ground to a fine dust using an agate mortar and pestle. About 50 mg of oven dried FTIR SpectroGrade TM KBr powder (International Crystal Laboratories, Garfield, NJ) were mixed with the ground midges using a stainless steel spatula. The mixture was immediately pressed using a KBr Quick Press (International Crystal Laboratories) and a die set to form a KBr disk in a 7mm stainless steel collar. Each disk was scanned in transmission mode immediately after preparation with the same spectrometer conditions used for ATR scanning. Transmission spectra of insects were collected for comparison with ATR spectra of insects to see if the ATR technique produces spectra of whole insects or only of the surface layer of the exoskeleton of insects. 2.4 Analysis of spectra The spectra collected by the Perkin Elmer instrument in .sp data format were imported and converted to .spc format for further spectral analysis by the GRAMS/AI 8.0 software package (Thermo Electron, Salem, NH). The absorbance spectra were exported to .xls format using the Excel Exchange program of GRAMS/AI 8.0. Spectra in .xls format were retrieved by Microsoft Excel 2007 to calculate selected spectral band peak absorption values of each spectrum. Peak height of ATR spectral bands were computed by subtracting the peak absorbance value by the absorbance at 2000 cm-1 (baseline absorbance) to account for the height gain due to baseline shift of the

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spectrum. The absorbance ratios (A peak1/Apeak2) of two spectral bands were calculated for all possible combinations of selected spectral bands. Those ratios were plotted against insect age with the objective of finding absorption ratios that systematically change with insect age.

3. Results 3.1 Characteristics of midge ATR spectra Preliminary tests were done to develop a scanning protocol to obtain ATR spectra of midges using both fresh and desiccated insects. Spectra of fresh insects showed a broader O-H stretch band around 3400 cm-1 with a baseline shift and higher absorption values compared to that of desiccated insects (Fig. 1). Desiccated insects yielded spectra only with a slight baseline shift, with spectral bands in the 3000-2800 cm-1 region well pronounced. Therefore, insects were desiccated by storing in plastic vials with silica gel before collecting the ATR spectra.

Fig. 1. Mid-infrared Attenuated Total Reflectance spectra of fresh and desiccated midges.

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Fig. 2. Mid-infrared Attenuated Total Reflectance absorbance spectra of 1, 4, 7, 10, 13and 16 days

old desiccated female midges. Individual insects were used to collect the ATR spectra. The average ATR spectra (n = 32-36) for each age class are presented in Fig. 2. The ATR spectra absorbance values were all below 0.50 absorbance units with a slight baseline shift of spectra. The spectra of insects in the transmission mode were collected via KBr pellets prepared using 12 desiccated insects per pellet. The transmission spectra of 1, 7, and 13d old insects are given in Fig. 3. Transmission spectra had higher absorbance values (up to 1.37 absorbance units at 1653 cm-1) with a highly variable baseline shift as compared with the ATR spectra. Comparison of spectra of midges acquired with ATR and transmission sampling techniques (Figs. 2 and 3) showed that all spectral bands in both ATR and transmission spectra were nearly similar. Transmission spectra of desiccated midges had a strong broad band (3700 - 3000 cm-1) similar to that of ATR spectra of fresh midges (Figs. 1 and 3). The notable spectral bands with peak positions as observed in ATR and transmission spectra of midges are presented in Table 1. A shift in the position of some spectral band peaks were noted between ATR and transmission spectra.

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Fig. 3. Fourier Transformed Infrared Transmission spectra when scanning KBr pellets comprised of 1, 7, and 13 day old desiccated female midges.

3.2 ATR spectral differences in relation to insect chronological age As a preliminary step to identify any spectral band height ratio(s) that systematically change in relation to insect age, absorption peak height of 16 selected ATR spectral bands and the absorbance ratios for 240 two-band combinations of those selected bands were computed. Those band height ratios were plotted against insect age and examined (data not shown). While most absorbance ratios fluctuated irregularly, a systematic relationship was noted in the ratio of Amide I to Amide II absorption with peaks at 1634 cm-1 (A1634) and 1540 cm-1 (A1540), respectively . The absorbance ratios A1634/A1540 computed for each insect spectrum in each age group and the average ratio is given in Fig. 4. The absorbance ratios were generally higher for younger (1, 3, and 7days) insects when compared to the absorbance ratios for older (10, 13, and 16 days) insects. Ratios of absorbance around these peaks gave similar classification results. Generally, insects less than 7 days had an absorbance ratio > 1.13 while older insects had a ratio < 1.13. Therefore, the proportion of insects in each age group with absorbance ratio above or below 1.13 was computed (Table 2). A high proportion (81.8 - 96.9 %) of midges aged 7 days or less had a

ratio >1.13 while 43.8 - 82.9 % of 10 days or older insects had an absorption ratio < 1.13.

To further validate these observations regarding the changes in the A1634/A1540 absorbance ratio of ATR spectra in relation to insect age, 10 female midges from each age group (1d to 16d) were scanned under the same spectrometer conditions as a test population. The absorption ratio A1634/A1540 was computed for each spectrum and plotted against insect age (Fig. 5). Results of this test population also showed that with increasing chronological age of insects, the absorption ratios decrease.

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Table 1 The major mid-infrared spectral bands of Attenuated Total Reflectance (ATR) and Transmission (T) spectra of desiccated midges.

Spectral

Band (cm-1)

ATR Band Peak (cm-1)

T Band Peak (cm-1)

Assignment Origin

3150-3650 ND* ~ 3450 OH Stretch chitin, water,

3150-3650 3275 3295 NH (As) Stretch chitin, protein,

2950-2970 2956 2960 CH3 (As) Stretch chitin, wax

2910-2940 2921 2927 CH2 (As) Stretch chitin, wax

2865-2880 2875 2876 CH3 (S) Stretch chitin, wax

2840-2860 2853 2853 CH2 (S) Stretch chitin, wax

1620-1650 1634 1653 C=O Stretch (Amide I) chitin, protein

1525-1550 1540 1542

Out-of-phase combination of N-H in plane bend and C-N Stretch (Amide II)

chitin, protein

1500-1525 1518 ND Amide II chitin, protein

1440-1460 1455 1454 CH2 bend chitin, wax

1390-1420 1404 1397 C-H, C-CH3 bend chitin, wax

1370-1390 1381

C-H, C-CH3 bend chitin, wax

1300-1320 1313 1314

In phase combination of N-H in plane bend and C-N Stretch (Amide III)

chitin, protein

1220-1250 1237 1237 N-H bend chitin, protein

1130-1170 1152 1154 C-O-C ring (As) Stretch chitin

940-1130 1075 1026 C-O Stretch chitin

450 -880 520 550 C-C bend chitin

* ND-These band peaks are not discernible in respective spectra. Table 2 Percentage of insects in each age group with an absorption peak ratio of A1634/A1540 above

or below 1.13.

Age (Days)

N

Percent insects, %

Abs 1634/1540

>1.13 <1.13

1 32 96.9 3.1

4 36

88.9

11.1

7 33

81.8

18.2

10 35

17.1

82.9

13 36

36.1

63.9

16 32 56.3 43.8

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Fig. 4. Absorbance ratio A1634/A1540 of 1-16d old female midges.

Fig. 5. Absorbance ratio A1634/A1540 of test population of 10 female midges from each age group.

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Fig. 6. Amide I and Amide II bands of ATR (left) and Transmission (right) spectra. 3.3 Characteristics of midge transmission spectra Transmission spectra had a Amide I peak at 1655 cm-1 and one Amide II peak at 1545 cm-1 (Fig. 6). The dual Amide II band peaks observed in ATR spectra were fused together as a single band in the transmission spectra of midges. The effect of the baseline shift in transmission spectra were corrected by subtracting peak absorbance value by the absorbance value at 2000 cm-1. The absorbance ratio A1655/A1545 of the midge transmission spectra for1d, 7d, and 13d midges were 1.464, 1.405, and 1.350, respectively, again showing a decreasing trend with increasing insect age.

4. Discussion Compared to ATR spectra of desiccated insects, the broader O-H stretch band of fresh insects around 3400 cm-1 with higher absorption values (Fig. 1) may most likely be due to the higher water content of fresh insects. The baseline shift and broadening observed among transmission spectra (Fig. 2), though the KBr disks were prepared using desiccated insects, may be due to the variations in path length resulting from minute differences in the thickness of KBr disks as well as the moisture absorbed by KBr powder during pellet preparation.

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The ATR technique is ideal for highly absorbing samples and for surfaces and thin film measurements, because MIR energy penetrates only a few microns into materials (Grdadolnik, 2002). ATR spectra had relatively stronger absorption bands at lower frequencies compared to those bands in the transmission spectra. Also a slight shift in the position of some band peaks was noted (Table 1). These changes in the strengths and position of bands may due to the differences in penetration depth which is influenced by the wavelength of the incidence radiation and refractive index of the sample (Grdadolnik, 2002; Griffiths and de Haseth, 2007). The size of the biting midge, C. sonorensis, is very small (~2 mm in length) and the desiccated insects are smaller than the scanning window which makes it possible to get the spectra of whole insects. When the desiccated insects were crushed by pressing onto the diamond crystal for ATR scanning, internal body parts of the insect are pressed against the ATR crystal. As a result, transmission spectra collected by homogenizing whole midges and ATR spectra collected by pressing the whole insect to the ATR crystal may provide information about the whole body composition of midges. Insect exoskeleton is made up of an epicuticle consisting of waxes and a procuticle composed mainly of chitin and protein matrix (Anderson, 1979). Various insect body muscles and organs under the exoskeleton may be predominately composed of proteins as the major building block. Insect wax is composed of a variety of saturated alkanes (C23 to C31) often with one or two methyl branches and wax esters, sterol esters, free fatty alcohols and acids (AOCS , 2012). Chitin, the major component of insect cuticles, is a long chain polymer of N-acetylglucosamine which has -CH3, -CH2, -C=O, -NH and C-O-C groups in its structure (Pillai et al., 2009). Therefore, the MIR spectra of midges are expected to show a combination of bands common to wax, chitin, proteins and water spectra. The major absorption bands of the midge spectra may have originated from the absorptions of compounds like chitin, proteins, wax and bound water in desiccated midges (Table 1). Similar band assignments have been reported in FTIR spectroscopic analysis of chitins (Brugnerotto et al., 2001; Cardenas et al., 2004), bee wax (Zimnicka and Hacura, 2006), and proteins (Barth and Zscherp, 2002; Cai and Singh, 2004). Studies have shown that proteins and enzymes of whole body homogenates of Culicoides midges change during insect morphogenesis (Nunamaker and McKinnon, 1989). Shih and Fallon (2001) showed changes in fat body proteins during gonotropic cycle of Aedes aegypti mosquitoes. C. sonorensis midges also respond to environmental stresses, such as subfreezing temperatures of 0C -15C, by producing various stress proteins (Nunamaker et al., 1996). It may also be possible that the sclerotization of cuticle (Hopkins and Kramer, 1992) changes with the age of insects. Amide I and Amide II bands observed in the midge spectra mainly originate from the vibrations of C=O, N-H found in proteins and chitins. Therefore, the observed changes in the absorbance ratios may be due to qualitative and/or quantitative changes in insect cuticle and body proteins with the physiological development respective to chronological age. The ability of the MIR spectroscopy to detect those systematic changes that occur with the aging of insects as demonstrated in this study makes this technique suitable for age determination of midges. In regard to spectral data collection techniques, the ATR technique can be conveniently used for rapidly scanning individual midges for age grading. ATR provides the spectral information of whole insect without the sample preparation that is required with transmission spectra. The ATR spectrum of an insect could be collected in about 30 seconds, enabling rapid evaluation of insects for age grading. It also takes some time to desiccate insects, but we have not yet evaluated

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the minimum time required to get insects desiccated enough to get a good spectrum. Typically, it takes 2-3 days to desiccate insects to collect spectra from insects that are stored in closed vials with silica gel. If it takes >2 days to bring the samples from the field, then perhaps the insects may be ready for scanning immediately after arrival. Other alternative desiccation methods to reduce time required from collection to scanning need to be studied in order to reduce the time required to determine the age distribution of an insect population. The results of this study showed that changes in the chemical composition of insects related to age could be detected due to the changes in the absorption of infrared radiation. A systematic change in absorption patterns were noted in the absorption bands in the 1650-1500 cm-1 spectral range. The Amide I and Amide II absorption bands of the midge spectra in this spectral region can be assigned to the infrared absorption by the fundamental vibrations of C=O and N-H atomic groups in compounds such as chitins, waxes and proteins abundantly found in cuticle and internal body parts of midges. Preparation of KBr disks for transmission spectroscopy is time consuming and often difficult with individual midges due to their small size. However, ATR spectroscopy could be used to collect spectra of individual insects without any sample preparation. The experimental results also showed that ATR spectral bands of midges are identical to the transmission spectral bands of midges, indicating that ATR spectra carries information related to the whole body composition of midges. This allows one to use ATR spectroscopy to develop rapid spectroscopic methods for age grading compared to use of transmission spectroscopy.

5. Conclusion The age classifications reported in this study used a selected absorption ratio of 1.13 to demarcate young from old insects. It used wavelengths at 1540 and 1634 cm-1 to demonstrate that age related systematic changes in infrared absorptions occur in midges. Therefore, infrared spectroscopy may be used to develop a rapid complementary method for age grading of C. sonorensis. Determination of the proportion of young and adult insects in a midge population by using such a technique will be important for rapidly assessing risk of vector population density and general arbovirus epidemiology.

Acknowledgements The authors thank James Kempert and William Yarnell (ABADRU, USDA) for providing midges.

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